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macromolecules in solution steadily undergo conformational changes at room temperature . various structural studies on diverse model systems have shown that the conformational plasticity of proteins plays a key role in molecular mechanisms such as catalytic activity , biomolecular recognition , and allosteric regulation . thus , characterizing dynamics of biological macromolecules is crucial to understanding their biological activity and function . molecular dynamics ( md ) simulations have proven to be an efficient tool to capture the flexibility of macromolecules . yet , state - of - the - art md simulations routinely capture dynamics on the nanosecond to microsecond time scale , while many biologically significant motions appear on the millisecond time scale or slower . inherent limitations in conformational sampling can either be overcome by usage of dedicated simulation hardware or by application of enhanced sampling algorithms . accelerated md ( amd ) is a promising enhanced sampling technique , which improves the efficiency of conventional md ( cmd ) simulation without a priori knowledge of the potential energy surface . introduction of a continuous , non - negative bias potential increases escape rates from local energy basins . thus , the conformational space is sampled more extensively at negligible computational overhead costs . the versatile applicability of amd simulations has repeatedly been proven on manifold macromolecular systems . current amd studies predominantly focus on analyzing the global dynamics of the obtained conformational ensembles . yet , for a comprehensive understanding of biomolecular properties , it is crucial to be able to localize flexibility in specific protein domains . current approaches estimating local dynamics in amd simulations are limited to expensive large - scale calculations of amide - order parameters from multiple amd trajectories on various acceleration levels . rather than approximations of nmr observables , a straightforward approach based on the thermodynamics of a system would be desirable . so far , no metric is available to directly quantify local flexibility from amd trajectories based on the captured thermodynamics of the system . we propose residue - wise dihedral entropy as the first methodology to efficiently characterize local dynamics of macromolecules from amd simulations . to confirm the validity and efficiency of our approach , we apply the metric to the model systems alanine dipeptide ( di - ala ) and bovine pancreatic trypsin inhibitor ( bpti ) . the conformational dynamics of bpti have been investigated thoroughly in nmr experiments as well as in large - scale computer simulations . it has already been demonstrated in previous studies that metrics for local flexibility in a 1 ms cmd simulation of bpti track the characteristic motions of bpti , known from nmr and global flexibility studies . here , we show that residue - wise dihedral entropies deriving from a 500 ns amd and a 1 ms cmd simulation of bpti correlate remarkably . application of our metric on amd trajectories provides a possibility to track low - frequency local dynamics on the millisecond time scale . additionally , we apply our metric to cmd and amd simulations of the major birch pollen allergen bet v 1.0101 ( bet v 1a ) . bet v 1a is a highly immunogenic storage protein and most prominent for causing seasonal pollen allergy in the northern hemisphere . despite a sequence similarity of more than 95% and minor differences in their 3d structures , the more than 13 reported isoforms of bet v 1a vary strongly in their immunogenicity . investigations on differences in proteolytic stability and ligand binding of different isoforms and mutants of bet v 1a suggest a linkage between immunogenic potential and conformational flexibility . the accuracy of our results is underlined by comparison to nmr data , displaying analogous trends in experimentally and computationally estimated flexibility . our results show that dihedral entropies from amd simulations are an efficient tool to describe local protein dynamics on the millisecond time scale . all structures were prepared in moe ( molecular operating environment , chemical computing group , version 2014.0901 ) using the protonate3d tool . with tleap of the ambertools15 package , all three systems were soaked into a truncated octahedral solvent box of tip3p water molecules . for di - ala , the minimum wall distance was set to 12 and for bpti and bet v 1a to 10 . parameters for all three systems derive from the amber force field 99sb - ildn . precedent cmd simulations as well as all amd simulations were performed in npt ensemble using pmemd.cuda . bonds involving hydrogen atoms were restrained by applying the shake algorithm , allowing a time step of 2.0 fs . atmospheric pressure of the system was preserved by weak coupling to an external bath using the berendsen algorithm . the langevin thermostat was used to maintain the temperature during simulations at 300 k for di - ala and bpti and 310 k for bet v 1a ( human body temperature ) . all amd simulations were performed using the dual - boost protocol implemented in pmemd.cuda . thereby the total potential is accelerated and an extra boosting is applied to the dihedral potential . it has been shown that dual - boost amd simulations sample the diffusive solvent motions more extensively . ensemble averages as well as entropy estimates converge faster than in dihedral - boost amd simulations . all simulations were analyzed using cpptraj in ambertools15 , the reweighting protocol provided by miao et al . , and in - house scripts . the free energy profile was reconstructed from the amd simulations via boltzmann reweighting using a maclaurin series expansion ( up to the tenth order ) as the approximation for the exponential term , as suggested in previous studies . the local backbone flexibility profiles were estimated from the resulting reweighted one - dimensional free energy profiles and state populations , respectively , of the backbone dihedrals and . the entropy is calculated by integration of the reweighted state populations of a given dihedral . a high entropy of a residue backbone dihedral indicates high local backbone flexibility . in the presented work , we prioritized dihedral entropies s over s in the representation protein dynamics as they captured the backbone dynamics more comprehensively . yet , s alone does not reflect the entire backbone dynamics , and dihedral entropies s were calculated as well ( si ) . for the reference cmd simulation of alanine dipeptide ( di - ala ) , a 10 s trajectory comprising 100,000 frames , previously performed in our group , was reanalyzed . residue - wise dihedral entropies of the reference cmd simulations were calculated from probability density functions reconstructed by nonparametric kernel density estimation . as proposed by botev et al . , we optimize the bandwidth of the kernel function by cross validation , resulting in a continuous probability density function for each dihedral . we periodically duplicate our data to minimize the overestimated flexibility at the boundaries . p(x ) log(p(x ) ) on its probability density function p(x ) as described in huber et al . the standard deviations were calculated by splitting the trajectory into 100 segments . for the amd calculations , the solvated and equilibrated di - ala system was simulated for 1 s and stored as 500,000 equal - spaced snapshots ( 2 ps spacing ) . the amd boosting parameters were calculated as suggested in previous studies ( see si for further information ) . to minimize the statistical noise ( and capture dynamics corresponding to 10 s of cmd ) , the resulting trajectory was divided into 200 segments of 5 ns each ( 2500 frames ) using the segments for averaging ( si , figure s4 ) . from the reweighted dihedral populations , a ramachadran plot was created to ensure sufficient and accurate sampling of conformational space ( si , figure s1 ) . to compare amd and cmd free energies , we calculated the free energy of the cmd trajectory using the reweighting protocol by miao et al . we investigated several different bin sizes , and the jaggedness of the cmd profile disappears only using a bin size above 20 ( si , figure s2 ) . , we observe a bin size of 6 to be a reasonable compromise between accuracy and statistical noise . d. e. shaw research kindly provided us with a long time scale 1.03 ms trajectory comprising 4,140,000 snapshots as a cmd reference . we used the same joint neutron / x - ray refined structure of bpti ( pdb : 5pti ) as shaw et al . as the starting structure for our large scale cmd and amd simulations . a 500 ns amd simulation was performed , and the trajectory was stored as 250,000 snapshots . the amd boosting parameters were calculated as suggested by pierce et al . and can be found in the supporting information . to localize the observed dynamic hot spots , we calculated dihedral entropies as described earlier for 1 ms and 1 s of cmd and 500 ns of amd sampling time . on the basis of the crystal structure of bet v 1a with pdb i d 4a88 , we sampled 3 s of cmd simulations on bet v 1a . suitable amd boosting parameters for bet v 1a were determined by a systematic search ( si ) . we performed a 1 s amd simulation stored as 100,000 equally spaced snapshots . the trajectory was split in 50 segments of 20 ns each ( 2000 snapshots ) and reweighted as described above . nmr order parameters were kindly provided by grutsch et al . ; the experimental setup has been described elsewhere . to establish our approach , we calculated backbone dihedral entropies for and of alanine dipeptide from 5 ns amd sampling and used a 10 s cmd simulation as a reference ( figure 1 ) . we find a significant reproduction of local minima and overall shape of the potential energy surface for the backbone dihedral of di - ala . as reported before , we also observe a shift in the extrema of backbone dihedral to smaller values in amd simulations . exemplary , in the cmd simulation , an energy minimum is found at 24 , while the corresponding minimum in the amd results is recovered at 42. still the overall energy landscape of the reweighted amd trajectory is in reliable agreement with the cmd results . from a reweighted amd trajectory to dihedral entropies of alanine dipeptide : state populations p are calculated from free energy profiles of the backbone dihedral of a 10 s cmd ( black ) and a reweighted 5 ns amd ( red ) trajectory . integration of the resulting state populations leads to the dihedral entropy s,cmd = 42.08 j/(mol k ) and s , amd = 45.42 j/(mol k ) . considering these errors , the resulting dihedral entropies , s,amd = 41.03 ( 2.95 ) j/(mol k ) and s,amd = 45.42 ( 3.25 ) j/(mol k ) , agree very well with those of the cmd ensemble , s,cmd = 40.20 ( 0.60 ) j/(mol k ) and s,cmd = 42.08 ( 0.39 ) j/(mol k ) . to benchmark our local flexibility metric , we analyzed a 500 ns amd simulation of bpti and compared it to a 1 ms cmd simulation provided by d. e. shaw research ( figure 2 ) . it has been demonstrated previously that 500 ns of bpti amd simulation cover a conformational space equivalent to a 1 ms cmd . in addition to the findings of pierce et al . , we observe equal assessment of local backbone flexibility for the 500 ns amd and 1 ms cmd simulation . in the 1 ms reference , cmd maxima of s are found in regions from residues 1020 and 3244 and the c - terminal residues from 5458 . each flexibility hot spot is reproduced in s from 500 ns of amd sampling . the high similarity of local flexibility in both simulation protocols is reflected in a spearman rank correlation over the protein length of r = 0.93 for s between cmd and amd . comparable agreement between the amd and reference cmd simulations when looking at dihedral entropies from a 1 s cmd simulation , notable differences are found for s in the regions of residues 1014 and 3244 . in 1 s of cmd sampling , no elevation in conformational plasticity is captured in these domains , though this is clearly observed on the millisecond time scale as well as in the amd - derived ensemble . the deviation of the local dynamics pattern results in a spearman rank correlation of r = 0.65 for s in 500 ns of amd and 1 s cmd sampling . comparing local flexibility of bpti captured in cmd and amd simulations . residue - wise dihedral entropies s from a 1 ms cmd simulation ( black ) and 500 ns amd simulation of bpti ( red ) show remarkable rank correlation ( r = 0.93 ) . local flexibility observed in a 1 s cmd simulation ( turquoise ) clearly differs from the amd results ( r = 0.65 ) . for the dihedral entropies s and s captured in 500 ns of amd , we find a correlation of r = 0.77 . the high correlation of s and s supports the assumption that a similar extent of flexibility is captured in both backbone dihedral angles . in figure 3 , the flexibility hot spots displayed in figure 2 are color coded and projected on the bpti fold . the differences in flexibility captured in 500 ns of amd ( b ) and 1 s cmd ( c ) are highlighted in ( d ) . here , s of the 1 s cmd simulation was subtracted from s resulting in 500 ns amd . positive values ( blue ) indicate domains , which are more flexible in the amd than in the cmd simulation of 1 s sampling length . when looking at the structure , the most prominent deviation between 500 ns amd and 1 s cmd simulations lies in the local flexibility of the loop regions . clearly , the dynamic nature of the loop involving residues 3244 found in the millisecond simulation is not adequately sampled in 1 s cmd but is accurately represented in amd sampling of 500 ns length . flexibility hot spots of bpti : residue - wise dihedral entropies for ( s ) from the 1 ms cmd ( a ) , 500 ns amd simulation ( b ) , and 1 s cmd ( c ) are projected on the structure of bpti ( pdb - id : 5pti ) . in panels a , b , and c , the color coding ranges from red ( s 30 j/(mol k ) ) via yellow to green ( s 45 j/(mol k ) ) . thus , the most rigid residues are pictured in red , whereas the most flexible ones are colored green . part d shows the differences in s between 500 ns amd and 1 s cmd ( s = b c ) . the color coding in d ranges from red ( s 15 j/(mol k ) ) to white to blue ( s 15 j/(mol k ) ) ; i.e. , blue indicates regions where the amd simulation captures a higher local flexibility . thus , the cmd simulation clearly underestimates the conformational dynamics of bpti in the region of residues 1014 and 3244 . with 159 residues , the major birch pollen allergen bet v 1a is the largest system in our study . a 1 s amd simulation was split into 50 segments , 20 ns each . a 3 s cmd simulation and order parameters s from backbone amide nmr relaxation experiments act as references for our metric to quantify local motions in amd simulations ( figure 4 ) . order parameters range from zero to one , indicating no or full constriction of internal mobility of backbone amide groups on the ps- to ns - time scale . thus , an anticorrelation is expected since high entropy indicates lower order . as observed for the bpti system , the amd - derived ensemble shows higher backbone flexibility for all residues compared to the cmd - derived one . the general flexibility patterns are conserved for most parts of the protein in both simulations . however , especially in the region from 1 to 2 ( residues 1545 ) , enhanced dynamics are visible in amd , which are not reflected in the cmd simulation . order parameters s show the expected anticorrelation with dihedral entropies of the core domains , i.e. , 3-helix and -sheets 47 ( residues 70159 ) . yet , in contrast to experimental findings , we obtain lowered local dynamics for the 1-helix and the 2-sheet , while for the loop region in between elevated flexibility is observed . these opposing qualitative observations are reflected in a spearman rank correlation between the dihedral entropies from amd simulations and amide order parameters s of r = 0.35 for the whole fold . when restricting the correlation analysis to the core helix 3- and -sheets 57 ( residues 70159 ) , the rank correlation is strengthened to r = 0.61 . local flexibility on different time scales in bet v 1a : dihedral entropies s from 20 ns amd ( red ) and 3 s of cmd ( black ) are compared to experimental ps / ns dynamics captured by nmr - derived backbone amide - order parameters s ( blue ) local flexibility is decisive for biomolecular recognition mechanisms like protein protein interactions and ligand binding , as well as for protein folding . it has been shown that the flexibility patterns of bet v 1 are linked to its fold - stability and allergenicity . furthermore , studies on the dynamics of proteases found a remarkable correlation between substrate specificity and local flexibility of protease active sites . the associated dynamics include motions from the nanosecond to the millisecond time scale . metrics to quantify the amount of global and local motions are indispensable for a holistic understanding of macromolecular interactions . several expedient metrics have been developed to account for global and local flexibility in cmd simulations , such as root - mean - square fluctuation , locally and globally aligned b - factors , or torsional entropies . dynamics from amd simulations are currently predominantly described on a global level only . in our study on three model systems of increasing size , we establish an alignment - free internal coordinate - based metric estimating local flexibility in amd simulations . as expected , enhanced local dynamics are captured using enhanced sampling . as demonstrated for global movements , we are able to describe local protein flexibility on the millisecond time scale after several hundred of nanoseconds of amd sampling . the residue - wise quantification of motion in a protein backbone is constructed from torsional free energy profiles of reweighted amd trajectories via calculation of dihedral entropies . investigations on the smallest system in our study , di - ala , illustrate the applied work flow . as already outlined in a previous study , we also find a shift of minima in the free energy landscape of in the reweighted amd trajectory compared to the cmd results . this deviation is most probably generated by the reweighting step when using the maclaurin series of the tenth order as approximation for the exponential . as shown by miao et al . for alanine dipeptide , reweighting using cumulant expansion to the second order reconstructs the free energy surface of amd simulations accurately . yet . this may be approximately the case for a small system like di - ala , but we observed that it is less successful for larger biomolecular systems such as bpti ( si , figure s7 ) . previous studies on bpti and other proteins showed accurate results for maclaurin series expansion of the tenth order . thus , we prefer to use maclaurin series expansion for all systems in our study for consistency . overall , a quantitative reproduction of the positions of the local extrema is not essential for our methodology since their exact location has no influence on the resulting entropies . the state probability distribution is generally broader in the amd ensemble but results in statistically equal dihedral entropies . thus , the same dynamic tendencies are estimated from the amd and reference cmd simulation . bpti is widely used as a test system for nmr and protein dynamics in general and has been investigated thoroughly over the last decades . the group of d. e. shaw performed large - scale computer simulations and extensive studies on the dynamics of this model system . we have demonstrated in previous studies that metrics of local flexibility in a 1 ms cmd simulation of bpti capture characteristic motions , known from nmr and global flexibility studies . these prominent movement motifs comprise the isomerization of a disulfide bridge involving cys-14 and cys-38 . with our metric , we quantified local flexibility for a 500 ns amd and a 1 ms cmd simulation of bpti with a striking spearman rank correlation of r = 0.93 for s. thus , we are able to quantify and localize millisecond dynamics with amd simulations of several hundred nanoseconds length . when compared to a 1 s cmd simulation , it is evident that these results can not be obtained with state - of - the - art simulation protocols of the same length ( r = 0.65 ) . the differences in captured molecular motions in 500 ns amd and 1 s cmd sampling can be traced back to the loop regions ( residue 1014 and 3244 ) , which comprise the switching disulfide bridge mentioned earlier ( si , figure s8 ) . the isomerization of this bond , which is described by nmr studies and the calculations of the shaw group , implies an enhancement of local flexibility in the surrounding region . the characteristic dynamics of the domain are evidently quantified by our metric in 500 ns amd sampling . these results show that the approach allows access to dynamics of low - frequency motions . additionally , our metric provides a tool to localize the origin of elevated flexibility thereby identifying domains with prominent dynamics and allowing a residue - wise interpretation . bet v 1a is the largest and thereby most challenging system in our study . the boosting parameters for the di - ala and bpti simulations were applied as suggested in previous studies . for bet v 1a , we set up six simulations on different levels of acceleration to test the limit of applied acceleration without unfolding the protein ( si ) . it has been shown that the choice of parameter has a crucial impact on the resulting trajectory and has to be evaluated carefully . to estimate the reliability of our findings , we compared a 20 ns amd trajectory to a conventional 3 s md simulation and nmr - derived nh order parameters s. flexibility patterns recovered from amd and cmd simulation are overall in good agreement . characteristic deviations are observed in the region from helix 1 to the sheet 2 ( residues 1545 ) . these observations are reflected by a spearman rank correlation of r = 0.51 between the amd and cmd simulations . we hypothesize that this rather low correlation can primarily be explained by the different time scales captured . extrapolating from previous studies on bpti , where 500 ns of amd sampling covers the dynamics of 1 ms cmd sampling , 20 ns of amd should correspond to dynamics of around 40 s . it can be assumed that these flexibilities clearly deviate from motions captured in only 3 s sampling time . comparison of the dihedral entropy profile to order parameters s of the backbone amide leads to similar findings . nmr order parameters s are sensitive to ps- to ns - dynamics , capturing much faster motions than those shown in amd data . we expect a coupling between slow backbone dynamics , profiled by amd simulation , and fast motions , captured in nmr data . thus , nmr order parameters s represent a method to experimentally probe protein backbone dynamics on residue resolution and an insightful reference to estimate the reliability of our approach . as expected , we observe an anticorrelation between the calculated diheral entropies and the experimental order parameters . again reasonable agreement is visible for the region reaching from residue 70 to the c - terminus , while for the domain from helix 1 to the sheet 2 ( residues 1545 ) almost opposing trends are found . these qualitative observations become apparent in a spearman rank correlation of r = 0.39 between the amd dihedral entropies and s when considering the whole protein . this is only a slight improvement over cmd simulations , where correlations of r = 0.23 for torsional entropies and order parameters are obtained . this might result from dynamics captured by amd being beyond the scope of the nmr time scale . it has been shown in previous studies that the region from 1 to 2 undergoes a noticeable rigidification upon ligand binding . order parameters and relaxation dispersion profiles of the apo protein confirm the flexible nature of bet v 1a on a pico- to nanosecond as well as on a micro- to millisecond time scale . residues from 1 to 2 show elevated dynamics in both experiments . for the remaining parts of the protein ( residue 70159 ) , a correlation of r = 0.61 is found between the amd dihedral entropies and s. here , the correlation of cmd simulations and order parameters is still notably lower with r = 0.35 . additionally , dihedral entropies were calculated from amd simulations of varying lengths ranging from 10 ns to 1 s ( si , figure s10 ) . again , the resulting entropies show similar flexibility profiles as experimental nmr studies , with exception of the discussed domain reaching from 1 to 2 . this emphasizes the presence of complex conformational dynamics on multiple time scales in this area . with the presented metric , we provide a tool to map low - frequency conformational dynamics of biomolecules at the residue level . with increasing system size , reproduction of the original flexibility profile the decorrelation time of amd and cmd data has been investigated extensively in previous studies . it has been shown that the amd generally reduces the statistical inefficiency of a simulation . an extensive probing of the acceleration parameter is crucial for the reliability of any amd trajectory . aggressive boosting enables extensive speedup in conformational exploration , but can lead to a substantial loss of accuracy . some approaches , like boosting of rotatable torsions only ( ramd ) , gaussian amd , or selectively applied amd , alleviate the impact of the reweighting error . with the present study , we introduce and validate a metric to characterize local protein dynamics on the millisecond time scale . accelerated md simulations provide access to time scales 3 orders of magnitude beyond state - of - the - art sampling time . subsequent calculation of dihedral entropies from amd trajectories quantifies backbone flexibility of each residue . the general functionality of our approach is shown on the model system di - ala . we calculated dihedral entropies from a 1 ms cmd simulation of bpti , serving as reference to validate and benchmark method and metric . we were able to show that dihedral entropies from only 500 ns of amd simulation identify the same flexibility hot spots , as observed in the 1 ms cmd trajectory . the results are supported by previous nmr studies , which observe local conformational changes in the same regions characterized as most flexible in our study . our study shows good agreement with local dynamics found in a 3 s cmd simulation as well as with nmr - derived amide - order parameters . we encourage the application of dihedral entropies as a local flexibility metric on different amd protocols . we anticipate our novel metric to facilitate characterizing and thus understanding the influence of molecular dynamics on biomolecular recognition and protein folding .

here , we demonstrate a method to capture local dynamics on a time scale 3 orders of magnitude beyond state - of - the - art simulation approaches . we apply accelerated molecular dynamics simulations for conformational sampling and extract reweighted backbone dihedral distributions . local dynamics are characterized by torsional probabilities , resulting in residue - wise dihedral entropies . our approach is successfully validated for three different protein systems of increasing size : alanine dipeptide , bovine pancreatic trypsin inhibitor ( bpti ) , and the major birch pollen allergen bet v 1a . we demonstrate excellent agreement of flexibility profiles with both large - scale computer simulations and nmr experiments . thus , our method provides efficient access to local protein dynamics on extended time scales of high biological relevance .

Introduction Methods Results Discussion Conclusion

various structural studies on diverse model systems have shown that the conformational plasticity of proteins plays a key role in molecular mechanisms such as catalytic activity , biomolecular recognition , and allosteric regulation . thus , characterizing dynamics of biological macromolecules is crucial to understanding their biological activity and function . molecular dynamics ( md ) simulations have proven to be an efficient tool to capture the flexibility of macromolecules . yet , state - of - the - art md simulations routinely capture dynamics on the nanosecond to microsecond time scale , while many biologically significant motions appear on the millisecond time scale or slower . inherent limitations in conformational sampling can either be overcome by usage of dedicated simulation hardware or by application of enhanced sampling algorithms . thus , the conformational space is sampled more extensively at negligible computational overhead costs . current approaches estimating local dynamics in amd simulations are limited to expensive large - scale calculations of amide - order parameters from multiple amd trajectories on various acceleration levels . we propose residue - wise dihedral entropy as the first methodology to efficiently characterize local dynamics of macromolecules from amd simulations . to confirm the validity and efficiency of our approach , we apply the metric to the model systems alanine dipeptide ( di - ala ) and bovine pancreatic trypsin inhibitor ( bpti ) . the conformational dynamics of bpti have been investigated thoroughly in nmr experiments as well as in large - scale computer simulations . here , we show that residue - wise dihedral entropies deriving from a 500 ns amd and a 1 ms cmd simulation of bpti correlate remarkably . application of our metric on amd trajectories provides a possibility to track low - frequency local dynamics on the millisecond time scale . additionally , we apply our metric to cmd and amd simulations of the major birch pollen allergen bet v 1.0101 ( bet v 1a ) . bet v 1a is a highly immunogenic storage protein and most prominent for causing seasonal pollen allergy in the northern hemisphere . despite a sequence similarity of more than 95% and minor differences in their 3d structures , the more than 13 reported isoforms of bet v 1a vary strongly in their immunogenicity . investigations on differences in proteolytic stability and ligand binding of different isoforms and mutants of bet v 1a suggest a linkage between immunogenic potential and conformational flexibility . our results show that dihedral entropies from amd simulations are an efficient tool to describe local protein dynamics on the millisecond time scale . all structures were prepared in moe ( molecular operating environment , chemical computing group , version 2014.0901 ) using the protonate3d tool . for di - ala , the minimum wall distance was set to 12 and for bpti and bet v 1a to 10 . parameters for all three systems derive from the amber force field 99sb - ildn . bonds involving hydrogen atoms were restrained by applying the shake algorithm , allowing a time step of 2.0 fs . atmospheric pressure of the system was preserved by weak coupling to an external bath using the berendsen algorithm . the langevin thermostat was used to maintain the temperature during simulations at 300 k for di - ala and bpti and 310 k for bet v 1a ( human body temperature ) . , and in - house scripts . the local backbone flexibility profiles were estimated from the resulting reweighted one - dimensional free energy profiles and state populations , respectively , of the backbone dihedrals and . a high entropy of a residue backbone dihedral indicates high local backbone flexibility . in the presented work , we prioritized dihedral entropies s over s in the representation protein dynamics as they captured the backbone dynamics more comprehensively . yet , s alone does not reflect the entire backbone dynamics , and dihedral entropies s were calculated as well ( si ) . for the reference cmd simulation of alanine dipeptide ( di - ala ) , a 10 s trajectory comprising 100,000 frames , previously performed in our group , was reanalyzed . residue - wise dihedral entropies of the reference cmd simulations were calculated from probability density functions reconstructed by nonparametric kernel density estimation . , we optimize the bandwidth of the kernel function by cross validation , resulting in a continuous probability density function for each dihedral . to minimize the statistical noise ( and capture dynamics corresponding to 10 s of cmd ) , the resulting trajectory was divided into 200 segments of 5 ns each ( 2500 frames ) using the segments for averaging ( si , figure s4 ) . to compare amd and cmd free energies , we calculated the free energy of the cmd trajectory using the reweighting protocol by miao et al . we investigated several different bin sizes , and the jaggedness of the cmd profile disappears only using a bin size above 20 ( si , figure s2 ) . , we observe a bin size of 6 to be a reasonable compromise between accuracy and statistical noise . d. e. shaw research kindly provided us with a long time scale 1.03 ms trajectory comprising 4,140,000 snapshots as a cmd reference . a 500 ns amd simulation was performed , and the trajectory was stored as 250,000 snapshots . and can be found in the supporting information . to localize the observed dynamic hot spots , we calculated dihedral entropies as described earlier for 1 ms and 1 s of cmd and 500 ns of amd sampling time . on the basis of the crystal structure of bet v 1a with pdb i d 4a88 , we sampled 3 s of cmd simulations on bet v 1a . suitable amd boosting parameters for bet v 1a were determined by a systematic search ( si ) . to establish our approach , we calculated backbone dihedral entropies for and of alanine dipeptide from 5 ns amd sampling and used a 10 s cmd simulation as a reference ( figure 1 ) . we find a significant reproduction of local minima and overall shape of the potential energy surface for the backbone dihedral of di - ala . as reported before , we also observe a shift in the extrema of backbone dihedral to smaller values in amd simulations . from a reweighted amd trajectory to dihedral entropies of alanine dipeptide : state populations p are calculated from free energy profiles of the backbone dihedral of a 10 s cmd ( black ) and a reweighted 5 ns amd ( red ) trajectory . considering these errors , the resulting dihedral entropies , s,amd = 41.03 ( 2.95 ) j/(mol k ) and s,amd = 45.42 ( 3.25 ) j/(mol k ) , agree very well with those of the cmd ensemble , s,cmd = 40.20 ( 0.60 ) j/(mol k ) and s,cmd = 42.08 ( 0.39 ) j/(mol k ) . to benchmark our local flexibility metric , we analyzed a 500 ns amd simulation of bpti and compared it to a 1 ms cmd simulation provided by d. e. shaw research ( figure 2 ) . , we observe equal assessment of local backbone flexibility for the 500 ns amd and 1 ms cmd simulation . in the 1 ms reference , cmd maxima of s are found in regions from residues 1020 and 3244 and the c - terminal residues from 5458 . the high similarity of local flexibility in both simulation protocols is reflected in a spearman rank correlation over the protein length of r = 0.93 for s between cmd and amd . comparable agreement between the amd and reference cmd simulations when looking at dihedral entropies from a 1 s cmd simulation , notable differences are found for s in the regions of residues 1014 and 3244 . in 1 s of cmd sampling , no elevation in conformational plasticity is captured in these domains , though this is clearly observed on the millisecond time scale as well as in the amd - derived ensemble . the deviation of the local dynamics pattern results in a spearman rank correlation of r = 0.65 for s in 500 ns of amd and 1 s cmd sampling . residue - wise dihedral entropies s from a 1 ms cmd simulation ( black ) and 500 ns amd simulation of bpti ( red ) show remarkable rank correlation ( r = 0.93 ) . for the dihedral entropies s and s captured in 500 ns of amd , we find a correlation of r = 0.77 . the high correlation of s and s supports the assumption that a similar extent of flexibility is captured in both backbone dihedral angles . here , s of the 1 s cmd simulation was subtracted from s resulting in 500 ns amd . flexibility hot spots of bpti : residue - wise dihedral entropies for ( s ) from the 1 ms cmd ( a ) , 500 ns amd simulation ( b ) , and 1 s cmd ( c ) are projected on the structure of bpti ( pdb - id : 5pti ) . in panels a , b , and c , the color coding ranges from red ( s 30 j/(mol k ) ) via yellow to green ( s 45 j/(mol k ) ) . thus , the most rigid residues are pictured in red , whereas the most flexible ones are colored green . thus , the cmd simulation clearly underestimates the conformational dynamics of bpti in the region of residues 1014 and 3244 . with 159 residues , the major birch pollen allergen bet v 1a is the largest system in our study . order parameters range from zero to one , indicating no or full constriction of internal mobility of backbone amide groups on the ps- to ns - time scale . thus , an anticorrelation is expected since high entropy indicates lower order . however , especially in the region from 1 to 2 ( residues 1545 ) , enhanced dynamics are visible in amd , which are not reflected in the cmd simulation . order parameters s show the expected anticorrelation with dihedral entropies of the core domains , i.e. yet , in contrast to experimental findings , we obtain lowered local dynamics for the 1-helix and the 2-sheet , while for the loop region in between elevated flexibility is observed . these opposing qualitative observations are reflected in a spearman rank correlation between the dihedral entropies from amd simulations and amide order parameters s of r = 0.35 for the whole fold . when restricting the correlation analysis to the core helix 3- and -sheets 57 ( residues 70159 ) , the rank correlation is strengthened to r = 0.61 . local flexibility on different time scales in bet v 1a : dihedral entropies s from 20 ns amd ( red ) and 3 s of cmd ( black ) are compared to experimental ps / ns dynamics captured by nmr - derived backbone amide - order parameters s ( blue ) local flexibility is decisive for biomolecular recognition mechanisms like protein protein interactions and ligand binding , as well as for protein folding . it has been shown that the flexibility patterns of bet v 1 are linked to its fold - stability and allergenicity . the associated dynamics include motions from the nanosecond to the millisecond time scale . dynamics from amd simulations are currently predominantly described on a global level only . in our study on three model systems of increasing size , we establish an alignment - free internal coordinate - based metric estimating local flexibility in amd simulations . as expected , enhanced local dynamics are captured using enhanced sampling . as demonstrated for global movements , we are able to describe local protein flexibility on the millisecond time scale after several hundred of nanoseconds of amd sampling . the residue - wise quantification of motion in a protein backbone is constructed from torsional free energy profiles of reweighted amd trajectories via calculation of dihedral entropies . as already outlined in a previous study , we also find a shift of minima in the free energy landscape of in the reweighted amd trajectory compared to the cmd results . for alanine dipeptide , reweighting using cumulant expansion to the second order reconstructs the free energy surface of amd simulations accurately . previous studies on bpti and other proteins showed accurate results for maclaurin series expansion of the tenth order . thus , we prefer to use maclaurin series expansion for all systems in our study for consistency . the state probability distribution is generally broader in the amd ensemble but results in statistically equal dihedral entropies . thus , the same dynamic tendencies are estimated from the amd and reference cmd simulation . bpti is widely used as a test system for nmr and protein dynamics in general and has been investigated thoroughly over the last decades . the group of d. e. shaw performed large - scale computer simulations and extensive studies on the dynamics of this model system . we have demonstrated in previous studies that metrics of local flexibility in a 1 ms cmd simulation of bpti capture characteristic motions , known from nmr and global flexibility studies . with our metric , we quantified local flexibility for a 500 ns amd and a 1 ms cmd simulation of bpti with a striking spearman rank correlation of r = 0.93 for s. thus , we are able to quantify and localize millisecond dynamics with amd simulations of several hundred nanoseconds length . when compared to a 1 s cmd simulation , it is evident that these results can not be obtained with state - of - the - art simulation protocols of the same length ( r = 0.65 ) . the differences in captured molecular motions in 500 ns amd and 1 s cmd sampling can be traced back to the loop regions ( residue 1014 and 3244 ) , which comprise the switching disulfide bridge mentioned earlier ( si , figure s8 ) . the isomerization of this bond , which is described by nmr studies and the calculations of the shaw group , implies an enhancement of local flexibility in the surrounding region . these results show that the approach allows access to dynamics of low - frequency motions . additionally , our metric provides a tool to localize the origin of elevated flexibility thereby identifying domains with prominent dynamics and allowing a residue - wise interpretation . bet v 1a is the largest and thereby most challenging system in our study . for bet v 1a , we set up six simulations on different levels of acceleration to test the limit of applied acceleration without unfolding the protein ( si ) . to estimate the reliability of our findings , we compared a 20 ns amd trajectory to a conventional 3 s md simulation and nmr - derived nh order parameters s. flexibility patterns recovered from amd and cmd simulation are overall in good agreement . we hypothesize that this rather low correlation can primarily be explained by the different time scales captured . we expect a coupling between slow backbone dynamics , profiled by amd simulation , and fast motions , captured in nmr data . thus , nmr order parameters s represent a method to experimentally probe protein backbone dynamics on residue resolution and an insightful reference to estimate the reliability of our approach . as expected , we observe an anticorrelation between the calculated diheral entropies and the experimental order parameters . these qualitative observations become apparent in a spearman rank correlation of r = 0.39 between the amd dihedral entropies and s when considering the whole protein . this might result from dynamics captured by amd being beyond the scope of the nmr time scale . order parameters and relaxation dispersion profiles of the apo protein confirm the flexible nature of bet v 1a on a pico- to nanosecond as well as on a micro- to millisecond time scale . for the remaining parts of the protein ( residue 70159 ) , a correlation of r = 0.61 is found between the amd dihedral entropies and s. here , the correlation of cmd simulations and order parameters is still notably lower with r = 0.35 . additionally , dihedral entropies were calculated from amd simulations of varying lengths ranging from 10 ns to 1 s ( si , figure s10 ) . again , the resulting entropies show similar flexibility profiles as experimental nmr studies , with exception of the discussed domain reaching from 1 to 2 . this emphasizes the presence of complex conformational dynamics on multiple time scales in this area . with the presented metric , we provide a tool to map low - frequency conformational dynamics of biomolecules at the residue level . some approaches , like boosting of rotatable torsions only ( ramd ) , gaussian amd , or selectively applied amd , alleviate the impact of the reweighting error . with the present study , we introduce and validate a metric to characterize local protein dynamics on the millisecond time scale . accelerated md simulations provide access to time scales 3 orders of magnitude beyond state - of - the - art sampling time . subsequent calculation of dihedral entropies from amd trajectories quantifies backbone flexibility of each residue . the general functionality of our approach is shown on the model system di - ala . we calculated dihedral entropies from a 1 ms cmd simulation of bpti , serving as reference to validate and benchmark method and metric . we were able to show that dihedral entropies from only 500 ns of amd simulation identify the same flexibility hot spots , as observed in the 1 ms cmd trajectory . our study shows good agreement with local dynamics found in a 3 s cmd simulation as well as with nmr - derived amide - order parameters . we encourage the application of dihedral entropies as a local flexibility metric on different amd protocols . we anticipate our novel metric to facilitate characterizing and thus understanding the influence of molecular dynamics on biomolecular recognition and protein folding .

bioethics is often thought to be subject to two competing aims , which we can refer to as the theoretical and the practical . the theoretical aim is to arrive at correct ( or at least well justified ) answers to normative questions such as whether it is ever ethically acceptable for a doctor to prescribe a deceptive placebo to a patient , or whether hard paternalism is ever permissible . the practical aim is to bring the world closer to some normatively justified state of affairs perhaps by making it easier to get compulsory licenses for essential medicines , or by implementing a particular policy to reduce health inequities . the two aims seem to pull bioethicists in different directions , because it is plausible to think that the most effective means of pursuing the theoretical aim will be relatively ineffective in pursuing the practical aim , and vice versa . this apparent conflict arises much more strongly for bioethics than it does , say , for political philosophy . in part this is because the questions that bioethics deals with are often much more obviously shaped by our present concerns than those in political philosophy , and in part it is because there is no more applied academic discipline than bioethics that stands in the relationship to it that political science and political theory stand to political philosophy . in brief , we tend to approach bioethics with the goal of improving our grip on real world ethical questions , and in working out what we should do all things considered , the buck stops with it . perhaps the most significant reason for the lack of congruence between the theoretical and the practical aims is that public ethical discourse relies much more on narratives and systems of analogies than on rigorous normative arguments . persuading others to think of a problem within a certain frame is usually much more practically effective than presenting a cogent normative argument that has no obvious connection to any existing policy frames or narratives.1 overwhelmingly , the frame that is put on an issue determines the arguments that will have political currency , rather than vice versa . for this reason , much political debate consists in telling stories that encourage citizens to apply one rather than another pre - existing frame to the problem seeing the situation for example as one of protecting our national security or of preventing the construction of a big brother state . however , the best means of supporting or refuting particular ethical claims appears to be rigorous normative arguments . unfortunately rigorous normative arguments are usually sufficiently complex , unintuitive , or contextually irrelevant , that they are largely ignored by citizens and by policymakers . moreover , both politicians and ordinary citizens tend to be suspicious of the idea that bioethicists have any expertise to which they ought to defer , either in regards to bioethical methodology or in the substantive conclusions they reach . insofar as theoretical bioethical claims do influence policy , the analysis that makes its way into policy will usually be markedly less sophisticated than what one would see in a high quality ethics journal , and will be deployed most often in the manner of means - end or specificatory reasoning , whereby the frame sets the end , and the argument draws out the implications of this framing for the issue at hand . a normative argument that is practically effective in one context , or in one community , will not necessarily gain policy traction in another . thus , successfully pursuing the practical aim requires a much greater attention to context than successfully pursuing the theoretical aim . the uk organ donation taskforce , of which montgomery was a member , decided not to recommend a change in policy from opt in to opt out for organ donation despite seeing the potential rationale for an opt - out policy . one of the factors that swayed them against recommending an opt - out policy was the fact that the then prime minister , gordon brown , made public his support for such a policy . unfortunately , brown was at the time so unpopular that his support for the policy made it markedly more difficult for the taskforce to recommend it . the taskforce came to the conclusion that if they advocated an opt out policy , seemingly on the back of brown s views , it was likely that this would undermine the consensus they needed to achieve for many of their other recommendations to be successful . they therefore decided against recommending an opt - out policy.2 pursuing the theoretical aim will typically involve making fine distinctions whose necessity will only become obvious once one has studied the area in depth . research questions in academic bioethics often thus have ( or can appear to have ) a fractal quality : however much we zoom into a particular debate , there is still further detail to be looked at and further subtle distinctions to be made . each distinction that is made provides the possibility for further disagreement , and so the net result is that academics naturally form themselves into a large number of camps , each with a very small number of people in them.3 such fragmentation on the basis of small differences is an anathema to achieving the kind of political or systemic changes that are usually required by the practical aim . as political movements typically require an agreed platform that many people from a variety of different perspectives can sign up to , successful political arguments are usually not too specific and are nearly always significantly vaguer than fully articulated positions in theoretical bioethics are.4 given this lack of congruence between the theoretical and the practical aims , it is natural to ask two questions . first , a question of scope : do both the practical and the theoretical aims fall within the scope of bioethics proper ? ( of course , even if it were agreed that at least the core of both aims did fall inside bioethics proper , there would be room for further debate about whether there are elements of each that fall outside , and belong , say , to normative ethical theory on the one side or to political lobbying on the other side . ) second , assuming that at least a core of both the theoretical and the practical aims fall within bioethics proper , which should have the higher priority ? questions such as these about the boundaries and purpose of disciplines embody only in a fairly superficial sense factual disputes about what most practitioners of the discipline would consider to be the boundaries of the discipline , and which types of questions they consider to be most important . in a deeper sense they are normative questions about how best to advance the discipline , considered as a a tradition in macintyre s sense , as an historically extended , socially embodied argument , and an argument precisely in part about the goods which constitute that tradition ( 1981 , p. 222 ) . bioethics was transformed by one such set of arguments in the early years of this century , expanding from a discipline that examined primarily promethean new technologies and relationships between healthcare professionals and lay persons in wealthy countries , to one that is global in its outlook and focuses as much on institutions as individuals , and as much on the social determinants of health as on healthcare.5 what was determinative in this case was the sense that existing bioethics frameworks had overlooked ( and were poorly equipped to deal with ) large swathes of ethical questions connected to health and healthcare , including the social determinants of health and disease , the ethics of healthcare rationing , and the ethics of infectious disease . this paper aims to start a different kind of reflexive conversation about the future of bioethics , focused not so much on which topics should be inside and which should be outside of bioethics , but about why we are doing bioethics , and what would count as doing it well . just as in the case of the shift to a more institutional and global focus , the changes required are unlikely to follow clearly from a conceptual analysis of the idea of bioethics alone , or from taking a poll of what current individual bioethicists think that it is for , but by the gradual accretion of evidence of unsolved problems and missed opportunities , and a careful construction of a new and more fruitful framework . hence , one of the main contributions of this article is to fashion an understanding of bioethics that allows us to see the theoretical and practical aims as interlinked and mutually supporting parts of a continuum rather than in antagonism to each other . a few preliminary comments are in order before we turn to the constructive task . first , regardless of the stance that one takes on the relative importance of the theoretical versus the practical aims , it should be common ground that much work in bioethics is in fact motivated by a desire to mitigate or resolve ethical problems that have been made salient by changes in the lived environment . perhaps a new procedure such as transplantation of mitochondrial dna becomes available , which plausibly raises ethical issues that have not so far been considered ; or it becomes apparent that antimicrobials are being ( and have for some time been ) used in an unsustainable way for some time , and many thousands will die unless the problem is brought under control.6 so whilst there may be reasons in other areas of philosophy for resisting the contamination of normative enquiry by practical concerns , and for framing normative enquiry as pure truth seeking , in bioethics at least , claiming only such a pure motivation would risk being disingenuous . although the answers we seek might conceivably be timeless and discoverable through a priori reasoning alone , the questions that catch our attention are heavily influenced by our current concerns . second , whichever way one answers our two fundamental questions about bioethics , it should also be common ground that responding wisely to challenging ethical questions that arise from changes in the lived environment is an important practical goal . even if we were to conclude that bioethics , properly considered , should have a purely theoretical aim , this would not make these pressing practical ethical problems go away ; it would simply mean that we then needed another discipline or discourse that could translate between theoretical bioethics and these practical problems . we can call this the translation problem . given that these practical ethical problems appear to be very significant , adequately addressing the translation problem would be important even if doing so fell outside of bioethics proper . the last thing to say by way of preliminary is that there are a set of deep questions about the nature of ethics that bear on our two fundamental questions about bioethics for example , whether we should view ethics as fundamentally a theoretical pursuit that aims at the discovery of normative truths , or whether we should think of ethics as a fundamentally practical endeavour that aims to solve various problems of coordination necessary for sustained and complex human life . i shall attempt to sidestep these deeper debates as far as is possible.7 my aim is to point out a set of problems that bioethics will need to solve regardless of whether we view ethics as fundamentally theoretical or fundamentally practical , and to provide a lens for thinking about these problems that will be attractive to thinkers from both perspectives . i shall argue that regardless of whether changing the world should form part of bioethics s core aims , any plausible conception of bioethics should be at least committed to informing ethical judgements about what ought to be done in richly described real life cases . as much of the rest of the article explores , getting individual real world cases right is likely to require the kind of messy , complex and contextual work for which more abstract levels of theorising can only provide limited guidance . so , even if one s orientation in bioethics is entirely towards the theoretical , questions about how to move between judgements based on simple cases and judgements in complex cases will be of central importance . the rest of the paper proceeds as follows : section 2 introduces various themes from debates on innovation in healthcare research in particular debates around how to best connect up blue skies basic research with practical innovations that can improve human lives as a way of reflecting on the relationship between the theoretical and the practical aims in bioethics . it explains how a simple top - down linear model of innovation has been replaced by a more complex translational model , which recognises that innovations move along different pathways both from the more basic. section 3 adapts the translational model to bioethics arguing that we should replace the idea of an opposition between the theoretical and the practical with the idea of a continuum from the most simple and abstract cases ( thought experiments ) to the most concrete and complex cases ( real world cases ) . insights need to travel in both directions along this continuum both from the simpler and more abstract to the more complex and from the more complex to the simpler and more abstract . section 4 examines some ways in which judgements that are correct in simple cases can fail to translate into helpful insights about more complex cases . section 4.1 focuses on thought experiments , whilst sect . section 5 concludes by reflecting on the implications of the analysis for the future of the discipline . questions about the relationship between theoretical advances and practical benefit are particularly urgent in healthcare research , owing to the ubiquity of death and suffering , the technical difficulty and expense of bringing new drugs to market , and governments own sizeable investments in funding both healthcare research and the successful treatments that result from it . so it is perhaps unsurprising that debate on the relationship between the theoretical and the practical is louder , older and more voluble in healthcare research than it is in bioethics , and contains a rich potential for cross - fertilisation . moreover , approaching the question of the relationship between theory and practice in bioethics via debates about translational research in healthcare has the advantage that it allows us to sidestep the dispute between fundamentally theoretical and fundamentally practical approaches to ethics . by starting from outside ethics , in a domain in which it is relatively uncontroversial that there are theoretical facts that inquiry aims to discover , it becomes apparent that even in this domain , it follows neither that more abstract theoretical truths are more worthy of pursuit than more concrete ones , nor that inquiry should be guided by the pursuit of theoretical truths alone.8 world war ii saw an unprecedented mobilisation of science for the war effort . the us set up the office of scientific research and development ( osrd ) in 1941 , whose goal was to coordinate , supervise , and conduct scientific research on the problems underlying the development , production , and use of mechanisms and devices of warfare , leading most notably to the manhattan project . as the war drew to a close , the us government faced a choice about the future of science funding : should it continue to funnel scientific research budgets towards short term solutions for centrally chosen goals , or would it be better to let scientists pursue their own endeavours without regard to practical application ? vannevar bush , the director of osrd , was tasked with answering this question . the resulting report , science : the endless frontier set the tone for much of the approach to science funding over the next thirty years . bush began from a distinction between basic and applied research : basic research is performed without thought of practical ends . this general knowledge provides the means of answering a large number of important practical problems , though it may not give a complete specific answer to any one of them . ( bush 1945 : chapter 3.3 ) bush argued forcefully that it is the role of governments to support basic research , and that basic research is best undertaken in universities . applied research , it was crucial to his account that investing in basic science will ( although in ways we can not yet predict ) have significant payoffs in the future : one of the peculiarities of basic science is the variety of paths which lead to productive advance . many of the most important discoveries have come as a result of experiments undertaken with very different purposes in mind . statistically it is certain that important and highly useful discoveries will result from some fraction of the undertakings in basic science ; but the results of any one particular investigation can not be predicted with accuracy . ( bush 1945 : chapter 3.3)the idea that theoretical work produces practical benefits but in unexpected ways that it would be counterproductive to attempt to second - guess is also at the heart of certain defences of philosophy that emphasise its long - term impact , but deny that this impact could usefully be measured or optimised in the short term.9 of course , one obvious disanalogy is that , when compared to science , there is a decided lack of concerted action in either the commercial sector or in civil society to translate the results of basic normative or other philosophical work into commercialisable or socially beneficial results ( though the use of game theory in management consultancy and elsewhere might be a possible analogue ) . this general knowledge provides the means of answering a large number of important practical problems , though it may not give a complete specific answer to any one of them . the function of applied research is to provide such complete answers . ( bush 1945 : chapter 3.3 ) one of the peculiarities of basic science is the variety of paths which lead to productive advance . many of the most important discoveries have come as a result of experiments undertaken with very different purposes in mind . statistically it is certain that important and highly useful discoveries will result from some fraction of the undertakings in basic science ; but the results of any one particular investigation can not be predicted with accuracy . ( bush 1945 : chapter 3.3 ) bush s approach to innovation was amplified and extended by a number of other figures over the next 20 years ( as summarised for example , by godin 2006 and balconi et al . 2010 ) , and the resulting approach to thinking about innovation has come to be known as the linear model . the linear model , at its most radical makes the following assumptions about how innovative products such as new drugs come to be invented:(1.1 ) a clear distinction can be drawn between basic ( scientific ) and applied ( technological and industrial ) research ( 1.2 ) basic or fundamental or prior scientific research is the main or rather the unique source of technical innovation ( 1.3 ) new knowledge acquired through basic research trickles down , almost automatically , to applied research , technology and innovations , even within short time spans . 2010 , p. 5 ) each of these elements of the linear model has been criticised . first , as stokes ( 1997 ) argues , scientific research projects can not be divided neatly into those that aim to discover fundamental truths about the ways things are , and those that aim at practical application . louis pasteur s work , which aimed to address practical questions such as how to avoid milk and beer spoiling , but answered these questions by making fundamental discoveries such as the germ theory of disease , is a key exemplar of this ( stokes 1997 , pp . 1213).10 ( 1.1 ) a clear distinction can be drawn between basic ( scientific ) and applied ( technological and industrial ) research ( 1.2 ) basic or fundamental or prior scientific research is the main or rather the unique source of technical innovation ( 1.3 ) new knowledge acquired through basic research trickles down , almost automatically , to applied research , technology and innovations , even within short time spans . 2010 , p. 5 ) the idea that advances in basic science are always necessary for improved technologies in bush s words , that whilst there are obvious cases where new therapies have been developed out of a bedrock of advances in basic science ( such as monoclonal antibodies ) , it is implausible to claim that healthcare innovation always starts from advances in basic science . indeed , influential innovation scholars such as kline and rosenberg ( 1986 , p. 288 ) argue that innovation led by basic research is the exception , rather than the rule . third , the idea that new basic science automatically leads to changes in medical practice that benefit patients is also highly questionable . the history of medical science is littered with examples of failures to properly connect basic and applied science . even a case such as the discovery of penicillin , which might initially seem to be an obvious success story , shows how contingent takeup of basic science into applied science can be.11 as a result of the shortcomings of the linear model , a consensus has grown within healthcare research that funders and researchers need to think in systemic terms about how the different stages of research from the most basic science to the most applied can best fit together , an approach to thinking that has come to be known as translational research . as translational research developed , it became increasingly apparent that optimising the benefits to patients from research spending requires a rigorous and systematic focus on the pathways and transitions between the different stages of healthcare research , looking for bottlenecks and other opportunities to improve research efficiency . worries about the lack of direct connection between progress in basic theory and progress in answering more concrete questions apply even more strongly in bioethics than in healthcare research . as section 1 explored , it is plausible to think that much high quality work in abstract normative theory has very little practical impact , and as we shall see , searching questions can also be asked about current efficiency in moving between ethical questions at different points of the continuum between the fully abstract and fully concrete . in order to be able to talk sensibly about efficiency in translation between basic and applied contexts in a domain , we need at least a rough model of the continuum of different levels at which research questions can be asked and answered in that domain , and different potential ways of moving between them . once we have such a model it is much easier to determine whether research insights are currently moving between these different levels as effectively as they could be . bioethicists do not often ask questions about research efficiency in their discipline , or explicitly address the question of how best to move between different levels of abstraction in ethical thinking , and so again the healthcare research literature will provide a starting - point . as table 1 indicates , we can divide the journey from the most basic science to interventions that actually benefit patients into five stages.12 if an intervention that benefits patients is to be derived from basic science , it must pass through each of these stages.13 given the uncontroversial focus in healthcare research prioritisation on the benefits to actual patients that are achieved at stage 5 , the model immediately raises questions about research funding priorities if it turns out that prioritising basic science is a markedly less efficient way of achieving patient benefit than , say , research - led improvement of doctors prescribing habits.table 1the translational continuum in healthcare research and in bioethicsstagehealthcare research stagebioethics equivalent1basic sciencediscussion of normative theory without any attempt to think about the applicability of moral theory to real life cases2proof of concept e.g . phase 0 trial , testing pharmacodynamics and pharmacokinetics of drug to see that it could in principle be an effective therapyworking out what ought to be done in thought experiments3proof of efficacy e.g . phase 2 and 3 trials , demonstrating that the drug has a clinical effect under idealised conditionsworking out what ought to be done in simplified but somewhat realistic cases4proof of effectiveness e.g. pragmatic trial , to determine the effectiveness of the drug in real world clinical settingsworking out what we should do , all things considered , in real world situations5implementation e.g. policy changes , supported by healthcare research to benefit patientspolicy changes or other actions to make the world closer to how it should be the translational continuum in healthcare research and in bioethics the model begins from the observation that much basic research for example the discovery that a particular protein has a role in regulating inflammation does not have obvious or immediate clinical application . it may take significant amounts of work to come up with a potential clinical application . the first main place in which there can be failures of translation is thus in moving from ( 1 ) basic science to ( 2 ) proof of concept in a potential clinical application . the journey from ( 2 ) proof of concept to ( 3 ) proof of efficacy , i.e. that the intervention works under idealised and controlled conditions , is long and arduous . there are many sites along this journey at which translation could potentially be improved , including better understanding of the limits of animal models , better research regulation , speedier marketing approval and so on . once there is proof of efficacy , the next main stage is proof that the intervention will be safe and be more effective than its competitors in real world settings ( 4 ) . so we can think of the final hurdle as the journey from proof of effectiveness ( 4 ) to patients being benefited by the intervention being adopted into standard clinical practice ( 5 ) . we can construct a roughly parallel trajectory for bioethics , tracing the kind of steps that would need to occur if new insights in basic normative theory were to lead to ethical improvements on the ground.14 as in the case of healthcare research , the idea is not to say that moral change inevitably follows this trajectory or that it should , but rather to allow us to use the model to ask questions about where time and other resources are currently being concentrated along this path , and whether transitions between different elements could be improved . as this is a first shot at constructing such a trajectory , it is likely that much could be improved . first , we can take discussion of normative theory without any attempt to think about its applicability to be analogous to basic science . normative theory of this kind will encompass questions such as what sorts of things are intrinsically valuable , the degree of commensurability of different intrinsic values , the range of different appropriate responses to things that are intrinsically valuable whether all should be promoted for instance , or whether there are some that should be respected.15 perhaps back in the early days of bioethics , it might have seemed plausible to think that we would be able to step straight from basic normative theory to claims about what should be done in the real world . but this possibility now seems fanciful.16 the continued deep and unresolved disagreements about matters of fundamental moral theory mean that even if it is clear what a particular moral theory would advise in a given case , knowing this does little to help us decide what to do when other undefeated moral theories recommend something else . moreover , it has also become clear that moral theories lack the specificity to give us a definitive steer on many practical questions : as in the case of basic science , it will often take considerable work to develop a claim in fundamental normative theory to a point where it has clear implications for practice , even in idealised cases . thought experiments play a key bridging role in crossing the continuum between basic normative theory and judgements about real world cases . thought experiments as i understand them here are toy philosophical cases that are designed to simplify a philosophical problem along a number of dimensions , thus making the problem more philosophically tractable . like real - world experiments , their aim is to test a hypothesis , or to establish a point of principle though the hypothesis or point of principle is an ethical one in the case of bioethical thought experiments . thought experiments can be used to try to support or to undermine claims in basic normative theory , but they are also used as a controlled test environment for judgements about more complex and more realistic cases . as frances kamm explains it , real - life cases often do not contain the relevant or solely the relevant characteristics to help in our search for principles . if our aim is to discover the relative weight of , say , two factors , we should consider cases that involve only these two factors , perhaps artificially , rather than distract ourselves with other factors and options i focus on some of the potential difficulties involved in moving from thought experiments to judgements about more complex cases , and from more complex cases to what should be done all things considered . however , it is important to notice that the main point of the model that i am proposing is that we need to think more rigorously and more systematically about how best to move between different levels of complexity in the analysis of ethical problems . similar questions can and should be asked about moving from simpler to more complex cases on other parts of the continuum , about moving from complex cases to other cases of similar complexity , and about the implications of judgements about richly described complex cases for judgements about simpler and more abstract cases points i return to in sect . 5 . thought experiments in bioethics are usually different in a number of salient aspects from the real world scenarios in which actual moral agents make their choices . thought experiments in bioethics typically make all of simplifications 14 , and may also make simplifications 5 and 6:authoritative ethical framing : the case raises the ethical question that the author of the thought experiment says it does . what the ethical issue is that the case raises is not subject to dispute.confined choices : choices must be made from a short predefined menu , with no ability to alter the terms of the problem . what counts as a viable response to the problem is stipulated by the author of the thought experiment , and is not up for discussion . the world of the thought experiment may operate according to laws that are plainly false of the real world . hence , responses that would be likely to be effective in real world analogues of the thought experiment may be stipulated not to work , and other responses , which would be unlikely to be effective in real world analogues , may be stipulated to be effective.certainty of effect : each of the defined choices will bring about its stipulated effect with certainty . it is possible to identify in advance who will benefit , and who will lose , from each of the predefined choices . where the choice is stipulated to bring about the desired effect a certain percentage of the time , these stipulated probabilities are entirely accurate.ceteris paribus : no morally relevant differences other than those which have been stipulated by the framer of the thought experiment apply to the situation.small numbers : the case is presented as one involving either single individuals , or groups of no more than five , even if the final desired aim is to make recommendations about situations involving large groups of people.atomicity : the case stands entirely on its own . there is no relevant history that affects judgements about the case , and no relevant future policy implications of making one decision rather than another . although the problem is not usually discussed in these terms , it is apparent that , just like clinical trials , the use of thought experiments can suffer from problems of internal and external validity . a clinical experiment is internally valid if it is designed in such a way that it correctly measures the causal effect of an independent variable or variables on one or more dependent variables . similarly , a thought experiment is internally valid to the extent that it allows its readers to make judgements that are confident and free of bias about the hypothesis or point of principle that it aims to test . thus for example , if the thought experiment requires comparing two cases which differ only in a single respect , we need to be confident that the cases do only differ in this one respect ; and if the framer of the thought experiment claims that only one of the stipulated choices in the case is morally permissible , we should be able to judge confidently that this is true . authoritative ethical framing : the case raises the ethical question that the author of the thought experiment says it does . confined choices : choices must be made from a short predefined menu , with no ability to alter the terms of the problem . what counts as a viable response to the problem is stipulated by the author of the thought experiment , and is not up for discussion . the world of the thought experiment may operate according to laws that are plainly false of the real world . hence , responses that would be likely to be effective in real world analogues of the thought experiment may be stipulated not to work , and other responses , which would be unlikely to be effective in real world analogues , may be stipulated to be effective . certainty of effect : each of the defined choices will bring about its stipulated effect with certainty . it is possible to identify in advance who will benefit , and who will lose , from each of the predefined choices . where the choice is stipulated to bring about the desired effect a certain percentage of the time , these stipulated probabilities are entirely accurate . ceteris paribus : no morally relevant differences other than those which have been stipulated by the framer of the thought experiment apply to the situation . small numbers : the case is presented as one involving either single individuals , or groups of no more than five , even if the final desired aim is to make recommendations about situations involving large groups of people . there is no relevant history that affects judgements about the case , and no relevant future policy implications of making one decision rather than another . an experiment trial has external validity in addition to internal validity to the extent that the causal effects demonstrated in it can be generalised to a wide range of other situations . within the fields of healthcare and public policy research , it is now a commonplace that an intervention that is efficacious in the highly idealised context of a controlled trial may not be effective in other contexts ( rothwell 2005 ) . as cartwright ( 2013 ) puts it , a randomised controlled trial ( rct ) can show that an intervention worked somewhere but not that it will work here . to describe a trial as lacking in external validity is not by itself to impugn its quality as research : it is in the nature of an rct that the rigour in experimental design that is necessary for internal validity sets limits on how , if at all , the results can be extrapolated to other contexts ( cartwright 2007 ) . however , research that lacks external validity will be useful for changing the world only in a narrow range of contexts . similarly , we can say that a thought experiment has external validity in addition to internal validity to the extent that points of principle that are established by it can be generalised to a wide range of other circumstances . thought experiments , and normative arguments more broadly , can lack external validity in at least two ways . first , if the principles they establish presuppose factual circumstances that are relevantly different from those that currently obtain . for example , nozick s entitlement theory of property rights is often thought to lack external validity on these grounds of factual irrelevance . the theory requires that property is justly held if and only if it was either ( a ) justly initially acquired or ( b ) legitimately transferred from someone else who was already entitled to it . even leaving on one side the kinds of difficulties that can be raised to nozick s account of just acquisition , it is clear that there have been massive and systematic ruptures in justice in transfer throughout human history , including wars , colonialism , slavery , and the genocidal destruction of first nation peoples . even if goods have been transferred legitimately since the time when they were unjustly acquired , nozick s view would not imply that they are now justly held . rather such goods would be subject to a principle of justice in rectification a principle that nozick does not specify . so despite initial appearances , nozick s account tells us virtually nothing about who is entitled to what , or what could be legitimately taken from whom and redistributed to others , in our world with its particular history . to his credit , nozick himself recognises the problem , and admits that his theory would require a full account of justice in rectification , which he is unable to offer , stating that in the absence of such a treatment applied to a particular society , one can not use the analysis and theory presented here to condemn any particular scheme of transfer payments , unless it is clear that no considerations of rectification of injustice could apply to justify it17 nozick ( 1975 , p. 231 ) . second , ethical principles and considerations are often claimed to interact with one another in holistic ways . on this view , there are scenarios in which ethical considerations that favour acting in certain ways in many or most cases no longer provide a reason in favour of acting in that way , and may even change polarity and provide a reason against acting in that way . for example , in usual circumstances , the fact that doing x will provide someone else with pleasure speaks in favour of it , but there are readily imaginable scenarios where the fact that something will create pleasure for someone would count against it ( suppose the pleasure is sadistic ) . this view has the implication that even if we have an internally valid thought experiment , it would not follow that the principle established by the thought experiment will make the same contribution in other cases . so even if we have two precisely matched cases that differ only in one respect say that one is a doing , and the other an allowing ( as rachels 1975 aims to do)and establish that in this precisely controlled pair of cases , the factor that contrasts between the two cases does not make a moral difference , it would not follow that this factor will not make a moral difference in other cases . the alternative to this holistic view seems to be to make the equally substantive and controversial assumption that each morally relevant factor makes its contribution to the overall ethical reasons in play independently from all other factors . such a view , which kagan ( 1988 ) calls the additive assumption , would ( if true ) provide a solid justification of the external validity of thought experiments in ethics . however , the fact that the additive assumption is itself highly controversial and has been subjected to many purported counterexamples , suggests that it would make work in bioethics less rather than more rigorous if bioethicists were to presuppose its truth in doing ethical reasoning . refusing to grant the additive assumption would place the external validity of thought experiments in ethics on a par with that of randomised clinical trials . in neither case would there be a logical entailment between internal validity and external validity . the failure of this logical entailment does not imply that randomised clinical trials can not be externally valid , and would not imply that thought experiments in ethics can not be externally valid . so to abandon the additive assumption is not say that the use of thought experiments should be ( even could be ) abandoned in bioethics , but rather to suggest that bioethicists need to be aware that even rigorously designed thought experiments may not show what their designers think they do and that consequently bioethicists need to be on the look out for , and to expect , defeaters that prevent translation from one context to another.18 one key question is whether there are some kinds of thought experiments that are more likely to lack external validity than others . elster ( 2011 ) argues convincingly that outlandish thought experiments ( involving , say , individuals with two - hundred arms ) are much more likely to fail to be externally valid . in order to be confident that the thought experiment is externally valid , its readers need to be able to imagine in sufficient detail not only the outlandish elements of the thought experiment , but also the implications of the outlandish elements for ethical norms and practice within the world of the thought experiment . given that thought experiments are usually very briefly described , there is much that can go wrong here : something that the author intended to be imaginable , such as a human being with arms so long they can reach from one end of india to the other19 may not really be really imaginable , or be imagined very differently by different readers.20 even if a world containing such creatures is genuinely and stably imaginable , it may well be that taking the outlandish elements of the thought experiment seriously would entail such profound changes to other aspects of ethical life that there is little reason to think that ethical claims that are true of that world are transferable to our world . much work in bioethics analyses cases that are richer than thought experiments but still involve a number of important simplifications when compared to the real world . presumably the hope is that whilst such cases are significantly simplified , it will nonetheless be reasonable to assume that the case discussed is realistic enough to have clear implications for what should be done all things considered in actual cases , and that the greater richness of the examples discussed will help to evade some of the problems of external validity that can affect thought experiments . examples of this strategy would be examining the moral permissibility of an action type such as organ sales in the abstract , or analysing the validity of a particular argument against commercial surrogacy ( say that it constitutes the selling of babies ) . one worry about the external validity of this type of strategy , as i explore in more detail in wilson ( 2009 ) , is that it is much more difficult to justify conclusions about what ought to be done all things considered on the basis of philosophical analysis of arguments than some bioethicists seem to assume . for example , even if it could be adequately established that given valid consent and appropriate background conditions , sale of one s organs is morally permissible , there is a long journey from this claim to the conclusion that therefore organ sale should be legalised . in scenarios closer to real life , the risks of exploitation , organ theft , crowding out of altruistic donation , transplant tourism or of black markets may be sufficiently high that we judge that it would not be justifiable to legalise organ sales , despite the fact that we can conceive of an idealised situation in which organ sales might be morally permissible.21 focusing on the validity and soundness of ethical arguments is an indispensable tool for bioethicists . my claim is not that focusing on the quality of arguments is a bad way of doing bioethics , but that analyses of ethical arguments can be rigorous in the sense of attending carefully to the meaning of the terms and the pattern of logical inferences used , but yet still fail to be informative about what should be done all things considered . just like thought experiments thought experiments in bioethics are usually different in a number of salient aspects from the real world scenarios in which actual moral agents make their choices . thought experiments in bioethics typically make all of simplifications 14 , and may also make simplifications 5 and 6:authoritative ethical framing : the case raises the ethical question that the author of the thought experiment says it does . what the ethical issue is that the case raises is not subject to dispute.confined choices : choices must be made from a short predefined menu , with no ability to alter the terms of the problem . what counts as a viable response to the problem is stipulated by the author of the thought experiment , and is not up for discussion . the world of the thought experiment may operate according to laws that are plainly false of the real world . hence , responses that would be likely to be effective in real world analogues of the thought experiment may be stipulated not to work , and other responses , which would be unlikely to be effective in real world analogues , may be stipulated to be effective.certainty of effect : each of the defined choices will bring about its stipulated effect with certainty . it is possible to identify in advance who will benefit , and who will lose , from each of the predefined choices . where the choice is stipulated to bring about the desired effect a certain percentage of the time , these stipulated probabilities are entirely accurate.ceteris paribus : no morally relevant differences other than those which have been stipulated by the framer of the thought experiment apply to the situation.small numbers : the case is presented as one involving either single individuals , or groups of no more than five , even if the final desired aim is to make recommendations about situations involving large groups of people.atomicity : the case stands entirely on its own . there is no relevant history that affects judgements about the case , and no relevant future policy implications of making one decision rather than another . although the problem is not usually discussed in these terms , it is apparent that , just like clinical trials , the use of thought experiments can suffer from problems of internal and external validity . a clinical experiment is internally valid if it is designed in such a way that it correctly measures the causal effect of an independent variable or variables on one or more dependent variables . similarly , a thought experiment is internally valid to the extent that it allows its readers to make judgements that are confident and free of bias about the hypothesis or point of principle that it aims to test . thus for example , if the thought experiment requires comparing two cases which differ only in a single respect , we need to be confident that the cases do only differ in this one respect ; and if the framer of the thought experiment claims that only one of the stipulated choices in the case is morally permissible , we should be able to judge confidently that this is true . authoritative ethical framing : the case raises the ethical question that the author of the thought experiment says it does . confined choices : choices must be made from a short predefined menu , with no ability to alter the terms of the problem . what counts as a viable response to the problem is stipulated by the author of the thought experiment , and is not up for discussion . the world of the thought experiment may operate according to laws that are plainly false of the real world . hence , responses that would be likely to be effective in real world analogues of the thought experiment may be stipulated not to work , and other responses , which would be unlikely to be effective in real world analogues , may be stipulated to be effective . certainty of effect : each of the defined choices will bring about its stipulated effect with certainty . it is possible to identify in advance who will benefit , and who will lose , from each of the predefined choices . where the choice is stipulated to bring about the desired effect a certain percentage of the time , these stipulated probabilities are entirely accurate . ceteris paribus : no morally relevant differences other than those which have been stipulated by the framer of the thought experiment apply to the situation . small numbers : the case is presented as one involving either single individuals , or groups of no more than five , even if the final desired aim is to make recommendations about situations involving large groups of people . there is no relevant history that affects judgements about the case , and no relevant future policy implications of making one decision rather than another . an experiment trial has external validity in addition to internal validity to the extent that the causal effects demonstrated in it can be generalised to a wide range of other situations . within the fields of healthcare and public policy research , it is now a commonplace that an intervention that is efficacious in the highly idealised context of a controlled trial may not be effective in other contexts ( rothwell 2005 ) . as cartwright ( 2013 ) puts it , a randomised controlled trial ( rct ) can show that an intervention worked somewhere but not that it will work here . to describe a trial as lacking in external validity is not by itself to impugn its quality as research : it is in the nature of an rct that the rigour in experimental design that is necessary for internal validity sets limits on how , if at all , the results can be extrapolated to other contexts ( cartwright 2007 ) . however , research that lacks external validity will be useful for changing the world only in a narrow range of contexts . similarly , we can say that a thought experiment has external validity in addition to internal validity to the extent that points of principle that are established by it can be generalised to a wide range of other circumstances . thought experiments , and normative arguments more broadly , can lack external validity in at least two ways . first , if the principles they establish presuppose factual circumstances that are relevantly different from those that currently obtain . for example , nozick s entitlement theory of property rights is often thought to lack external validity on these grounds of factual irrelevance . the theory requires that property is justly held if and only if it was either ( a ) justly initially acquired or ( b ) legitimately transferred from someone else who was already entitled to it . even leaving on one side the kinds of difficulties that can be raised to nozick s account of just acquisition , it is clear that there have been massive and systematic ruptures in justice in transfer throughout human history , including wars , colonialism , slavery , and the genocidal destruction of first nation peoples . even if goods have been transferred legitimately since the time when they were unjustly acquired , nozick s view would not imply that they are now justly held . rather such goods would be subject to a principle of justice in rectification a principle that nozick does not specify . so despite initial appearances , nozick s account tells us virtually nothing about who is entitled to what , or what could be legitimately taken from whom and redistributed to others , in our world with its particular history . to his credit , nozick himself recognises the problem , and admits that his theory would require a full account of justice in rectification , which he is unable to offer , stating that in the absence of such a treatment applied to a particular society , one can not use the analysis and theory presented here to condemn any particular scheme of transfer payments , unless it is clear that no considerations of rectification of injustice could apply to justify it17 nozick ( 1975 , p. 231 ) . second , ethical principles and considerations are often claimed to interact with one another in holistic ways . on this view , there are scenarios in which ethical considerations that favour acting in certain ways in many or most cases no longer provide a reason in favour of acting in that way , and may even change polarity and provide a reason against acting in that way . for example , in usual circumstances , the fact that doing x will provide someone else with pleasure speaks in favour of it , but there are readily imaginable scenarios where the fact that something will create pleasure for someone would count against it ( suppose the pleasure is sadistic ) . this view has the implication that even if we have an internally valid thought experiment , it would not follow that the principle established by the thought experiment will make the same contribution in other cases . so even if we have two precisely matched cases that differ only in one respect say that one is a doing , and the other an allowing ( as rachels 1975 aims to do)and establish that in this precisely controlled pair of cases , the factor that contrasts between the two cases does not make a moral difference , it would not follow that this factor will not make a moral difference in other cases . the alternative to this holistic view seems to be to make the equally substantive and controversial assumption that each morally relevant factor makes its contribution to the overall ethical reasons in play independently from all other factors . such a view , which kagan ( 1988 ) calls the additive assumption , would ( if true ) provide a solid justification of the external validity of thought experiments in ethics . however , the fact that the additive assumption is itself highly controversial and has been subjected to many purported counterexamples , suggests that it would make work in bioethics less rather than more rigorous if bioethicists were to presuppose its truth in doing ethical reasoning . refusing to grant the additive assumption would place the external validity of thought experiments in ethics on a par with that of randomised clinical trials . in neither case would there be a logical entailment between internal validity and external validity . the failure of this logical entailment does not imply that randomised clinical trials can not be externally valid , and would not imply that thought experiments in ethics can not be externally valid . so to abandon the additive assumption is not say that the use of thought experiments should be ( even could be ) abandoned in bioethics , but rather to suggest that bioethicists need to be aware that even rigorously designed thought experiments may not show what their designers think they do and that consequently bioethicists need to be on the look out for , and to expect , defeaters that prevent translation from one context to another.18 one key question is whether there are some kinds of thought experiments that are more likely to lack external validity than others . elster ( 2011 ) argues convincingly that outlandish thought experiments ( involving , say , individuals with two - hundred arms ) are much more likely to fail to be externally valid . in order to be confident that the thought experiment is externally valid , its readers need to be able to imagine in sufficient detail not only the outlandish elements of the thought experiment , but also the implications of the outlandish elements for ethical norms and practice within the world of the thought experiment . given that thought experiments are usually very briefly described , there is much that can go wrong here : something that the author intended to be imaginable , such as a human being with arms so long they can reach from one end of india to the other19 may not really be really imaginable , or be imagined very differently by different readers.20 even if a world containing such creatures is genuinely and stably imaginable , it may well be that taking the outlandish elements of the thought experiment seriously would entail such profound changes to other aspects of ethical life that there is little reason to think that ethical claims that are true of that world are transferable to our world . much work in bioethics analyses cases that are richer than thought experiments but still involve a number of important simplifications when compared to the real world . presumably the hope is that whilst such cases are significantly simplified , it will nonetheless be reasonable to assume that the case discussed is realistic enough to have clear implications for what should be done all things considered in actual cases , and that the greater richness of the examples discussed will help to evade some of the problems of external validity that can affect thought experiments . examples of this strategy would be examining the moral permissibility of an action type such as organ sales in the abstract , or analysing the validity of a particular argument against commercial surrogacy ( say that it constitutes the selling of babies ) . one worry about the external validity of this type of strategy , as i explore in more detail in wilson ( 2009 ) , is that it is much more difficult to justify conclusions about what ought to be done all things considered on the basis of philosophical analysis of arguments than some bioethicists seem to assume . for example , even if it could be adequately established that given valid consent and appropriate background conditions , sale of one s organs is morally permissible , there is a long journey from this claim to the conclusion that therefore organ sale should be legalised . in scenarios closer to real life , the risks of exploitation , organ theft , crowding out of altruistic donation , transplant tourism or of black markets may be sufficiently high that we judge that it would not be justifiable to legalise organ sales , despite the fact that we can conceive of an idealised situation in which organ sales might be morally permissible.21 focusing on the validity and soundness of ethical arguments is an indispensable tool for bioethicists . my claim is not that focusing on the quality of arguments is a bad way of doing bioethics , but that analyses of ethical arguments can be rigorous in the sense of attending carefully to the meaning of the terms and the pattern of logical inferences used , but yet still fail to be informative about what should be done all things considered . just like thought experiments , more realistic cases can also suffer from problems of external validity . we started from an apparent tension between the theoretical and the practical aims of bioethics , and two questions . first , a question about whether both the practical and the theoretical aims should fall within the scope of bioethics proper . the first question can be answered briefly . normative theory without any thought to application ( stage 1 ) is not a part of bioethics but of normative ethics more abstractly conceived . any attempt to make normative judgements about cases ( from thought experiments ( stage 2 ) to real world cases ( stage 4 ) ) should uncontroversially be . making correct ( or at least well justified ) moral judgements about cases but this complexity seems no reason to draw the boundaries of bioethics in such a way that the questions it deals with are always simple . the world in which we act as moral agents is , after all , complex ; and bioethics gains much of its practical impetus from the attempt to help provide normative guidance in this world . so there is little reason to think that the boundaries of bioethics should stop short of including making wise judgements about complex individual cases . once one has determined what is ethically required all things considered in response to a real world situation , and if one has at least some power to influence the situation , it would be odd to think that this had no implications for what one should do . i thus find it difficult to deny that if a bioethicist comes to a particular conclusion about a real world case on the basis of a careful analysis of the relevant reasons , and then seeks to change the world on the basis of this analysis ( by means such as arguing for the view publicly ) that they do what they do as a bioethicist . so , at least where the means used are rational persuasion , stage 5 is very plausibly a core part of bioethics . on the second question , we can now see that the supposed opposition between the theoretical and the practical aims of bioethics from which we started is misleading . theoretical questions in ethics form a continuum from the simplest questions such as whether knowledge or pleasure is valuable in itself , to the most complex , such as what ought to be done , and by whom , to combat the rise of antimicrobial resistance.22 working out what ethical duties are held and by whom in a real world case is still a theoretical question , and like the simplest questions about cases involving one value in isolation , requires us to bring together relevant theoretical claims with situational judgement . the main difference is that as we travel across the continuum in one way , more and more of the messiness and complexity of the real world is bracketed for simplicity , and in the other way , more and more of the messiness and complexity of the real world is revealed . hence , even focusing entirely on bioethics as a theoretical rather than a practical pursuit gives no reason in itself to think that the examination of simpler and more abstract cases is more important than messier and more complex ones . a further argument the key question could be rephrased as whether the messiness and complexity of the real world is something that stands in the way of us getting a grip on what is really going on , ethically speaking , or whether this messiness and complexity is what is going on , ethically speaking . many philosophers assume , implicitly if not explicitly , that the former view is correct.23 this assumption makes it seem natural that , other things being equal , work on simpler and more abstract problems will be more fruitful for the discipline , given that such work appears to give rise to principles that will apply to a wider variety of cases , whilst analyses of richly described real world cases seem to tell us only about that particular case . we have seen that , given the implausibility of the linear model , things are likely to be more complicated than this . analysis of rich cases can themselves have important theoretical implications : for example , they may bring out problems or contradictions that went unnoticed in a more abstract model . analysis of real world cases can also be more widely useful to the practice of bioethics through the power of example : seeing how complex and contested empirical material bears on what should be done in one ethical problem may be useful in determining how complex and contested empirical material bears on a different ethical problem.24 as in the case of healthcare research , it seems plausible to assume that determining which are the better , and which the worse , ways of moving between simpler and more complex research problems is itself a complex research problem a problem that this paper has introduced , but not solved . perhaps the main lesson is that work in bioethics can fail to be rigorous in two distinct ways . in explaining this first , work in bioethics can be shoddy on its own terms : thought experiments can be poorly equalised , distinctions made inexactly , arguments vague or fallacious . second , work that is rigorous on its own terms can be unhelpful , or misleading , if authors extrapolate its lessons to other cases without due attention to the factors that could undermine external validity . bioethics journals are full of attempts to do research that is rigorous in the first sense , and to expose cases where others have done research that is not rigorous in this sense . but as yet there is very little written on , and little attention to , rigour in the second sense . if bioethics is going provide a useful basis for improving the world , this needs to change . first , bioethicists should be clearer and more explicit about the implications ( if any ) that they think that their arguments about simpler cases have for other more complex cases . they should not make claims that their analysis has policy implications unless they are willing for those policy implications to be taken seriously.25 second , bioethics needs a literature on how best to move between different levels of complexity , both on what counts as good practice in this area , and what is required to train the next generation of bioethicists .

this paper reflects on the relationship between theory and practice in bioethics , by using various concepts drawn from debates on innovation in healthcare research in particular debates around how best to connect up blue skies basic research with practical innovations that can improve human lives . it argues that it is a mistake to assume that the most difficult and important questions in bioethics are the most abstract ones , and also a mistake to assume that getting clear about abstract cases will automatically be of much help in getting clear about more complex cases . it replaces this implicitly linear model with a more complex one that draws on the idea of translational research in healthcare . on the translational model , there is a continuum of cases from the most simple and abstract ( thought experiments ) to the most concrete and complex ( real world cases ) . insights need to travel in both directions along this continuum from the more abstract to the more concrete and from the more concrete to the more abstract . the paper maps out some difficulties in moving from simpler to more complex cases , and in doing so makes recommendations about the future of bioethics .

Introduction The linear model in healthcare research Bioethics and the translational research paradigm Moving from simpler to more complex cases Internal and external validity in thought experiments Ethical arguments and what should be done all things considered Conclusion: embracing complexity

the two aims seem to pull bioethicists in different directions , because it is plausible to think that the most effective means of pursuing the theoretical aim will be relatively ineffective in pursuing the practical aim , and vice versa . in part this is because the questions that bioethics deals with are often much more obviously shaped by our present concerns than those in political philosophy , and in part it is because there is no more applied academic discipline than bioethics that stands in the relationship to it that political science and political theory stand to political philosophy . in brief , we tend to approach bioethics with the goal of improving our grip on real world ethical questions , and in working out what we should do all things considered , the buck stops with it . each distinction that is made provides the possibility for further disagreement , and so the net result is that academics naturally form themselves into a large number of camps , each with a very small number of people in them.3 such fragmentation on the basis of small differences is an anathema to achieving the kind of political or systemic changes that are usually required by the practical aim . in a deeper sense they are normative questions about how best to advance the discipline , considered as a a tradition in macintyre s sense , as an historically extended , socially embodied argument , and an argument precisely in part about the goods which constitute that tradition ( 1981 , p. 222 ) . this paper aims to start a different kind of reflexive conversation about the future of bioethics , focused not so much on which topics should be inside and which should be outside of bioethics , but about why we are doing bioethics , and what would count as doing it well . just as in the case of the shift to a more institutional and global focus , the changes required are unlikely to follow clearly from a conceptual analysis of the idea of bioethics alone , or from taking a poll of what current individual bioethicists think that it is for , but by the gradual accretion of evidence of unsolved problems and missed opportunities , and a careful construction of a new and more fruitful framework . so , even if one s orientation in bioethics is entirely towards the theoretical , questions about how to move between judgements based on simple cases and judgements in complex cases will be of central importance . the rest of the paper proceeds as follows : section 2 introduces various themes from debates on innovation in healthcare research in particular debates around how to best connect up blue skies basic research with practical innovations that can improve human lives as a way of reflecting on the relationship between the theoretical and the practical aims in bioethics . it explains how a simple top - down linear model of innovation has been replaced by a more complex translational model , which recognises that innovations move along different pathways both from the more basic. section 3 adapts the translational model to bioethics arguing that we should replace the idea of an opposition between the theoretical and the practical with the idea of a continuum from the most simple and abstract cases ( thought experiments ) to the most concrete and complex cases ( real world cases ) . insights need to travel in both directions along this continuum both from the simpler and more abstract to the more complex and from the more complex to the simpler and more abstract . questions about the relationship between theoretical advances and practical benefit are particularly urgent in healthcare research , owing to the ubiquity of death and suffering , the technical difficulty and expense of bringing new drugs to market , and governments own sizeable investments in funding both healthcare research and the successful treatments that result from it . so it is perhaps unsurprising that debate on the relationship between the theoretical and the practical is louder , older and more voluble in healthcare research than it is in bioethics , and contains a rich potential for cross - fertilisation . moreover , approaching the question of the relationship between theory and practice in bioethics via debates about translational research in healthcare has the advantage that it allows us to sidestep the dispute between fundamentally theoretical and fundamentally practical approaches to ethics . by starting from outside ethics , in a domain in which it is relatively uncontroversial that there are theoretical facts that inquiry aims to discover , it becomes apparent that even in this domain , it follows neither that more abstract theoretical truths are more worthy of pursuit than more concrete ones , nor that inquiry should be guided by the pursuit of theoretical truths alone.8 world war ii saw an unprecedented mobilisation of science for the war effort . as the war drew to a close , the us government faced a choice about the future of science funding : should it continue to funnel scientific research budgets towards short term solutions for centrally chosen goals , or would it be better to let scientists pursue their own endeavours without regard to practical application ? ( bush 1945 : chapter 3.3 ) bush argued forcefully that it is the role of governments to support basic research , and that basic research is best undertaken in universities . ( bush 1945 : chapter 3.3)the idea that theoretical work produces practical benefits but in unexpected ways that it would be counterproductive to attempt to second - guess is also at the heart of certain defences of philosophy that emphasise its long - term impact , but deny that this impact could usefully be measured or optimised in the short term.9 of course , one obvious disanalogy is that , when compared to science , there is a decided lack of concerted action in either the commercial sector or in civil society to translate the results of basic normative or other philosophical work into commercialisable or socially beneficial results ( though the use of game theory in management consultancy and elsewhere might be a possible analogue ) . even a case such as the discovery of penicillin , which might initially seem to be an obvious success story , shows how contingent takeup of basic science into applied science can be.11 as a result of the shortcomings of the linear model , a consensus has grown within healthcare research that funders and researchers need to think in systemic terms about how the different stages of research from the most basic science to the most applied can best fit together , an approach to thinking that has come to be known as translational research . worries about the lack of direct connection between progress in basic theory and progress in answering more concrete questions apply even more strongly in bioethics than in healthcare research . as section 1 explored , it is plausible to think that much high quality work in abstract normative theory has very little practical impact , and as we shall see , searching questions can also be asked about current efficiency in moving between ethical questions at different points of the continuum between the fully abstract and fully concrete . bioethicists do not often ask questions about research efficiency in their discipline , or explicitly address the question of how best to move between different levels of abstraction in ethical thinking , and so again the healthcare research literature will provide a starting - point . as table 1 indicates , we can divide the journey from the most basic science to interventions that actually benefit patients into five stages.12 if an intervention that benefits patients is to be derived from basic science , it must pass through each of these stages.13 given the uncontroversial focus in healthcare research prioritisation on the benefits to actual patients that are achieved at stage 5 , the model immediately raises questions about research funding priorities if it turns out that prioritising basic science is a markedly less efficient way of achieving patient benefit than , say , research - led improvement of doctors prescribing habits.table 1the translational continuum in healthcare research and in bioethicsstagehealthcare research stagebioethics equivalent1basic sciencediscussion of normative theory without any attempt to think about the applicability of moral theory to real life cases2proof of concept e.g . policy changes , supported by healthcare research to benefit patientspolicy changes or other actions to make the world closer to how it should be the translational continuum in healthcare research and in bioethics the model begins from the observation that much basic research for example the discovery that a particular protein has a role in regulating inflammation does not have obvious or immediate clinical application . we can construct a roughly parallel trajectory for bioethics , tracing the kind of steps that would need to occur if new insights in basic normative theory were to lead to ethical improvements on the ground.14 as in the case of healthcare research , the idea is not to say that moral change inevitably follows this trajectory or that it should , but rather to allow us to use the model to ask questions about where time and other resources are currently being concentrated along this path , and whether transitions between different elements could be improved . thought experiments play a key bridging role in crossing the continuum between basic normative theory and judgements about real world cases . if our aim is to discover the relative weight of , say , two factors , we should consider cases that involve only these two factors , perhaps artificially , rather than distract ourselves with other factors and options i focus on some of the potential difficulties involved in moving from thought experiments to judgements about more complex cases , and from more complex cases to what should be done all things considered . however , it is important to notice that the main point of the model that i am proposing is that we need to think more rigorously and more systematically about how best to move between different levels of complexity in the analysis of ethical problems . similar questions can and should be asked about moving from simpler to more complex cases on other parts of the continuum , about moving from complex cases to other cases of similar complexity , and about the implications of judgements about richly described complex cases for judgements about simpler and more abstract cases points i return to in sect . thought experiments in bioethics are usually different in a number of salient aspects from the real world scenarios in which actual moral agents make their choices . thought experiments in bioethics typically make all of simplifications 14 , and may also make simplifications 5 and 6:authoritative ethical framing : the case raises the ethical question that the author of the thought experiment says it does . there is no relevant history that affects judgements about the case , and no relevant future policy implications of making one decision rather than another . so even if we have two precisely matched cases that differ only in one respect say that one is a doing , and the other an allowing ( as rachels 1975 aims to do)and establish that in this precisely controlled pair of cases , the factor that contrasts between the two cases does not make a moral difference , it would not follow that this factor will not make a moral difference in other cases . however , the fact that the additive assumption is itself highly controversial and has been subjected to many purported counterexamples , suggests that it would make work in bioethics less rather than more rigorous if bioethicists were to presuppose its truth in doing ethical reasoning . so to abandon the additive assumption is not say that the use of thought experiments should be ( even could be ) abandoned in bioethics , but rather to suggest that bioethicists need to be aware that even rigorously designed thought experiments may not show what their designers think they do and that consequently bioethicists need to be on the look out for , and to expect , defeaters that prevent translation from one context to another.18 one key question is whether there are some kinds of thought experiments that are more likely to lack external validity than others . in order to be confident that the thought experiment is externally valid , its readers need to be able to imagine in sufficient detail not only the outlandish elements of the thought experiment , but also the implications of the outlandish elements for ethical norms and practice within the world of the thought experiment . given that thought experiments are usually very briefly described , there is much that can go wrong here : something that the author intended to be imaginable , such as a human being with arms so long they can reach from one end of india to the other19 may not really be really imaginable , or be imagined very differently by different readers.20 even if a world containing such creatures is genuinely and stably imaginable , it may well be that taking the outlandish elements of the thought experiment seriously would entail such profound changes to other aspects of ethical life that there is little reason to think that ethical claims that are true of that world are transferable to our world . much work in bioethics analyses cases that are richer than thought experiments but still involve a number of important simplifications when compared to the real world . presumably the hope is that whilst such cases are significantly simplified , it will nonetheless be reasonable to assume that the case discussed is realistic enough to have clear implications for what should be done all things considered in actual cases , and that the greater richness of the examples discussed will help to evade some of the problems of external validity that can affect thought experiments . one worry about the external validity of this type of strategy , as i explore in more detail in wilson ( 2009 ) , is that it is much more difficult to justify conclusions about what ought to be done all things considered on the basis of philosophical analysis of arguments than some bioethicists seem to assume . for example , even if it could be adequately established that given valid consent and appropriate background conditions , sale of one s organs is morally permissible , there is a long journey from this claim to the conclusion that therefore organ sale should be legalised . my claim is not that focusing on the quality of arguments is a bad way of doing bioethics , but that analyses of ethical arguments can be rigorous in the sense of attending carefully to the meaning of the terms and the pattern of logical inferences used , but yet still fail to be informative about what should be done all things considered . just like thought experiments thought experiments in bioethics are usually different in a number of salient aspects from the real world scenarios in which actual moral agents make their choices . thought experiments in bioethics typically make all of simplifications 14 , and may also make simplifications 5 and 6:authoritative ethical framing : the case raises the ethical question that the author of the thought experiment says it does . there is no relevant history that affects judgements about the case , and no relevant future policy implications of making one decision rather than another . so even if we have two precisely matched cases that differ only in one respect say that one is a doing , and the other an allowing ( as rachels 1975 aims to do)and establish that in this precisely controlled pair of cases , the factor that contrasts between the two cases does not make a moral difference , it would not follow that this factor will not make a moral difference in other cases . however , the fact that the additive assumption is itself highly controversial and has been subjected to many purported counterexamples , suggests that it would make work in bioethics less rather than more rigorous if bioethicists were to presuppose its truth in doing ethical reasoning . so to abandon the additive assumption is not say that the use of thought experiments should be ( even could be ) abandoned in bioethics , but rather to suggest that bioethicists need to be aware that even rigorously designed thought experiments may not show what their designers think they do and that consequently bioethicists need to be on the look out for , and to expect , defeaters that prevent translation from one context to another.18 one key question is whether there are some kinds of thought experiments that are more likely to lack external validity than others . given that thought experiments are usually very briefly described , there is much that can go wrong here : something that the author intended to be imaginable , such as a human being with arms so long they can reach from one end of india to the other19 may not really be really imaginable , or be imagined very differently by different readers.20 even if a world containing such creatures is genuinely and stably imaginable , it may well be that taking the outlandish elements of the thought experiment seriously would entail such profound changes to other aspects of ethical life that there is little reason to think that ethical claims that are true of that world are transferable to our world . much work in bioethics analyses cases that are richer than thought experiments but still involve a number of important simplifications when compared to the real world . presumably the hope is that whilst such cases are significantly simplified , it will nonetheless be reasonable to assume that the case discussed is realistic enough to have clear implications for what should be done all things considered in actual cases , and that the greater richness of the examples discussed will help to evade some of the problems of external validity that can affect thought experiments . one worry about the external validity of this type of strategy , as i explore in more detail in wilson ( 2009 ) , is that it is much more difficult to justify conclusions about what ought to be done all things considered on the basis of philosophical analysis of arguments than some bioethicists seem to assume . for example , even if it could be adequately established that given valid consent and appropriate background conditions , sale of one s organs is morally permissible , there is a long journey from this claim to the conclusion that therefore organ sale should be legalised . my claim is not that focusing on the quality of arguments is a bad way of doing bioethics , but that analyses of ethical arguments can be rigorous in the sense of attending carefully to the meaning of the terms and the pattern of logical inferences used , but yet still fail to be informative about what should be done all things considered . any attempt to make normative judgements about cases ( from thought experiments ( stage 2 ) to real world cases ( stage 4 ) ) should uncontroversially be . i thus find it difficult to deny that if a bioethicist comes to a particular conclusion about a real world case on the basis of a careful analysis of the relevant reasons , and then seeks to change the world on the basis of this analysis ( by means such as arguing for the view publicly ) that they do what they do as a bioethicist . theoretical questions in ethics form a continuum from the simplest questions such as whether knowledge or pleasure is valuable in itself , to the most complex , such as what ought to be done , and by whom , to combat the rise of antimicrobial resistance.22 working out what ethical duties are held and by whom in a real world case is still a theoretical question , and like the simplest questions about cases involving one value in isolation , requires us to bring together relevant theoretical claims with situational judgement . the main difference is that as we travel across the continuum in one way , more and more of the messiness and complexity of the real world is bracketed for simplicity , and in the other way , more and more of the messiness and complexity of the real world is revealed . hence , even focusing entirely on bioethics as a theoretical rather than a practical pursuit gives no reason in itself to think that the examination of simpler and more abstract cases is more important than messier and more complex ones . many philosophers assume , implicitly if not explicitly , that the former view is correct.23 this assumption makes it seem natural that , other things being equal , work on simpler and more abstract problems will be more fruitful for the discipline , given that such work appears to give rise to principles that will apply to a wider variety of cases , whilst analyses of richly described real world cases seem to tell us only about that particular case . analysis of real world cases can also be more widely useful to the practice of bioethics through the power of example : seeing how complex and contested empirical material bears on what should be done in one ethical problem may be useful in determining how complex and contested empirical material bears on a different ethical problem.24 as in the case of healthcare research , it seems plausible to assume that determining which are the better , and which the worse , ways of moving between simpler and more complex research problems is itself a complex research problem a problem that this paper has introduced , but not solved . they should not make claims that their analysis has policy implications unless they are willing for those policy implications to be taken seriously.25 second , bioethics needs a literature on how best to move between different levels of complexity , both on what counts as good practice in this area , and what is required to train the next generation of bioethicists .

it causes progressive atrophy of the optic disc resulting in typical defects in the visual field.1 it can lead to total loss of vision if left untreated.2,3 indeed glaucoma is the second most common cause of blindness worldwide.3,4 unfortunately , the pathogenesis of this disorder is not fully explained . because elevated intraocular pressure ( iop ) is known to be the main risk factor for development and progression of glaucoma , its therapy relies nowadays on iop reduction.510 topical use of antiglaucoma drugs is probably the most commonly used tool for this . among the many topical hypotensive medications , prostaglandin analogs are proved to be the most potent in lowering iop and with very few systemic side effects.11,12 for these reasons , they are recommended as first - line therapy for this disease.3,11,13,14 prostaglandin analogs were first proposed for glaucoma treatment by camras and brito.1416 nowadays , derivatives of prostaglandin f2 , ie , latanoprost , travoprost , unoprostone , and a prostamide , bimatoprost , are commercially available . in van der valk s meta - analysis , latanoprost reduced iop by 28%31% from baseline , travoprost by 29%31% , and bimatoprost by 28%33%.12 latanoprost and travoprost are selective prostanoid fp receptor agonists , and by binding to these receptors they exert their iop - lowering effect.1721 bimatoprost is a prostamide , the exact molecular mode of action of which is not clearly understood.14,18,21 all these compounds decrease iop by increasing aqueous outflow , mainly through the uveoscleral ( unconventional ) route.14,22 in young individuals , up to 30% of aqueous fluid flows out through the unconventional route , but this drops during aging.22 hypotensive lipids can increase aqueous fluid outflow by up to 50%.22 moreover , they do not have an effect on aqueous production.14 they probably act by increasing the activity of matrix metalloproteinases and widening the spaces between ciliary muscle bundles.14 it has been found that the increase in uveoscleral outflow may also be due to relaxation of the ciliary muscle , leading to widening spaces between muscle bundles.14 ester forms of pgf2 derivatives penetrate the cornea much better . this is why all prostanoids except for bimatoprost are administered topically as prodrugs . after entering the cornea , they are hydrolyzed by corneal esterases to their active carboxylic acid forms.17 prostaglandin derivatives have a strong , long - lasting , stable iop - lowering effect with few systemic side effects.4,14,17 unfortunately , they may induce local adverse side effects , including conjunctival and ocular hyperemia , iris and periocular skin pigmentation , and eyelash growth.4,17,23 there have been efforts to find other prostanoid fd receptor agonists which are more potent in reducing iop but with fewer and milder side effects.17,21,24 in preclinical in vitro and in vivo studies , nakajama et al tested different pgf2 derivatives and found that 15,15 difluoro pgf2 derivatives retained iris constrictor activity , decreased iop in conscious monkeys , but did not increase the melanin content of cultured b16-f10 melanoma cells.17 this last observation was confirmed by tagaki et al , who also demonstrated an ocular hypotensive effect for this compound . the effect was dose - dependent , with a peak at eight hours after administration.24 moreover , the effect was stronger and more continuous than that induced by latanoprost.24 reduction of iop was dose - dependent in the eyes of ddy mice.19 this effect was similar to the effect of travoprost , but stronger and more longer - lasting than after administration of latanoprost.19 in this animal model , ota et al also found that tafluprost lowered iop via prostanoid fp receptors , and that part of the hypotensive effect may be related to fp receptor - mediated prostaglandin production and stimulation of ep3 receptors.20 this iop - lowering effect in mice was confirmed by akaishi et al.25 it has been postulated that alterations in optic nerve head blood flow may be involved in the pathogenesis of glaucoma.1,26 for this reason , improvement in ocular blood flow by glaucoma therapy may be helpful . studies assessing the influence of the available prostaglandin analogs on ocular blood flow have yielded both positive and negative results.14 in a study by izumi et al tafluprost 0.0015% significantly increased retinal blood flow and blood velocity as measured by laser doppler velocimetry in cats.29 this observation was then confirmed by akaishi et al27 who found that optic nerve head blood flow increased in rabbit eyes after 28 days administration of any of the three f2 prostaglandin analogs , ie , tafluprost , latanoprost , or travoprost . moreover , the increase induced by tafluprost was greater than that induced by latanoprost or travoprost.27 tafluprost also increased optic nerve head blood flow in both normal and experimental glaucomatous eyes in monkeys.28 dong et al wanted to determine if tafluprost could relax precontracted rabbit ciliary arteries , and found that it induced concentration - dependent relaxation but by a mechanism that was independent of endothelial - derived factor.26 in the future , these findings are likely to be shown to be real additional effects of antiglaucoma drugs . at this time we do not possess reliable tools for blood flow measurement , and thus the beneficial role of improvement in ocular blood flow can not as yet be ascertained.29,30 another interesting question concerning antiglaucoma agents concerns their neuroprotective properties . a direct antiapoptotic effect in cultured retinal ganglion cells ( rgc-5s ) and rat retinal ganglion cells with optic nerve crush was identified by kanamori et al . in an in vitro model , tafluprost promoted cell viability in a dose - dependent manner , significantly reducing the number of caspase 3-positive cells and suppressing [ ca ] evoked by exogenous glutamate . in an in vivo rat model , tafluprost increased the survival rate of ganglion cells in eyes treated with this drug for 14 days after optic nerve crush.31 these observations suggest that tafluprost possesses an antiapoptotic effect in retinal ganglion cells in rats . tafluprost ( 1-methylethyl ( 5z)-7-[(1r,2r,3r,5s)-2-[(1e)-3,3-difluoro-4-phenoxy-1-butenyl]-3,5-dihydroxycyclopentyl]-5- heptenoate)24,31 is a 16-phenoxy analog of pgf2 , with a 15,15-difluoro substitution.21 it is presently available in two formulations , ie , with benzalkonium chloride ( bak)-tapros in japan ( santen pharmaceutical co. ltd . , osaka , japan ) and preservative - free in europe , ie , taflotan ( santen oy , finland ) , both in 0.0015% ( 15 g / ml ) concentrations.21 tafluprost differs from the other prostanoids available on the market because it possesses two fluorine atoms at the carbon 15 position , instead of the hydroxyl group present in latanoprost , travoprost , and bimatiprost.14,22 it is an isopropyl ester ( afp-168 ) and , like other prostaglandin analogs , is rapidly hydrolyzed by corneal esterases to the free acid of tafluprost ( afp-172 ) , which is its active form.14,21,24 afp- 172 is a very potent fp receptor agonist , with a ki of 0.4b nm.17,24 its affinity for the human prostanoid fp receptor is 12 times that of the carboxylic acid in latanoprost and 1700 times that in unoprostone . it also has a 126-fold higher affinity for fp receptors than for ep3 receptors , and negligible affinity to other prostanoid receptors ( dp , ep2 , ip , tp).24 tafluprost increases in vivo uveoscleral outflow as measured by fluorophotometry.24 after either single or repeated topical dosing , the plasma concentration of tafluprost is low . its active form , ie , tafluprost acid , can be detected in plasma for up to one hour after topical administration , with a peak at 10 minutes.3 it is thought that the pharmacologic profile of this compound is similar to that of other prostaglandins available on the market.32 the pharmacodynamics , safety , and tolerability of tafluprost in healthy volunteers were assessed in a clinical , masked , placebo - controlled phase i study.23 tafluprost 0.0025% and 0.005% , latanoprost 0.005% , and placebo were given for seven days . the decline in iop from baseline was 4.3 mmhg for tafluprost 0.0025% , 6.8 mmhg for tafluprost 0.005% , 5.3 mmhg for latanoprost , and 3.1 mmhg for placebo . the decrease in iop values versus baseline was significant for all treatment groups , and superior with tafluprost 0.005% to values with placebo and latanoprost 0.005% . in another placebo - controlled phase i study , healthy volunteers were given sequential ascending doses of tafluprost , ie , 0.0001% , 0.0005% , 0.0025% , and 0.005%.4 for all these doses , a decreasing iop effect was present as compared with placebo . the effect was dose - dependent and significant for concentrations of 0.0005% , 0.0025% , and 0.005% . the effect was maximal at 12 hours after administration and lasted throughout the duration of treatment.4 the therapeutic concentration of tafluprost was set at 0.0015% as a result of a phase ii dose - response study performed in japan.21 in a randomized , double - masked , controlled , multicenter , multinational phase ii study , traverso et al assessed the duration and stability of the iop - lowering effect and tolerability of tafluprost 0.0015% compared with latanoprost 0.005% in patients with primary open - angle glaucoma , exfoliation glaucoma , or ocular hypertension.33 they observed that maximum reduction of iop was reached by day 7 of treatment and sustained until day 42 . they showed that tafluprost 0.0015% decreased iop from baseline by 9.7 3.3 mmhg and latanoprost 0.005% by 8.8 4.3 mmhg.33 phase iii clinical studies were conducted in japan and in europe . in the first one , the efficacy of tafluprost with that of latanoprost in 109 patients with open - angle glaucoma and ocular hypertension were compared.21 after four weeks of administration , they observed reduction in iop by 6.6 2.5 mmhg ( 27.6 9.6% ) in the tafluprost group and by 6.2 2.5 mmhg ( 25.9 9.7% ) in the latanoprost group.21 a second phase iii study involving 351 japanese patients with open - angle glaucoma or ocular hypertension showed that the reduction in iop from baseline was stable throughout 52 weeks and varied from 4.9 to 5.7 mmhg.21 a potent effect of tafluprost 0.0015% on iop and the safety of this medication were also demonstrated in a randomized , parallel - group , double - masked european phase iii study conducted in 49 centers in eight countries for up to 24 months.34 the objective of this study was to compare the efficacy and safety profiles of tafluprost 0.0015% and latanoprost 0.005% in 533 patients with open - angle glaucoma and ocular hypertension . patients had not only primary open - angle glaucoma but also pigmentary and exfoliative disease . both drugs exerted a potent and significant iop - lowering effect throughout the study , with average decreases of 68 mmhg ( 27%31% ) for tafluprost and 79 mmhg ( 29%35% ) for latanoprost . these results are in line with those of other prostaglandins , as shown in the van der valk meta - analysis.12 after 24 months , the mean decrease in iop from baseline was 7.1 mmhg ( 29.1% ) in the group treated with tafluprost and 7.7 mmhg ( 32.2% ) in the group treated with latanoprost.34 the primary aim of the randomized , investigator - masked , multicenter , crossover phase iii study described by hamacher et al was to evaluate the pharmacodynamics and safety of preserved and preservative - free tafluprost 0.0015% in patients with open - angle glaucoma or ocular hypertension . these investigators observed similar reductions in iop of > 5 mmhg with preservative - free and preserved formulations.11 a preservative - free tafluprost 0.0015% formulation also lowered iop effectively in a population with poor iop control with prior medications . mean iop reduction in 544 screened patients was from 19.4 5.0 mmhg at baseline to 15.3 3.5 mmhg after 12 weeks of tafluprost treatment . in total , 79.5% of eyes treated with the preservative - free formulation of tafluprost achieved iop 18 mmhg 12 weeks after switching medication.35 other phase iii studies indicated that tafluprost , like other prostaglandin analogs , may also exert an additional iop - lowering effect when administered together with the -blocker , timolol , in cases when monotherapy is not adequate.36 in 2007 , sutton et al observed in a phase i clinical study that systemic safety was similar for tafluprost , latanoprost , and placebo when administered to eyes . investigators did not observe clinically significant changes in laboratory parameters , vital signs , or electrocardiographic parameters in any of 49 participants throughout the course of the study . this was more frequent after administration of tafluprost in concentrations of 0.0025% or 0.005% than after administration of latanoprost 0.005%.23 the incidence of photophobia was greater in the tafluprost group than in the latanoprost group . it is noteworthy that the doses used in this study exceeded the dose in the currently available preparations , ie , 0.0015% . the most frequently observed adverse effect was mild , concentration - dependent hyperemia.4 mild chemosis was also observed in two of 16 patients receiving tafluprost 0.0001% . interestingly , the authors stated that rates of adverse effects were similar for the tafluprost 0.0001% and 0.0025% and latanoprost 0.005% groups , but rates of ocular hyperemia was significantly lower in eyes receiving latanoprost.4 these authors did not find either cells or flare in the anterior chamber or abnormalities of the iris , lens , or vitreous humor in any eyes during the study.4 all adverse events described in the two aforementioned studies were mild to moderate and did not result in treatment discontinuation.4,23 similar observations were made by uusitalo et al who administered preserved and preservative - free formulations of tafluprost . ocular adverse events were of mild or moderate severity , with the most prevalent being ocular hyperemia.3 a phase iii study described by uusitalo analyzed the safety of tafluprost 0.0015% versus latanoprost 0.005% over 24 months in a representative group of 533 patients.34 both drugs were well tolerated . reported adverse events were only mild to moderate . the authors found that during the 24 month study period , at least one adverse event was reported by 176 of 264 ( 66.7% ) of patients receiving tafluprost , and by 162 of 264 ( 61.4% ) of patients receiving latanoprost . nonocular adverse events were reported by 133 ( 50.4% ) patients treated with tafluprost , and by 114 ( 43.2% ) with latanoprost , but only 11 in the tafluprost group and nine in the latanoprost group , respectively , were considered to be related to treatment.34 the authors did not find any clinically significant changes in blood pressure or heart rate during the 24 month study period , or in laboratory parameters up to 12 months . ocular adverse effects were reported by 48.1% of patients in the tafluprost group and by 44.3% patients in the latanoprost group . most frequently reported were conjunctival hyperemia and ocular redness . the stimulating effect on eyelash growth was absent or mild in > 90% of patients after month 24 in both the tafluprost and latanoprost groups . a slight overall tendency towards corneal thinning was observed in both groups of patients during the study , but the changes were comparable between the groups.34 it is known that pgf2 derivatives cause an increase in iris and periocular skin pigmentation.17,21 this was observed in 43%56% of patients receiving latanoprost for longer than one year.37 moreover , the pigmentation increases with prolonged therapy.23,37 latanoprost was reported to increase melanogenesis in cultured melanoma cells . the carboxylic acid in latanoprost increased the melanin content of cultured b16b10 melanoma cells in a dose - dependent manner , but derivatives possessing two fluorine atoms at the 15-position as afp-172 ( carboxylic acid of tafluprost ) did not , even at the maximal dose.17 the same results were obtained in 2004 by tagaki et al.24 such observations led to the suggestion that tafluprost may induce a lower incidence of iris and periocular skin pigmentation than latanoprost.24 in a long - term phase iii study , uusitalo et al reported slightly more cases of iris pigmentation in the group treated with latanoprost ( 28% ) than in those treated with tafluprost ( 26.1% ) , but these differences were not significant.34 toxic adverse reactions to antiglaucoma topical medication may be only minimal or be very severe . the cytotoxic effects of these drugs cause damage and death of conjunctival and corneal epithelial cells . inflammation may be the first sign of a toxic drug effect on superficial tissues of the eye . this is caused by activation of the inflammatory response in the conjunctiva , either acute or chronic . this could be papillary , with generalized injection , or follicular , caused by proliferation of lymphocytes and plasma cells.38 unfortunately , such inflammatory changes create a potential risk for failure of further glaucoma filtration surgery.39,40 these changes may also lead to keratinization and conjunctival scarring , with symblepharon formation , known as drug - induced pseudopemphigoid.3,11,38 many of these undesirable effects are connected with the preservatives present in eye drop formulations.2 the adverse influence of preservative - containing topical antiglaucoma medications on cells and tissues on the eye surface is well documented in in vitro and in vivo studies.2,32,38,41 bak is the most commonly used preservative in eye drops.42 it has already been found that this compound exerts cytotoxic ( proapoptotic and pronecrotic ) effects on the ocular surface and trabecular meshwork cells.4345 it also causes reduction of cellular viability , infiltration of conjunctival stroma , and overexpression of inflammation- or apoptosis - related molecules , such as apo-2,7 , fas ( cd45 ) , hla - dr , icam-1.32,38,4652 moreover , decreased expression of muc5ac on the conjunctival cells were found in impression cytology specimens taken from patients treated locally with antiglaucoma drugs containing bak . this phenomenon may explain the high prevalence of dry eye syndrome in patients after prolonged antiglaucoma therapy.34,50,53 solutions which are preservative - free , have lower bak concentrations , or contain alternative preservatives were introduced into topical glaucoma therapy to minimize side effects.2 among the widely used prostaglandin analogs , only tafluprost is available in a bak - free formulation.3,42 a preservative - free solution of tafluprost showed reduced toxicity in human conjunctival epithelial cell lines when compared with preserved latanoprost , travoprost , and bimatoprost.54 tafluprost had low proapoptotic / pro - oxidative effects in vitro when compared with preservative - containing formulations.54 liang et al assessed conjunctival and corneal reactions to preservative - free tafluprost , commercially available latanoprost , and benzalkonium chloride 0.02% in in vivo studies.45 corneal epithelium confocal microscopy in vivo revealed partial desquamation of epithelial cells , irregular cell shapes , anisocytosis and loss of cell borders , abnormal reflectivity patterns , swollen cells , and inflammatory infiltrations in rabbit eyes treated with latanoprost and bak solution . rabbit corneas treated with preservative - free tafluprost were almost normal , with the epithelium having a regular polygonal mosaic appearance , brightly reflective nuclei , and no obvious desquamation or swelling . a slight inflammatory infiltrate in the anterior corneal stroma was observed only after administration of bak . inflammatory infiltrations in the peripheral cornea and the limbus area were noted after exposure to latanoprost or bak , but not after exposure to preservative - free tafluprost.45 similarly , conjunctival stroma vessels showed rolling of inflammatory cells after installation of bak or latanoprost . after installation of preservative - free tafluprost , normal blood vessels were observed , without any rolling of inflammatory cells . moreover , significant inflammatory infiltration in specimens taken from eyes exposed to latanoprost and abundant inflammatory cell patches in those exposed to bak were observed in conjunctival impression cytology specimens.45 specimens taken from eyes exposed to latanoprost and bak showed a higher expression of cd45 + cells than those exposed to tafluprost without preservative . similarly , expression of tumor necrosis factor receptor 1 in conjunctival impression cytology samples was highest in patients treated with bak or latanoprost . only a few tunel+ cells were observed after installation of preservative - free tafluprost but more of these cells were present after installation of latanoprost or bak.45 these observations established the lower toxicity of the preservative - free formulation to the anterior segment of the eye.45 bak is thought to be an ocular penetration enhancer for topically administered drugs , because it increases the corneal permeability of pharmacologic agents.42,55 pellinen and lokkila evaluated corneal penetration of preserved and preservative - free tafluprost 0.0015% into rabbit aqueous humor after topical application . they noticed that there were no significant between - group differences in mean concentrations of tafluprost acid in the aqueous humor . they concluded that bak at the concentration used in the tafluprost formulations did not affect corneal penetration of this drug into rabbit aqueous humor.55 it is possible that tafluprost has its own high corneal penetrating ability and bak would not enhance it.55 in a phase i study evaluating the pharmacokinetics and efficacy of preserved and preservative - free tafluprost , uusitalo et al did not observe any significant differences in pharmacokinetic parameters between the formulations , after either single or repeated dosing.3 ocular hyperemia occurred with the same frequency in both groups , but was predominantly of moderate severity in eyes treated with preserved tafluprost , and of mild severity in those treated with the preservative - free formulation.3 the safety of preserved and preservative - free tafluprost was also assessed in a phase iii study.11 in contrast with the findings of uusitalo et al it was shown that conjunctival hyperemia was reported more often by people using preservative - free tafluprost.11 the aforementioned studies showed that iop reduction obtained by preservative - free tafluprost is equivalent to that achieved by the preserved formulation . it seems that removing the preservative bak from the tafluprost formulation does not change the iop - lowering properties of preparation.11,42 because the aim of designing a preservative - free prostaglandin formulation was to reduce toxic effects and clinical complications in patients treated for glaucoma and ocular hypertension , it was reasonable to check if preservative - free tafluprost was a genuine alternative for patients receiving prostaglandins other than tafluprost . uusitalo et al investigated the hypotensive effect and tolerability of preservative - free tafluprost in patients with open - angle glaucoma and ocular hypertension exhibiting ocular surface side effects during latanoprost treatment.32 twelve weeks after switching from preserved latanoprost to preservative - free tafluprost , iop was maintained at the same level . iop values were 16.8 2.5 mmhg at baseline and 16.4 2.7 mmhg at weeks 2 , 6 , and 12 of tafluprost treatment.32 the number of patients with ocular side effects , ie , conjunctival hyperemia , and corneal and conjunctival fluorescein staining , was reduced by 50% after switching the drugs . the same was reported for other ocular symptoms , including itching , tearing , irritation , burning , stinging and foreign body sensation . after 12 weeks of tafluprost preservative- free treatment , fluorescein break - up time increased from 4.5 2.5 seconds at baseline to 7.8 4.9 seconds ( p < 0.001).32 results of immunocytologic testing of impression cytology samples revealed that , after 12 weeks of treatment with preservative - free tafluprost in patients previously using latanoprost , there was a significant reduction in those expressing abnormal levels ( < 7% ) of muc5ac - positive goblet cells and abnormal levels ( > 40% ) of hla - dr - positive epithelial cells.32 this observations may indicate a less harmful influence of preservative - free tafluprost on the conjunctiva than preserved latanoprost.32 surprisingly , change in schirmer s test scores was smaller and statistically significant only at week 6 of treatment with the preservative - free tafluprost formulation.32 this is probably the reason for the relative lack of data on patient satisfaction and compliance . in the uusitalo study , the comparison of ophthalmic medications or tolerability questionnaire devised by barbel et al was used to assess drop discomfort in patients treated with preserved latanoprost and switched to preservative - free tafluprost.32,56 as the authors described , among patients receiving the commercially available latanoprost formulation , 30% experienced no negative effect on quality of life , 59% experienced a little or some , 10% quite a bit or very much , and 1% extremely negative.32 after switching to preservative - free tafluprost , no negative effect on quality of life was reported by 52% of enrolled patients , 46% reported a little or some , 2% quite a bit and 0% very much or extremely after 12 weeks of this therapy . the percentage of latanoprost patients who were totally satisfied with therapy at baseline was 16% , and 36% were very satisfied . at week 12 of treatment with preservative - free tafluprost , 32% of patients were totally satisfied and 45% were very satisfied.32 in another study , patients with poor local tolerance of their medications noticed improvement of subjective symptoms and clinical signs after changing their therapy to preservative - free tafluprost 0.0015%.35 similar analyses are needed to compare safety , tolerability , patient compliance , and comfort between tafluprost and the other prostaglandin analogs . both preserved and preservative - free formulations of tafluprost are relatively new regimens for glaucoma treatment . the results of existing clinical studies of tafluprost use are very promising . despite several studies concerning its efficacy , safety , and tolerability ,

glaucoma is one of the most common neuropathies of the optic nerve . an elevated intraocular pressure ( iop ) is a well documented risk factor for the development and progression of this disease . until now , iop reduction is the only well documented successful method of glaucoma treatment . among the many hypotensive drugs , prostaglandin analogs are proved to be the most potent antiglaucoma agents , with very few systemic side effects . a new prostanoid fp receptor analog , tafluprost , has been introduced into the medical treatment of glaucoma and ocular hypertension . many studies have shown that it is an efficient iop - lowering drug , and that it is safe and well tolerated . a preservative - free tafluprost formulation is as potent as a preserved one , but it has fewer and milder toxic effects on the eye .

Introduction Preclinical and animal studies Pharmacology, pharmacokinetics, and clinical efficacy Safety and tolerability Toxic adverse effects Patient compliance and comfort Conclusion

it causes progressive atrophy of the optic disc resulting in typical defects in the visual field.1 it can lead to total loss of vision if left untreated.2,3 indeed glaucoma is the second most common cause of blindness worldwide.3,4 unfortunately , the pathogenesis of this disorder is not fully explained . because elevated intraocular pressure ( iop ) is known to be the main risk factor for development and progression of glaucoma , its therapy relies nowadays on iop reduction.510 topical use of antiglaucoma drugs is probably the most commonly used tool for this . among the many topical hypotensive medications , prostaglandin analogs are proved to be the most potent in lowering iop and with very few systemic side effects.11,12 for these reasons , they are recommended as first - line therapy for this disease.3,11,13,14 prostaglandin analogs were first proposed for glaucoma treatment by camras and brito.1416 nowadays , derivatives of prostaglandin f2 , ie , latanoprost , travoprost , unoprostone , and a prostamide , bimatoprost , are commercially available . in van der valk s meta - analysis , latanoprost reduced iop by 28%31% from baseline , travoprost by 29%31% , and bimatoprost by 28%33%.12 latanoprost and travoprost are selective prostanoid fp receptor agonists , and by binding to these receptors they exert their iop - lowering effect.1721 bimatoprost is a prostamide , the exact molecular mode of action of which is not clearly understood.14,18,21 all these compounds decrease iop by increasing aqueous outflow , mainly through the uveoscleral ( unconventional ) route.14,22 in young individuals , up to 30% of aqueous fluid flows out through the unconventional route , but this drops during aging.22 hypotensive lipids can increase aqueous fluid outflow by up to 50%.22 moreover , they do not have an effect on aqueous production.14 they probably act by increasing the activity of matrix metalloproteinases and widening the spaces between ciliary muscle bundles.14 it has been found that the increase in uveoscleral outflow may also be due to relaxation of the ciliary muscle , leading to widening spaces between muscle bundles.14 ester forms of pgf2 derivatives penetrate the cornea much better . after entering the cornea , they are hydrolyzed by corneal esterases to their active carboxylic acid forms.17 prostaglandin derivatives have a strong , long - lasting , stable iop - lowering effect with few systemic side effects.4,14,17 unfortunately , they may induce local adverse side effects , including conjunctival and ocular hyperemia , iris and periocular skin pigmentation , and eyelash growth.4,17,23 there have been efforts to find other prostanoid fd receptor agonists which are more potent in reducing iop but with fewer and milder side effects.17,21,24 in preclinical in vitro and in vivo studies , nakajama et al tested different pgf2 derivatives and found that 15,15 difluoro pgf2 derivatives retained iris constrictor activity , decreased iop in conscious monkeys , but did not increase the melanin content of cultured b16-f10 melanoma cells.17 this last observation was confirmed by tagaki et al , who also demonstrated an ocular hypotensive effect for this compound . the effect was dose - dependent , with a peak at eight hours after administration.24 moreover , the effect was stronger and more continuous than that induced by latanoprost.24 reduction of iop was dose - dependent in the eyes of ddy mice.19 this effect was similar to the effect of travoprost , but stronger and more longer - lasting than after administration of latanoprost.19 in this animal model , ota et al also found that tafluprost lowered iop via prostanoid fp receptors , and that part of the hypotensive effect may be related to fp receptor - mediated prostaglandin production and stimulation of ep3 receptors.20 this iop - lowering effect in mice was confirmed by akaishi et al.25 it has been postulated that alterations in optic nerve head blood flow may be involved in the pathogenesis of glaucoma.1,26 for this reason , improvement in ocular blood flow by glaucoma therapy may be helpful . studies assessing the influence of the available prostaglandin analogs on ocular blood flow have yielded both positive and negative results.14 in a study by izumi et al tafluprost 0.0015% significantly increased retinal blood flow and blood velocity as measured by laser doppler velocimetry in cats.29 this observation was then confirmed by akaishi et al27 who found that optic nerve head blood flow increased in rabbit eyes after 28 days administration of any of the three f2 prostaglandin analogs , ie , tafluprost , latanoprost , or travoprost . , osaka , japan ) and preservative - free in europe , ie , taflotan ( santen oy , finland ) , both in 0.0015% ( 15 g / ml ) concentrations.21 tafluprost differs from the other prostanoids available on the market because it possesses two fluorine atoms at the carbon 15 position , instead of the hydroxyl group present in latanoprost , travoprost , and bimatiprost.14,22 it is an isopropyl ester ( afp-168 ) and , like other prostaglandin analogs , is rapidly hydrolyzed by corneal esterases to the free acid of tafluprost ( afp-172 ) , which is its active form.14,21,24 afp- 172 is a very potent fp receptor agonist , with a ki of 0.4b nm.17,24 its affinity for the human prostanoid fp receptor is 12 times that of the carboxylic acid in latanoprost and 1700 times that in unoprostone . its active form , ie , tafluprost acid , can be detected in plasma for up to one hour after topical administration , with a peak at 10 minutes.3 it is thought that the pharmacologic profile of this compound is similar to that of other prostaglandins available on the market.32 the pharmacodynamics , safety , and tolerability of tafluprost in healthy volunteers were assessed in a clinical , masked , placebo - controlled phase i study.23 tafluprost 0.0025% and 0.005% , latanoprost 0.005% , and placebo were given for seven days . the effect was maximal at 12 hours after administration and lasted throughout the duration of treatment.4 the therapeutic concentration of tafluprost was set at 0.0015% as a result of a phase ii dose - response study performed in japan.21 in a randomized , double - masked , controlled , multicenter , multinational phase ii study , traverso et al assessed the duration and stability of the iop - lowering effect and tolerability of tafluprost 0.0015% compared with latanoprost 0.005% in patients with primary open - angle glaucoma , exfoliation glaucoma , or ocular hypertension.33 they observed that maximum reduction of iop was reached by day 7 of treatment and sustained until day 42 . in the first one , the efficacy of tafluprost with that of latanoprost in 109 patients with open - angle glaucoma and ocular hypertension were compared.21 after four weeks of administration , they observed reduction in iop by 6.6 2.5 mmhg ( 27.6 9.6% ) in the tafluprost group and by 6.2 2.5 mmhg ( 25.9 9.7% ) in the latanoprost group.21 a second phase iii study involving 351 japanese patients with open - angle glaucoma or ocular hypertension showed that the reduction in iop from baseline was stable throughout 52 weeks and varied from 4.9 to 5.7 mmhg.21 a potent effect of tafluprost 0.0015% on iop and the safety of this medication were also demonstrated in a randomized , parallel - group , double - masked european phase iii study conducted in 49 centers in eight countries for up to 24 months.34 the objective of this study was to compare the efficacy and safety profiles of tafluprost 0.0015% and latanoprost 0.005% in 533 patients with open - angle glaucoma and ocular hypertension . these results are in line with those of other prostaglandins , as shown in the van der valk meta - analysis.12 after 24 months , the mean decrease in iop from baseline was 7.1 mmhg ( 29.1% ) in the group treated with tafluprost and 7.7 mmhg ( 32.2% ) in the group treated with latanoprost.34 the primary aim of the randomized , investigator - masked , multicenter , crossover phase iii study described by hamacher et al was to evaluate the pharmacodynamics and safety of preserved and preservative - free tafluprost 0.0015% in patients with open - angle glaucoma or ocular hypertension . in total , 79.5% of eyes treated with the preservative - free formulation of tafluprost achieved iop 18 mmhg 12 weeks after switching medication.35 other phase iii studies indicated that tafluprost , like other prostaglandin analogs , may also exert an additional iop - lowering effect when administered together with the -blocker , timolol , in cases when monotherapy is not adequate.36 in 2007 , sutton et al observed in a phase i clinical study that systemic safety was similar for tafluprost , latanoprost , and placebo when administered to eyes . interestingly , the authors stated that rates of adverse effects were similar for the tafluprost 0.0001% and 0.0025% and latanoprost 0.005% groups , but rates of ocular hyperemia was significantly lower in eyes receiving latanoprost.4 these authors did not find either cells or flare in the anterior chamber or abnormalities of the iris , lens , or vitreous humor in any eyes during the study.4 all adverse events described in the two aforementioned studies were mild to moderate and did not result in treatment discontinuation.4,23 similar observations were made by uusitalo et al who administered preserved and preservative - free formulations of tafluprost . this could be papillary , with generalized injection , or follicular , caused by proliferation of lymphocytes and plasma cells.38 unfortunately , such inflammatory changes create a potential risk for failure of further glaucoma filtration surgery.39,40 these changes may also lead to keratinization and conjunctival scarring , with symblepharon formation , known as drug - induced pseudopemphigoid.3,11,38 many of these undesirable effects are connected with the preservatives present in eye drop formulations.2 the adverse influence of preservative - containing topical antiglaucoma medications on cells and tissues on the eye surface is well documented in in vitro and in vivo studies.2,32,38,41 bak is the most commonly used preservative in eye drops.42 it has already been found that this compound exerts cytotoxic ( proapoptotic and pronecrotic ) effects on the ocular surface and trabecular meshwork cells.4345 it also causes reduction of cellular viability , infiltration of conjunctival stroma , and overexpression of inflammation- or apoptosis - related molecules , such as apo-2,7 , fas ( cd45 ) , hla - dr , icam-1.32,38,4652 moreover , decreased expression of muc5ac on the conjunctival cells were found in impression cytology specimens taken from patients treated locally with antiglaucoma drugs containing bak . this phenomenon may explain the high prevalence of dry eye syndrome in patients after prolonged antiglaucoma therapy.34,50,53 solutions which are preservative - free , have lower bak concentrations , or contain alternative preservatives were introduced into topical glaucoma therapy to minimize side effects.2 among the widely used prostaglandin analogs , only tafluprost is available in a bak - free formulation.3,42 a preservative - free solution of tafluprost showed reduced toxicity in human conjunctival epithelial cell lines when compared with preserved latanoprost , travoprost , and bimatoprost.54 tafluprost had low proapoptotic / pro - oxidative effects in vitro when compared with preservative - containing formulations.54 liang et al assessed conjunctival and corneal reactions to preservative - free tafluprost , commercially available latanoprost , and benzalkonium chloride 0.02% in in vivo studies.45 corneal epithelium confocal microscopy in vivo revealed partial desquamation of epithelial cells , irregular cell shapes , anisocytosis and loss of cell borders , abnormal reflectivity patterns , swollen cells , and inflammatory infiltrations in rabbit eyes treated with latanoprost and bak solution . rabbit corneas treated with preservative - free tafluprost were almost normal , with the epithelium having a regular polygonal mosaic appearance , brightly reflective nuclei , and no obvious desquamation or swelling . inflammatory infiltrations in the peripheral cornea and the limbus area were noted after exposure to latanoprost or bak , but not after exposure to preservative - free tafluprost.45 similarly , conjunctival stroma vessels showed rolling of inflammatory cells after installation of bak or latanoprost . after installation of preservative - free tafluprost , normal blood vessels were observed , without any rolling of inflammatory cells . only a few tunel+ cells were observed after installation of preservative - free tafluprost but more of these cells were present after installation of latanoprost or bak.45 these observations established the lower toxicity of the preservative - free formulation to the anterior segment of the eye.45 bak is thought to be an ocular penetration enhancer for topically administered drugs , because it increases the corneal permeability of pharmacologic agents.42,55 pellinen and lokkila evaluated corneal penetration of preserved and preservative - free tafluprost 0.0015% into rabbit aqueous humor after topical application . they concluded that bak at the concentration used in the tafluprost formulations did not affect corneal penetration of this drug into rabbit aqueous humor.55 it is possible that tafluprost has its own high corneal penetrating ability and bak would not enhance it.55 in a phase i study evaluating the pharmacokinetics and efficacy of preserved and preservative - free tafluprost , uusitalo et al did not observe any significant differences in pharmacokinetic parameters between the formulations , after either single or repeated dosing.3 ocular hyperemia occurred with the same frequency in both groups , but was predominantly of moderate severity in eyes treated with preserved tafluprost , and of mild severity in those treated with the preservative - free formulation.3 the safety of preserved and preservative - free tafluprost was also assessed in a phase iii study.11 in contrast with the findings of uusitalo et al it was shown that conjunctival hyperemia was reported more often by people using preservative - free tafluprost.11 the aforementioned studies showed that iop reduction obtained by preservative - free tafluprost is equivalent to that achieved by the preserved formulation . it seems that removing the preservative bak from the tafluprost formulation does not change the iop - lowering properties of preparation.11,42 because the aim of designing a preservative - free prostaglandin formulation was to reduce toxic effects and clinical complications in patients treated for glaucoma and ocular hypertension , it was reasonable to check if preservative - free tafluprost was a genuine alternative for patients receiving prostaglandins other than tafluprost . uusitalo et al investigated the hypotensive effect and tolerability of preservative - free tafluprost in patients with open - angle glaucoma and ocular hypertension exhibiting ocular surface side effects during latanoprost treatment.32 twelve weeks after switching from preserved latanoprost to preservative - free tafluprost , iop was maintained at the same level . after 12 weeks of tafluprost preservative- free treatment , fluorescein break - up time increased from 4.5 2.5 seconds at baseline to 7.8 4.9 seconds ( p < 0.001).32 results of immunocytologic testing of impression cytology samples revealed that , after 12 weeks of treatment with preservative - free tafluprost in patients previously using latanoprost , there was a significant reduction in those expressing abnormal levels ( < 7% ) of muc5ac - positive goblet cells and abnormal levels ( > 40% ) of hla - dr - positive epithelial cells.32 this observations may indicate a less harmful influence of preservative - free tafluprost on the conjunctiva than preserved latanoprost.32 surprisingly , change in schirmer s test scores was smaller and statistically significant only at week 6 of treatment with the preservative - free tafluprost formulation.32 this is probably the reason for the relative lack of data on patient satisfaction and compliance . in the uusitalo study , the comparison of ophthalmic medications or tolerability questionnaire devised by barbel et al was used to assess drop discomfort in patients treated with preserved latanoprost and switched to preservative - free tafluprost.32,56 as the authors described , among patients receiving the commercially available latanoprost formulation , 30% experienced no negative effect on quality of life , 59% experienced a little or some , 10% quite a bit or very much , and 1% extremely negative.32 after switching to preservative - free tafluprost , no negative effect on quality of life was reported by 52% of enrolled patients , 46% reported a little or some , 2% quite a bit and 0% very much or extremely after 12 weeks of this therapy . at week 12 of treatment with preservative - free tafluprost , 32% of patients were totally satisfied and 45% were very satisfied.32 in another study , patients with poor local tolerance of their medications noticed improvement of subjective symptoms and clinical signs after changing their therapy to preservative - free tafluprost 0.0015%.35 similar analyses are needed to compare safety , tolerability , patient compliance , and comfort between tafluprost and the other prostaglandin analogs .

hepatitis c virus ( hcv ) infection is the most common chronic bloodborne infection in the united states , with approximately 3.2 million americans chronically infected . moreover , this number is likely an underestimate , as groups with a high prevalence of hcv ( incarcerated and homeless persons ) are not reflected in these data . infection with hepatitis c is a leading cause of death from liver disease and the most common indication for liver transplant in the united states . therefore , use of contaminated needles for injection drug use is currently the most common method of hcv transmission in the united states . although much less frequent , occupational , perinatal , health care , and sexual exposures can result in the transmission of hcv . since the implementation of routine blood donor screening in 1992 , transmission of hcv via blood products is now extremely rare in the developed world . six major hcv genotypes ( named 16 ) are known to exist with variable geographic distribution . genotype 1 ( including subtypes 1a and 1b ) is the most common in the united states and worldwide , accounting for 60% of global infections , followed by genotypes 2 and 3 . genotypes 46 are less common in the united states but occur frequently in other countries . genotype 4 is seen most frequently in egypt , the middle east , and central africa . genotype 5 is found in south africa , and genotype 6 is found in asia . hcv genotypes play an important role in hepatitis c treatment evaluation , as genotypes can be classified as more or less responsive to antiviral therapy with pegylated interferon alpha and ribavirin ( pr ) . genotypes 1 and 4 are less responsive to pr , and genotypes 2 and 3 are more responsive . with the advent of protease inhibitor - based hepatitis c therapy in 2011 , hcv genotype determination has become especially important , as hcv protease inhibitors are only indicated for genotype 1 hcv infection . in addition to genotyping , the recently described il28b polymorphism is also important in predicting treatment success . a genome - wide association study published in 2012 found that a single nucleotide polymorphism at the il28b location is associated with the likelihood of response to hepatitis c treatment . persons with the cc allele have the highest chance of sustained virologic response ( svr ) or cure , those with ct have intermediate response , and those with tt allele have the lowest svr rates . this discovery offers some explanation as to the relatively low response rates of african americans to hepatitis c treatment compared with those patients of european ancestry . analysis of worldwide distribution of il28b polymorphisms show that the tt allele is most prevalent in persons of african descent , whereas the cc allele is most common in persons of european descent . in contrast to hepatitis a and b infections , most acute hcv infections are asymptomatic , with less than 20% of persons developing jaundice . thus , hepatitis c has been referred to as the silent killer , as most infected persons develop progressive liver fibrosis over years without developing any symptoms . when symptoms of chronic hepatitis c develop , they are often nonspecific , including fatigue , abdominal pain , depression , and joint pain . extrahepatic manifestations , such as glomerulonephritis , mixed cryoglobulinemia , vasculitis , and neuropathy , are also possible . because of the asymptomatic nature of acute and chronic infection and the suboptimal application of risk factor - based screening for hcv infection , the majority of hcv - infected persons ( 50%85% ) are unaware of their infection . in an effort to increase the number of persons aware of their hcv infection , the centers for disease control and prevention recently revised their recommendation for hcv screening , now recommending that all persons born between 1945 and 1965 receive a one - time hcv antibody test . this is in addition to the previous recommendation that persons in high - risk groups , such as injection drug users ( past or present ) ; those who received blood products or organ transplants before 1992 ; those with hiv , a history of hemodialysis , or elevated alanine transaminase levels ; children born to hcv - infected mothers ; and health care workers with recognized exposures be screened for hcv infection . the goal of enhanced screening is to make more hcv - infected persons aware of their infection so they can get needed care and evaluation for treatment . on the basis of prediction models , rates of cirrhosis and end - stage liver disease resulting from hcv infection are expected to continue to rise until they peak in 2030 , with 45% of those with hepatitis c having cirrhosis compared with 25% in 2010 . one way to positively affect these numbers is to get more infected persons diagnosed and treated , so that our current efficacious hepatitis c treatment can achieve effectiveness . in this review , we then describe the wide range of novel drugs in development for hcv treatment that are likely to become the new standard of care treatment in the near future . historically , treatment success rates have been low , and treatment regimens have been associated with significant adverse effects . from 2001 through 2011 , the standard - of - care therapy for hepatitis c consisted of pr , with duration of therapy ranging from 24 weeks for genotypes 2 and 3 ( with 70%80% svr rates ) to 48 weeks for genotype 1 ( with 40%50% svr rates ) . this regimen was associated with a variety of adverse effects , including influenza - like symptoms , depression , rash , and cytopenias . in 2011 , treatment was greatly advanced with the introduction of two novel hcv ns3/4a serine protease inhibitors : telaprevir and boceprevir . telaprevir and boceprevir were the first direct - acting antivirals ( daas ) directly targeting hcv to be introduced . triple - therapy regimen that includes pegylated interferon alpha and ribavirin has become the current standard - of - care treatment for genotype 1 hcv infection . for all other genotypes , standard - of - care therapy remains pegylated interferon alpha and ribavirin alone . during the hcv viral lifecycle , the hcv viral genome is translated by host ribosomes into a viral polypeptide approximately 3,000 amino acids in length . this polypeptide is subsequently cleaved by viral protease ns3/4a into nonstructural proteins , including ns5a and ns5b rna - dependent rna polymerase ( figure 1 ) . these nonstructural proteins form the viral replication complex that is required for hcv viral propagation . drugs in the protease inhibitor ( pi ) class inhibit hcv propagation by binding to the catalytic site of the ns3/4a protease enzyme . the pi class inhibits viral replication at an early stage and is a highly potent class of drugs . however , this class has a much lower barrier to resistance ( especially in genotype 1a ) , and cross - drug resistance can occur ( table 1 ) . genotype 1a virus requires only a single mutation ( the amino acid mutation r155 k ) to generate pi resistance , whereas genotype 1b virus requires two mutations . resistance is an important new aspect of hcv therapy with pis ( and other novel daas ) , as resistance was not a feature of pr therapy . the efficacy and safety of telaprevir were evaluated in two major phase iii trials for chronic genotype 1 hcv infection : one for treatment - nave patients ( advance ) , and the other for treatment - experienced patients ( realize ) . in the advance trial , patients were treated with 12 weeks of telaprevir plus pr , followed by an additional 12 weeks of pr for those with favorable early virologic response ( response - guided therapy ) , or an additional 36 weeks of pr for those without a favorable early virologic response . those in the telaprevir group had a 75% svr rate compared with a 44% svr rate for the pr control group . fifty - eight percent of patients were eligible for response - guided therapy ( demonstrating undetectable hcv rna at 4 and 12 weeks of therapy ) and thus were able to complete all treatment at 24 weeks . adverse effects of anemia , gastrointestinal adverse effects , and rash occurred more commonly in the telaprevir group compared with the pr group . however , rates of discontinuation were similar , at 10% for the telaprevir group and 7% in the pr group . in the realize trial , patients were divided by prior treatment response into null responders , partial responders , and relapsers . prior null responders who were treated with telaprevir plus pr for 12 weeks followed by pr for 36 weeks had a 29% svr rate ( compared with 5% for pr ) ; partial responders had a 59% svr ( compared with 15% for pr ) ; and prior relapsers had an 83% svr rate ( compared with 24% for pr ) . again , adverse effects including anemia , anorectal symptoms , and skin rashes were more common in the telaprevir group than the pr group . the efficacy and safety of boceprevir was evaluated in two major phase iii trials : one for treatment - nave patients ( sprint-2 ) and the other for treatment - experienced patients ( respond-2 ) . in the sprint-2 trial , patients received a 4-week lead - in period of pr treatment followed by 24 weeks of boceprevir plus pr if hcv rna was undetectable at weeks 8 and 24 ( response - guided therapy ) , or 24 weeks of boceprevir plus pr plus an additional 20 weeks of pr if hcv rna was detectable at either week 8 or 24 . in sprint-2 , a total of 67% of nonblack patients achieved svr compared with 40% of pr - treated controls . a total of 42% of black patients achieved svr compared with 23% of pr - treated controls . forty - four percent of patients qualified for response - guided therapy in this trial . boceprevir was added to pr therapy in patients who did not have an svr to prior pr therapy . this trial enrolled patients who were partial responders and relapsers to pr , but did not enroll prior null responders . again , a 4-week pr lead - in was used , followed by boceprevir plus pr for 32 weeks , plus an additional 12 weeks of pr if hcv rna was detectable at 8 weeks . a second arm used boceprevir plus pr for an additional 44 weeks after lead - in . overall svr rates were 59% in the first group and 66% in the second group , compared with 21% in the pr control group . anemia , dysgeusia , rash , and dry skin were reported more commonly in the boceprevir groups than the control group . the advent of ns3/4a protease inhibitor - based triple therapy for hcv infection has ushered in a new era for hepatitis c treatment . svr rates are higher and treatment durations potentially shorter than they have ever been for genotype 1 hcv infection . first , pis are only approved for genotype 1 hcv infection , leaving many thousands of non - genotype 1 patients awaiting more effective therapies . in addition , unlike pr therapy , pi - based therapy is associated with the development of hcv resistance . for this reason , patients who fail treatment with boceprevir or telaprevir can not be re - treated with the same or the other pi , leaving no re - treatment option for this group . another drawback is the additional , often severe , adverse effects associated with pi therapy , most significantly the anemia associated with both telaprevir and boceprevir therapy . finally , the cost and complexity of this therapy make it unavailable to many hcv - infected patients . during the hcv viral lifecycle , the hcv viral genome is translated by host ribosomes into a viral polypeptide approximately 3,000 amino acids in length . this polypeptide is subsequently cleaved by viral protease ns3/4a into nonstructural proteins , including ns5a and ns5b rna - dependent rna polymerase ( figure 1 ) . these nonstructural proteins form the viral replication complex that is required for hcv viral propagation . drugs in the protease inhibitor ( pi ) class inhibit hcv propagation by binding to the catalytic site of the ns3/4a protease enzyme . the pi class inhibits viral replication at an early stage and is a highly potent class of drugs . however , this class has a much lower barrier to resistance ( especially in genotype 1a ) , and cross - drug resistance can occur ( table 1 ) . genotype 1a virus requires only a single mutation ( the amino acid mutation r155 k ) to generate pi resistance , whereas genotype 1b virus requires two mutations . resistance is an important new aspect of hcv therapy with pis ( and other novel daas ) , as resistance was not a feature of pr therapy . the efficacy and safety of telaprevir were evaluated in two major phase iii trials for chronic genotype 1 hcv infection : one for treatment - nave patients ( advance ) , and the other for treatment - experienced patients ( realize ) . in the advance trial , patients were treated with 12 weeks of telaprevir plus pr , followed by an additional 12 weeks of pr for those with favorable early virologic response ( response - guided therapy ) , or an additional 36 weeks of pr for those without a favorable early virologic response . those in the telaprevir group had a 75% svr rate compared with a 44% svr rate for the pr control group . fifty - eight percent of patients were eligible for response - guided therapy ( demonstrating undetectable hcv rna at 4 and 12 weeks of therapy ) and thus were able to complete all treatment at 24 weeks . adverse effects of anemia , gastrointestinal adverse effects , and rash occurred more commonly in the telaprevir group compared with the pr group . however , rates of discontinuation were similar , at 10% for the telaprevir group and 7% in the pr group . in the realize trial , patients were divided by prior treatment response into null responders , partial responders , and relapsers . prior null responders who were treated with telaprevir plus pr for 12 weeks followed by pr for 36 weeks had a 29% svr rate ( compared with 5% for pr ) ; partial responders had a 59% svr ( compared with 15% for pr ) ; and prior relapsers had an 83% svr rate ( compared with 24% for pr ) . again , adverse effects including anemia , anorectal symptoms , and skin rashes were more common in the telaprevir group than the pr group . the efficacy and safety of boceprevir was evaluated in two major phase iii trials : one for treatment - nave patients ( sprint-2 ) and the other for treatment - experienced patients ( respond-2 ) . in the sprint-2 trial , patients received a 4-week lead - in period of pr treatment followed by 24 weeks of boceprevir plus pr if hcv rna was undetectable at weeks 8 and 24 ( response - guided therapy ) , or 24 weeks of boceprevir plus pr plus an additional 20 weeks of pr if hcv rna was detectable at either week 8 or 24 . in sprint-2 , a total of 67% of nonblack patients achieved svr compared with 40% of pr - treated controls . a total of 42% of black patients achieved svr compared with 23% of pr - treated controls . forty - four percent of patients qualified for response - guided therapy in this trial . boceprevir was added to pr therapy in patients who did not have an svr to prior pr therapy . this trial enrolled patients who were partial responders and relapsers to pr , but did not enroll prior null responders . again , a 4-week pr lead - in was used , followed by boceprevir plus pr for 32 weeks , plus an additional 12 weeks of pr if hcv rna was detectable at 8 weeks . a second arm used boceprevir plus pr for an additional 44 weeks after lead - in . overall svr rates were 59% in the first group and 66% in the second group , compared with 21% in the pr control group . anemia , dysgeusia , rash , and dry skin were reported more commonly in the boceprevir groups than the control group . the advent of ns3/4a protease inhibitor - based triple therapy for hcv infection has ushered in a new era for hepatitis c treatment . svr rates are higher and treatment durations potentially shorter than they have ever been for genotype 1 hcv infection . first , pis are only approved for genotype 1 hcv infection , leaving many thousands of non - genotype 1 patients awaiting more effective therapies . in addition , unlike pr therapy , pi - based therapy is associated with the development of hcv resistance . for this reason , patients who fail treatment with boceprevir or telaprevir can not be re - treated with the same or the other pi , leaving no re - treatment option for this group . another drawback is the additional , often severe , adverse effects associated with pi therapy , most significantly the anemia associated with both telaprevir and boceprevir therapy . finally , the cost and complexity of this therapy make it unavailable to many hcv - infected patients . fortunately , new drug development for hcv infection has skyrocketed since the release of boceprevir and telaprevir , and an unprecedented number of new daas are in various stages of development ( table 2 ) . these new daas have significant promise to further advance the field of hepatitis c treatment with higher svr rates , shorter durations of therapy , fewer adverse events , and efficacy against hcv genotypes other than genotype 1 . this section discusses drug class , mechanism of action , pharmacokinetics , potency , efficacy , and tolerability of select hcv daas under development , and highlights some specific examples within each class . of note , when reporting efficacy , most trials use a new svr definition ( svr12 ) , defined as maintenance of undetectable hcv rna 12 weeks after completion of treatment ( as opposed to the standard 24 weeks ) . simeprevir ( formerly tmc-435 ; janssen research and development , raritan , nj , usa ) is a macrocyclic ns3/4a pi that has activity against hcv genotypes 1 , 2 , 4 , 5 , and 6 but is not active against genotype 3 ( table 2 ) . simeprevir , metabolized by the cyp3a enzyme , has a half - life of 40 hours , which allows for once - daily dosing . quest-2 , a phase iii clinical trial , assessed simeprevir 150 mg daily plus pr for 12 weeks , followed by pr alone for either 12 or 36 weeks ( response - guided therapy ) in treatment - nave , genotype 1 hcv infection . for those in the simeprevir group , 81% achieved svr12 ; response rates were higher in those with il28b polymorphism cc ( 96% svr12 ) and in those with lower - stage ( metavir 0 to 2 ) liver fibrosis ( svr12 85% ) . the adverse effects that occurred more frequently in patients receiving simeprevir were rash ( 23% versus 11% ) and indirect hyperbilirubinemia . a summary of the phase ii clinical trials of simeprevir has been published . a new drug application was submitted on behalf of simeprevir to the us food and drug administration on march 28 , 2013 . faldaprevir ( formerly bi 201335 ) is a potent pi that is dosed once daily ( table 2 ) . in the efficacy and safety of bi 201335 ( faldaprevir ) in combination with pegylated interferon - alpha and ribavirin in treatment - nave genotype 1 hepatitis c infected patients ( startverso ) study , genotype 1 , treatment - nave patients were given either 120 mg or 240 mg faldaprevir with pr for 12 weeks . overall , around 80% achieved svr12 , with higher responses seen in those with genotype 1b and the cc genotype of the il28b polymorphism . adverse events that caused treatment discontinuation , including indirect hyperbilirubinemia , occurred in 5% of patients who received faldaprevir 240 mg . in the silen - c2 trial , genotype 1 patients with prior nonresponse to therapy were given faldaprevir and pr and 2150% achieved svr . faldaprevir has also been combined with bi 207127 and ribavirin in a small , interferon - free trial of treatment - nave , genotype 1 patients that found that 73%94% of the patients had undetectable hcv rna levels at 4 weeks . danoprevir ( formerly rg7227 ; roche , basel , switzerland ) is a potent , macrocyclic pi with activity against hcv genotypes 1 , 4 , and 6 , which has been studied in coadministration with ritonavir ( table 2 ) . similar to the hiv protease inhibitor drugs , it has been shown that combining danoprevir with 100 mg of ritonavir daily improves both the pharmacokinetics and safety profile of danoprevir . in the dauphine phase iib clinical trial of genotype 1/4 , treatment - nave , noncirrhotic adults , danoprevir was combined with ritonavir plus pr for 12 weeks or 24 weeks ( response - guided therapy ) , resulting in svr12 rates of 77% and 86% . abt-450 is another pi that has been combined with ritonavir to enhance its pharmacokinetics profile . asunaprevir ( formerly bms-650032 ) is a highly active pi that has been studied in combination with a potent ns5a replication complex inhibitor in interferon - free trials of noncirrhotic , prior null responders , with genotype 1 chronic hcv infection ( table 2 ) . other pi drugs under development include narlaprevir ( sch 900518 ) and vaniprevir ( mk-7009 ) . the exact function of the ns5a protein is unknown , but it is a protein with pleiotropic functions and is important in hcv replication ( table 1 ) . daclatasvir ( formerly bms-790052 ; bristol - myers squibb , new york , ny , usa ) is a ns5a replication complex inhibitor that is dosed daily and potently inhibits hcv replication across all genotypes . it works by inhibiting the formation of a functional viral replication complex by binding to the n - terminus of the nonenzymatic ns5a protein . its efficacy has been demonstrated in combination with pr in genotype 1 , treatment - nave , noncirrhotic patients and in interferon - free trials ( table 2 ) . daclatasvir was combined with asunaprevir and a nonnucleoside ns5b polymerase inhibitor ( bms-791325 ) in treatment - nave , genotype 1 , noncirrhotic patients for 12 and 24 weeks , and 88% and 94% achieved svr , respectively . the most frequently reported adverse effects were headache , weakness , diarrhea , nausea , and abdominal pain . no serious changes in liver enzymes ( alanine transaminase or aspartate transaminase ) , bilirubin , or blood cell counts were observed . daclatasvir was used successfully in combination with sofosbuvir ( see ns5b rna - dependent rna polymerase inhibitor section below ) in a liver transplant patient with severe recurrent cholestatic hcv infection and in combination with pr in another liver transplant patient . caution is warranted , however , when combining daclatasvir with ns5b inhibitors , given the death of one patient and severe cardiotoxicity in other patients in a study of daclatasvir and bms-986094 . nucleoside / nucleotide inhibitors ( nis ) act as chain terminators blocking the nascent hcv rna elongation by hcv polymerase ( table 1 ) . the structure and function of the catalytic site of the polymerase is highly conserved among hcv genotypes . in addition , the nis have the highest barrier to resistance ( because a mutation in the polymerase active site results in a major loss in viral fitness ) . although potentially potent , this class has a low barrier to resistance , and preliminary studies have demonstrated high rates of viral breakthrough and relapse . sofosbuvir ( formerly gs7977 ; gilead , foster city , ca , usa ) is a uridine analogue in the ni class that is dosed once daily and has demonstrated clinical efficacy against genotypes 1 , 2 , 3 , 4 , 5 , and 6 ( table 2 ) . gs-7977 with peginterferon alfa 2a and ribavirin for 12 weeks in treatment - nave subjects with chronic genotype 1 , 4 , 5 , or 6 hcv infection ( neutrino ) , a phase iii clinical trial of treatment - nave ( 17% with cirrhosis ) genotype 1 ( 89% ) , 4 ( 9% ) , and 5/6 ( 2% ) patients treated with sofosbuvir plus pr for 12 weeks found svr12 rates of 90% ( 92% for genotype 1a , 82% for genotype 1b , 96% for genotype 4 , 100% for genotype 5/6 ) . very few ( 2% ) the phase iii study of psi-7977 and ribavirin ( fission ) trial assessed the efficacy of sofosbuvir plus ribavirin for 12 weeks in genotype 2 or 3 treatment - nave ( 20% with cirrhosis ) patients and found it was noninferior to standard - of - care pr therapy , with svr12 rates of 67% in both groups . in those with genotype 2 gs-7977 + ribavirin for 12 weeks in subjects with chronic genotype 2 or 3 hcv infection who are interferon intolerant , interferon ineligible or unwilling to take interferon ( positron ) enrolled treatment - nave genotype 2 or 3 patients for whom interferon therapy was not an option ( contraindication , unacceptable adverse effects ) and found a 78% svr12 in patients treated with 12 weeks of sofosbuvir and ribavirin . the gs-7977 + ribavirin for 12 or 16 weeks in treatment experienced subjects with chronic genotype 2 or 3 hcv infection ( fusion ) study enrolled genotype 2 or 3 patients who had not responded to prior interferon therapy ( 34% had cirrhosis ) . among those treated with sofosbuvir and ribavirin for 12 weeks , 50% achieved svr12 , and in those treated for 16 weeks , 73% had an svr12 . the most common adverse events in all phase iii sofosbuvir trials were fatigue , headache , nausea , and insomnia . the only known sofosbuvir resistance variant ( s282 t mutation ) was not detected on deep - sequencing assays in any patient who received sofosbuvir in these studies . another ni under development is mericitabine ( formerly r7128 ; hoffmann - la roche , basel , switzerland ) , a cytidine analogue with activity against genotypes 1 , 2 , and 3 ( table 1 ) . pegylated interferon lambda is a type 3 interferon that has been shown to inhibit hcv viral replication in vitro . however , unlike interferon alpha , lambda has a more restricted cell and tissue distribution . lambda is expressed by hepatocytes but is minimally expressed by the cells of hematopoietic lineage . in proof - of - concept trials , pegylated interferon lambda is much better tolerated without the influenza - like symptoms , musculoskeletal complaints , and neutropenia often experienced by patients receiving type 1 interferon alpha . efficacy and safety study of peg - ril-29 plus ribavirin to treat chronic hepatitis c virus infection ( emerge ) , a phase ii clinical trial , compared lambda with alpha interferon in genotype 2 and 3 patients and found similar svr rates but fewer interferon - related adverse events . despite promising early results from interferon - free trials , difficult - to - treat patients will likely need interferon , and having a better tolerated interferon option will be critical . micrornas are small ( ~22 nucleotides ) rnas that interact with messenger rna and reduce translated protein , regulating host gene expression at the posttranscriptional level . mir-122 is a liver - specific microrna required for the stability and propagation of hcv rna . miravirsen ( santaris pharma , hrsholm , denmark ) is an antagomir to mir-122 that has been shown to have significant anti - hcv viral activity in a randomized , double - blind , placebo - controlled , ascending multiple dose - ranging phase iia study . miravirsen was given subcutaneously in weekly doses during a 29-day period ( at doses of 3 mg / kg , 5 mg / kg , or 7 mg / kg ) and resulted in at least a 2 log10 reduction in hcv rna in six of nine patients in the 5 mg / kg and 7 mg / kg groups . the use of miravirsen alone resulted in undetectable hcv rna levels in five patients , but viral rebound occurred in all but one . one patient had a syncopal episode after a miravirsen injection , and some patients had injection site reactions . miravirsen , a host - targeting agent , could potentially complement the direct acting antiviral agents in the treatment of chronic hcv infection , but more research is needed . simeprevir ( formerly tmc-435 ; janssen research and development , raritan , nj , usa ) is a macrocyclic ns3/4a pi that has activity against hcv genotypes 1 , 2 , 4 , 5 , and 6 but is not active against genotype 3 ( table 2 ) . simeprevir , metabolized by the cyp3a enzyme , has a half - life of 40 hours , which allows for once - daily dosing . quest-2 , a phase iii clinical trial , assessed simeprevir 150 mg daily plus pr for 12 weeks , followed by pr alone for either 12 or 36 weeks ( response - guided therapy ) in treatment - nave , genotype 1 hcv infection . for those in the simeprevir group , 81% achieved svr12 ; response rates were higher in those with il28b polymorphism cc ( 96% svr12 ) and in those with lower - stage ( metavir 0 to 2 ) liver fibrosis ( svr12 85% ) . the adverse effects that occurred more frequently in patients receiving simeprevir were rash ( 23% versus 11% ) and indirect hyperbilirubinemia . a new drug application was submitted on behalf of simeprevir to the us food and drug administration on march 28 , 2013 . faldaprevir ( formerly bi 201335 ) is a potent pi that is dosed once daily ( table 2 ) . in the efficacy and safety of bi 201335 ( faldaprevir ) in combination with pegylated interferon - alpha and ribavirin in treatment - nave genotype 1 hepatitis c infected patients ( startverso ) study , genotype 1 , treatment - nave patients were given either 120 mg or 240 mg faldaprevir with pr for 12 weeks . overall , around 80% achieved svr12 , with higher responses seen in those with genotype 1b and the cc genotype of the il28b polymorphism . adverse events that caused treatment discontinuation , including indirect hyperbilirubinemia , occurred in 5% of patients who received faldaprevir 240 mg . in the silen - c2 trial , genotype 1 patients with prior nonresponse to therapy were given faldaprevir and pr and 2150% achieved svr . faldaprevir has also been combined with bi 207127 and ribavirin in a small , interferon - free trial of treatment - nave , genotype 1 patients that found that 73%94% of the patients had undetectable hcv rna levels at 4 weeks . danoprevir ( formerly rg7227 ; roche , basel , switzerland ) is a potent , macrocyclic pi with activity against hcv genotypes 1 , 4 , and 6 , which has been studied in coadministration with ritonavir ( table 2 ) . similar to the hiv protease inhibitor drugs , it has been shown that combining danoprevir with 100 mg of ritonavir daily improves both the pharmacokinetics and safety profile of danoprevir . in the dauphine phase iib clinical trial of genotype 1/4 , treatment - nave , noncirrhotic adults , danoprevir was combined with ritonavir plus pr for 12 weeks or 24 weeks ( response - guided therapy ) , resulting in svr12 rates of 77% and 86% . abt-450 is another pi that has been combined with ritonavir to enhance its pharmacokinetics profile . asunaprevir ( formerly bms-650032 ) is a highly active pi that has been studied in combination with a potent ns5a replication complex inhibitor in interferon - free trials of noncirrhotic , prior null responders , with genotype 1 chronic hcv infection ( table 2 ) . other pi drugs under development include narlaprevir ( sch 900518 ) and vaniprevir ( mk-7009 ) . the exact function of the ns5a protein is unknown , but it is a protein with pleiotropic functions and is important in hcv replication ( table 1 ) . daclatasvir ( formerly bms-790052 ; bristol - myers squibb , new york , ny , usa ) is a ns5a replication complex inhibitor that is dosed daily and potently inhibits hcv replication across all genotypes . it works by inhibiting the formation of a functional viral replication complex by binding to the n - terminus of the nonenzymatic ns5a protein . its efficacy has been demonstrated in combination with pr in genotype 1 , treatment - nave , noncirrhotic patients and in interferon - free trials ( table 2 ) . daclatasvir was combined with asunaprevir and a nonnucleoside ns5b polymerase inhibitor ( bms-791325 ) in treatment - nave , genotype 1 , noncirrhotic patients for 12 and 24 weeks , and 88% and 94% achieved svr , respectively . the most frequently reported adverse effects were headache , weakness , diarrhea , nausea , and abdominal pain . no serious changes in liver enzymes ( alanine transaminase or aspartate transaminase ) , bilirubin , or blood cell counts were observed . daclatasvir was used successfully in combination with sofosbuvir ( see ns5b rna - dependent rna polymerase inhibitor section below ) in a liver transplant patient with severe recurrent cholestatic hcv infection and in combination with pr in another liver transplant patient . caution is warranted , however , when combining daclatasvir with ns5b inhibitors , given the death of one patient and severe cardiotoxicity in other patients in a study of daclatasvir and bms-986094 . nucleoside / nucleotide inhibitors ( nis ) act as chain terminators blocking the nascent hcv rna elongation by hcv polymerase ( table 1 ) . the structure and function of the catalytic site of the polymerase in addition , the nis have the highest barrier to resistance ( because a mutation in the polymerase active site results in a major loss in viral fitness ) . although potentially potent , this class has a low barrier to resistance , and preliminary studies have demonstrated high rates of viral breakthrough and relapse . sofosbuvir ( formerly gs7977 ; gilead , foster city , ca , usa ) is a uridine analogue in the ni class that is dosed once daily and has demonstrated clinical efficacy against genotypes 1 , 2 , 3 , 4 , 5 , and 6 ( table 2 ) . gs-7977 with peginterferon alfa 2a and ribavirin for 12 weeks in treatment - nave subjects with chronic genotype 1 , 4 , 5 , or 6 hcv infection ( neutrino ) , a phase iii clinical trial of treatment - nave ( 17% with cirrhosis ) genotype 1 ( 89% ) , 4 ( 9% ) , and 5/6 ( 2% ) patients treated with sofosbuvir plus pr for 12 weeks found svr12 rates of 90% ( 92% for genotype 1a , 82% for genotype 1b , 96% for genotype 4 , 100% for genotype 5/6 ) . very few ( 2% ) the phase iii study of psi-7977 and ribavirin ( fission ) trial assessed the efficacy of sofosbuvir plus ribavirin for 12 weeks in genotype 2 or 3 treatment - nave ( 20% with cirrhosis ) patients and found it was noninferior to standard - of - care pr therapy , with svr12 rates of 67% in both groups . in those with genotype 2 , 97% achieved an svr12 compared with 56% in those with genotype 3 . gs-7977 + ribavirin for 12 weeks in subjects with chronic genotype 2 or 3 hcv infection who are interferon intolerant , interferon ineligible or unwilling to take interferon ( positron ) enrolled treatment - nave genotype 2 or 3 patients for whom interferon therapy was not an option ( contraindication , unacceptable adverse effects ) and found a 78% svr12 in patients treated with 12 weeks of sofosbuvir and ribavirin . the gs-7977 + ribavirin for 12 or 16 weeks in treatment experienced subjects with chronic genotype 2 or 3 hcv infection ( fusion ) study enrolled genotype 2 or 3 patients who had not responded to prior interferon therapy ( 34% had cirrhosis ) . among those treated with sofosbuvir and ribavirin for 12 weeks , 50% achieved svr12 , and in those treated for 16 weeks , 73% had an svr12 . the most common adverse events in all phase iii sofosbuvir trials were fatigue , headache , nausea , and insomnia . the only known sofosbuvir resistance variant ( s282 t mutation ) was not detected on deep - sequencing assays in any patient who received sofosbuvir in these studies . another ni under development is mericitabine ( formerly r7128 ; hoffmann - la roche , basel , switzerland ) , a cytidine analogue with activity against genotypes 1 , 2 , and 3 ( table 1 ) . pegylated interferon lambda is a type 3 interferon that has been shown to inhibit hcv viral replication in vitro . however , unlike interferon alpha , lambda has a more restricted cell and tissue distribution . lambda is expressed by hepatocytes but is minimally expressed by the cells of hematopoietic lineage . in proof - of - concept trials , pegylated interferon lambda is much better tolerated without the influenza - like symptoms , musculoskeletal complaints , and neutropenia often experienced by patients receiving type 1 interferon alpha . efficacy and safety study of peg - ril-29 plus ribavirin to treat chronic hepatitis c virus infection ( emerge ) , a phase ii clinical trial , compared lambda with alpha interferon in genotype 2 and 3 patients and found similar svr rates but fewer interferon - related adverse events . despite promising early results from interferon - free trials , difficult - to - treat patients will likely need interferon , and having a better tolerated interferon option will be critical . micrornas are small ( ~22 nucleotides ) rnas that interact with messenger rna and reduce translated protein , regulating host gene expression at the posttranscriptional level . mir-122 is a liver - specific microrna required for the stability and propagation of hcv rna . miravirsen ( santaris pharma , hrsholm , denmark ) is an antagomir to mir-122 that has been shown to have significant anti - hcv viral activity in a randomized , double - blind , placebo - controlled , ascending multiple dose - ranging phase iia study . miravirsen was given subcutaneously in weekly doses during a 29-day period ( at doses of 3 mg / kg , 5 mg / kg , or 7 mg / kg ) and resulted in at least a 2 log10 reduction in hcv rna in six of nine patients in the 5 mg / kg and 7 mg / kg groups . the use of miravirsen alone resulted in undetectable hcv rna levels in five patients , but viral rebound occurred in all but one . one patient had a syncopal episode after a miravirsen injection , and some patients had injection site reactions . miravirsen , a host - targeting agent , could potentially complement the direct acting antiviral agents in the treatment of chronic hcv infection , but more research is needed . hepatitis c continues to be an emerging public health threat , and morbidity and mortality from hepatitis c - related liver disease have yet to peak in the united states . there is , however , opportunity for positively affecting these projections by both diagnosing more persons who are unaware of their hcv infection and treating with antiviral therapy those who are known to be infected . although current standard - of - care antiviral therapy with pegylated interferon , ribavirin , and a protease inhibitor is efficacious for genotype 1 hcv , this therapy has many drawbacks , including complicated dosing regimens , a long duration of therapy , multiple and potentially serious adverse effects , and approval for genotype 1 hcv infection only . this review outlines many promising new daas in development that offer superior efficacy , improved tolerability , and shorter duration of therapy . many show pan - genotypic activity , so that patients with difficult - to - treat non-1 genotypes will also be eligible for treatment with these new agents . in addition , interferon - free regimens , which have long been the holy grail of hepatitis c treatment , are now within reach . we are entering an exciting new era of hepatitis c treatment that promises an improved hepatitis c treatment experience and outcome both for the patient and the provider .

backgroundhepatitis c virus ( hcv ) infection is the most common chronic bloodborne infection in the united states , with approximately 3.2 million americans being chronically infected . rates of hcv - related end - stage liver disease and its associated morbidity and mortality have yet to peak , so there is a pressing need for more effective and tolerable hcv treatment . hcv genotypes 1 , 4 , 5 , and 6 are considered difficult to treat , and the need for improved therapies is especially great for persons infected with these genotypes.current strategies for hcv treatmentcurrent therapy for genotype 1 hcv infection includes triple therapy with pegylated interferon , ribavirin , and a ns3/4a protease inhibitor . sustained virologic response ( svr ) rates with triple therapy range from 42% to 75% , a vast improvement over pegylated interferon and ribavirin therapy alone . however , response rates remain suboptimal , and triple therapy is associated with significant adverse effects and is only indicated for genotype 1 hcv infection.novel drugs for hcv treatmenthcv drug development is proceeding at a rapid pace to meet this need . novel direct acting antiviral agents in several classes , including new ns3/4a serine protease inhibitors , ns5a replication complex inhibitors , ns5b polymerase inhibitors , interferon lambda , and micrornas , are in varying stages of development . these new therapeutic agents promise svr rates of up to 100% with durations as short as 12 weeks and , often , fewer adverse effects.conclusionnew drug development in hcv is proceeding at an unprecedented pace . novel agents promise higher svr rates , shorter duration of therapy , and fewer adverse effects than have been possible with hcv therapy to date .

Introduction Overview of current therapeutic strategies Mechanism of action of NS3/4A serine protease inhibitors Telaprevir-based triple therapy Boceprevir-based triple therapy DAAs under development Novel NS3/4A serine protease inhibitors NS5A replication complex inhibitors NS5B RNA-dependent RNA polymerase inhibitors Other therapeutic options under exploration/host targets Conclusion

hepatitis c virus ( hcv ) infection is the most common chronic bloodborne infection in the united states , with approximately 3.2 million americans chronically infected . infection with hepatitis c is a leading cause of death from liver disease and the most common indication for liver transplant in the united states . therefore , use of contaminated needles for injection drug use is currently the most common method of hcv transmission in the united states . genotype 1 ( including subtypes 1a and 1b ) is the most common in the united states and worldwide , accounting for 60% of global infections , followed by genotypes 2 and 3 . genotypes 46 are less common in the united states but occur frequently in other countries . hcv genotypes play an important role in hepatitis c treatment evaluation , as genotypes can be classified as more or less responsive to antiviral therapy with pegylated interferon alpha and ribavirin ( pr ) . with the advent of protease inhibitor - based hepatitis c therapy in 2011 , hcv genotype determination has become especially important , as hcv protease inhibitors are only indicated for genotype 1 hcv infection . persons with the cc allele have the highest chance of sustained virologic response ( svr ) or cure , those with ct have intermediate response , and those with tt allele have the lowest svr rates . because of the asymptomatic nature of acute and chronic infection and the suboptimal application of risk factor - based screening for hcv infection , the majority of hcv - infected persons ( 50%85% ) are unaware of their infection . this is in addition to the previous recommendation that persons in high - risk groups , such as injection drug users ( past or present ) ; those who received blood products or organ transplants before 1992 ; those with hiv , a history of hemodialysis , or elevated alanine transaminase levels ; children born to hcv - infected mothers ; and health care workers with recognized exposures be screened for hcv infection . on the basis of prediction models , rates of cirrhosis and end - stage liver disease resulting from hcv infection are expected to continue to rise until they peak in 2030 , with 45% of those with hepatitis c having cirrhosis compared with 25% in 2010 . in this review , we then describe the wide range of novel drugs in development for hcv treatment that are likely to become the new standard of care treatment in the near future . historically , treatment success rates have been low , and treatment regimens have been associated with significant adverse effects . from 2001 through 2011 , the standard - of - care therapy for hepatitis c consisted of pr , with duration of therapy ranging from 24 weeks for genotypes 2 and 3 ( with 70%80% svr rates ) to 48 weeks for genotype 1 ( with 40%50% svr rates ) . this regimen was associated with a variety of adverse effects , including influenza - like symptoms , depression , rash , and cytopenias . in 2011 , treatment was greatly advanced with the introduction of two novel hcv ns3/4a serine protease inhibitors : telaprevir and boceprevir . triple - therapy regimen that includes pegylated interferon alpha and ribavirin has become the current standard - of - care treatment for genotype 1 hcv infection . drugs in the protease inhibitor ( pi ) class inhibit hcv propagation by binding to the catalytic site of the ns3/4a protease enzyme . the pi class inhibits viral replication at an early stage and is a highly potent class of drugs . the efficacy and safety of telaprevir were evaluated in two major phase iii trials for chronic genotype 1 hcv infection : one for treatment - nave patients ( advance ) , and the other for treatment - experienced patients ( realize ) . in the advance trial , patients were treated with 12 weeks of telaprevir plus pr , followed by an additional 12 weeks of pr for those with favorable early virologic response ( response - guided therapy ) , or an additional 36 weeks of pr for those without a favorable early virologic response . adverse effects of anemia , gastrointestinal adverse effects , and rash occurred more commonly in the telaprevir group compared with the pr group . the advent of ns3/4a protease inhibitor - based triple therapy for hcv infection has ushered in a new era for hepatitis c treatment . svr rates are higher and treatment durations potentially shorter than they have ever been for genotype 1 hcv infection . first , pis are only approved for genotype 1 hcv infection , leaving many thousands of non - genotype 1 patients awaiting more effective therapies . in addition , unlike pr therapy , pi - based therapy is associated with the development of hcv resistance . another drawback is the additional , often severe , adverse effects associated with pi therapy , most significantly the anemia associated with both telaprevir and boceprevir therapy . drugs in the protease inhibitor ( pi ) class inhibit hcv propagation by binding to the catalytic site of the ns3/4a protease enzyme . the pi class inhibits viral replication at an early stage and is a highly potent class of drugs . resistance is an important new aspect of hcv therapy with pis ( and other novel daas ) , as resistance was not a feature of pr therapy . the efficacy and safety of telaprevir were evaluated in two major phase iii trials for chronic genotype 1 hcv infection : one for treatment - nave patients ( advance ) , and the other for treatment - experienced patients ( realize ) . in the advance trial , patients were treated with 12 weeks of telaprevir plus pr , followed by an additional 12 weeks of pr for those with favorable early virologic response ( response - guided therapy ) , or an additional 36 weeks of pr for those without a favorable early virologic response . adverse effects of anemia , gastrointestinal adverse effects , and rash occurred more commonly in the telaprevir group compared with the pr group . again , adverse effects including anemia , anorectal symptoms , and skin rashes were more common in the telaprevir group than the pr group . the advent of ns3/4a protease inhibitor - based triple therapy for hcv infection has ushered in a new era for hepatitis c treatment . svr rates are higher and treatment durations potentially shorter than they have ever been for genotype 1 hcv infection . first , pis are only approved for genotype 1 hcv infection , leaving many thousands of non - genotype 1 patients awaiting more effective therapies . in addition , unlike pr therapy , pi - based therapy is associated with the development of hcv resistance . another drawback is the additional , often severe , adverse effects associated with pi therapy , most significantly the anemia associated with both telaprevir and boceprevir therapy . fortunately , new drug development for hcv infection has skyrocketed since the release of boceprevir and telaprevir , and an unprecedented number of new daas are in various stages of development ( table 2 ) . these new daas have significant promise to further advance the field of hepatitis c treatment with higher svr rates , shorter durations of therapy , fewer adverse events , and efficacy against hcv genotypes other than genotype 1 . simeprevir ( formerly tmc-435 ; janssen research and development , raritan , nj , usa ) is a macrocyclic ns3/4a pi that has activity against hcv genotypes 1 , 2 , 4 , 5 , and 6 but is not active against genotype 3 ( table 2 ) . quest-2 , a phase iii clinical trial , assessed simeprevir 150 mg daily plus pr for 12 weeks , followed by pr alone for either 12 or 36 weeks ( response - guided therapy ) in treatment - nave , genotype 1 hcv infection . for those in the simeprevir group , 81% achieved svr12 ; response rates were higher in those with il28b polymorphism cc ( 96% svr12 ) and in those with lower - stage ( metavir 0 to 2 ) liver fibrosis ( svr12 85% ) . in the efficacy and safety of bi 201335 ( faldaprevir ) in combination with pegylated interferon - alpha and ribavirin in treatment - nave genotype 1 hepatitis c infected patients ( startverso ) study , genotype 1 , treatment - nave patients were given either 120 mg or 240 mg faldaprevir with pr for 12 weeks . faldaprevir has also been combined with bi 207127 and ribavirin in a small , interferon - free trial of treatment - nave , genotype 1 patients that found that 73%94% of the patients had undetectable hcv rna levels at 4 weeks . danoprevir ( formerly rg7227 ; roche , basel , switzerland ) is a potent , macrocyclic pi with activity against hcv genotypes 1 , 4 , and 6 , which has been studied in coadministration with ritonavir ( table 2 ) . in the dauphine phase iib clinical trial of genotype 1/4 , treatment - nave , noncirrhotic adults , danoprevir was combined with ritonavir plus pr for 12 weeks or 24 weeks ( response - guided therapy ) , resulting in svr12 rates of 77% and 86% . asunaprevir ( formerly bms-650032 ) is a highly active pi that has been studied in combination with a potent ns5a replication complex inhibitor in interferon - free trials of noncirrhotic , prior null responders , with genotype 1 chronic hcv infection ( table 2 ) . daclatasvir was combined with asunaprevir and a nonnucleoside ns5b polymerase inhibitor ( bms-791325 ) in treatment - nave , genotype 1 , noncirrhotic patients for 12 and 24 weeks , and 88% and 94% achieved svr , respectively . the most frequently reported adverse effects were headache , weakness , diarrhea , nausea , and abdominal pain . sofosbuvir ( formerly gs7977 ; gilead , foster city , ca , usa ) is a uridine analogue in the ni class that is dosed once daily and has demonstrated clinical efficacy against genotypes 1 , 2 , 3 , 4 , 5 , and 6 ( table 2 ) . gs-7977 with peginterferon alfa 2a and ribavirin for 12 weeks in treatment - nave subjects with chronic genotype 1 , 4 , 5 , or 6 hcv infection ( neutrino ) , a phase iii clinical trial of treatment - nave ( 17% with cirrhosis ) genotype 1 ( 89% ) , 4 ( 9% ) , and 5/6 ( 2% ) patients treated with sofosbuvir plus pr for 12 weeks found svr12 rates of 90% ( 92% for genotype 1a , 82% for genotype 1b , 96% for genotype 4 , 100% for genotype 5/6 ) . very few ( 2% ) the phase iii study of psi-7977 and ribavirin ( fission ) trial assessed the efficacy of sofosbuvir plus ribavirin for 12 weeks in genotype 2 or 3 treatment - nave ( 20% with cirrhosis ) patients and found it was noninferior to standard - of - care pr therapy , with svr12 rates of 67% in both groups . in those with genotype 2 gs-7977 + ribavirin for 12 weeks in subjects with chronic genotype 2 or 3 hcv infection who are interferon intolerant , interferon ineligible or unwilling to take interferon ( positron ) enrolled treatment - nave genotype 2 or 3 patients for whom interferon therapy was not an option ( contraindication , unacceptable adverse effects ) and found a 78% svr12 in patients treated with 12 weeks of sofosbuvir and ribavirin . among those treated with sofosbuvir and ribavirin for 12 weeks , 50% achieved svr12 , and in those treated for 16 weeks , 73% had an svr12 . the most common adverse events in all phase iii sofosbuvir trials were fatigue , headache , nausea , and insomnia . another ni under development is mericitabine ( formerly r7128 ; hoffmann - la roche , basel , switzerland ) , a cytidine analogue with activity against genotypes 1 , 2 , and 3 ( table 1 ) . pegylated interferon lambda is a type 3 interferon that has been shown to inhibit hcv viral replication in vitro . in proof - of - concept trials , pegylated interferon lambda is much better tolerated without the influenza - like symptoms , musculoskeletal complaints , and neutropenia often experienced by patients receiving type 1 interferon alpha . efficacy and safety study of peg - ril-29 plus ribavirin to treat chronic hepatitis c virus infection ( emerge ) , a phase ii clinical trial , compared lambda with alpha interferon in genotype 2 and 3 patients and found similar svr rates but fewer interferon - related adverse events . miravirsen , a host - targeting agent , could potentially complement the direct acting antiviral agents in the treatment of chronic hcv infection , but more research is needed . simeprevir ( formerly tmc-435 ; janssen research and development , raritan , nj , usa ) is a macrocyclic ns3/4a pi that has activity against hcv genotypes 1 , 2 , 4 , 5 , and 6 but is not active against genotype 3 ( table 2 ) . quest-2 , a phase iii clinical trial , assessed simeprevir 150 mg daily plus pr for 12 weeks , followed by pr alone for either 12 or 36 weeks ( response - guided therapy ) in treatment - nave , genotype 1 hcv infection . for those in the simeprevir group , 81% achieved svr12 ; response rates were higher in those with il28b polymorphism cc ( 96% svr12 ) and in those with lower - stage ( metavir 0 to 2 ) liver fibrosis ( svr12 85% ) . in the efficacy and safety of bi 201335 ( faldaprevir ) in combination with pegylated interferon - alpha and ribavirin in treatment - nave genotype 1 hepatitis c infected patients ( startverso ) study , genotype 1 , treatment - nave patients were given either 120 mg or 240 mg faldaprevir with pr for 12 weeks . danoprevir ( formerly rg7227 ; roche , basel , switzerland ) is a potent , macrocyclic pi with activity against hcv genotypes 1 , 4 , and 6 , which has been studied in coadministration with ritonavir ( table 2 ) . in the dauphine phase iib clinical trial of genotype 1/4 , treatment - nave , noncirrhotic adults , danoprevir was combined with ritonavir plus pr for 12 weeks or 24 weeks ( response - guided therapy ) , resulting in svr12 rates of 77% and 86% . asunaprevir ( formerly bms-650032 ) is a highly active pi that has been studied in combination with a potent ns5a replication complex inhibitor in interferon - free trials of noncirrhotic , prior null responders , with genotype 1 chronic hcv infection ( table 2 ) . daclatasvir was combined with asunaprevir and a nonnucleoside ns5b polymerase inhibitor ( bms-791325 ) in treatment - nave , genotype 1 , noncirrhotic patients for 12 and 24 weeks , and 88% and 94% achieved svr , respectively . sofosbuvir ( formerly gs7977 ; gilead , foster city , ca , usa ) is a uridine analogue in the ni class that is dosed once daily and has demonstrated clinical efficacy against genotypes 1 , 2 , 3 , 4 , 5 , and 6 ( table 2 ) . gs-7977 with peginterferon alfa 2a and ribavirin for 12 weeks in treatment - nave subjects with chronic genotype 1 , 4 , 5 , or 6 hcv infection ( neutrino ) , a phase iii clinical trial of treatment - nave ( 17% with cirrhosis ) genotype 1 ( 89% ) , 4 ( 9% ) , and 5/6 ( 2% ) patients treated with sofosbuvir plus pr for 12 weeks found svr12 rates of 90% ( 92% for genotype 1a , 82% for genotype 1b , 96% for genotype 4 , 100% for genotype 5/6 ) . very few ( 2% ) the phase iii study of psi-7977 and ribavirin ( fission ) trial assessed the efficacy of sofosbuvir plus ribavirin for 12 weeks in genotype 2 or 3 treatment - nave ( 20% with cirrhosis ) patients and found it was noninferior to standard - of - care pr therapy , with svr12 rates of 67% in both groups . gs-7977 + ribavirin for 12 weeks in subjects with chronic genotype 2 or 3 hcv infection who are interferon intolerant , interferon ineligible or unwilling to take interferon ( positron ) enrolled treatment - nave genotype 2 or 3 patients for whom interferon therapy was not an option ( contraindication , unacceptable adverse effects ) and found a 78% svr12 in patients treated with 12 weeks of sofosbuvir and ribavirin . the most common adverse events in all phase iii sofosbuvir trials were fatigue , headache , nausea , and insomnia . another ni under development is mericitabine ( formerly r7128 ; hoffmann - la roche , basel , switzerland ) , a cytidine analogue with activity against genotypes 1 , 2 , and 3 ( table 1 ) . in proof - of - concept trials , pegylated interferon lambda is much better tolerated without the influenza - like symptoms , musculoskeletal complaints , and neutropenia often experienced by patients receiving type 1 interferon alpha . efficacy and safety study of peg - ril-29 plus ribavirin to treat chronic hepatitis c virus infection ( emerge ) , a phase ii clinical trial , compared lambda with alpha interferon in genotype 2 and 3 patients and found similar svr rates but fewer interferon - related adverse events . miravirsen , a host - targeting agent , could potentially complement the direct acting antiviral agents in the treatment of chronic hcv infection , but more research is needed . hepatitis c continues to be an emerging public health threat , and morbidity and mortality from hepatitis c - related liver disease have yet to peak in the united states . although current standard - of - care antiviral therapy with pegylated interferon , ribavirin , and a protease inhibitor is efficacious for genotype 1 hcv , this therapy has many drawbacks , including complicated dosing regimens , a long duration of therapy , multiple and potentially serious adverse effects , and approval for genotype 1 hcv infection only . this review outlines many promising new daas in development that offer superior efficacy , improved tolerability , and shorter duration of therapy . many show pan - genotypic activity , so that patients with difficult - to - treat non-1 genotypes will also be eligible for treatment with these new agents .

since the introduction of antiretroviral therapy in 1996 , the clinical management of hiv infection revolves around a complex assemblage of potent pharmacological molecules that inhibit viral replication . the goal of this lifelong treatment regimen is to achieve ( and maintain ) viral suppression and to reach a threshold where copies of the virus are no longer detectable in the blood the lowest level of detection generally considered 4075 copies / ml ( hhs 2011 ) . an undetectable plasma viral load is widely interpreted as the marker of individual therapeutic success and is also used extensively to conceptualize health in the context of hiv ( persson et al . the person living with hiv who is able to achieve this kind of viral suppression through strict adherence can be understood as a good patient and deserving of the ongoing investment in their health , irrespective of the complexity of their lives and the numerous barriers to adherence many individuals face ( remien and mellins 2007 ) . however , in recent years , plasma viral load testing has become more than just a clinical tool to monitor treatment response at the individual level . increasingly , individual plasma viral load testing is being reported to public health agencies and is used to inform epidemiological surveillance and monitor the presence of the virus collectively ( terzian et al . in particular need of critical appraisal is the development of techniques to measure , map and monitor community viral load. this new epidemiological technology blurs the traditional boundaries of clinical care and public health work , thus raising questions about its implications . for the purpose of this article , we argue that community viral load should be examined in relation to the theoretical framework of foucault ( 1984 ) coined this concept to account for the ways through which capillary forms of power insert themselves into actions , attitudes , and discourses to produce a particular kind of person and population - productive , but docile and easily governed . miller and rose ( 2008 , p. 63 ) describe governmentality as a domain of strategies , techniques and procedures through which different forces seek to render programmes operable , and by means of which a multitude of connections are established between the aspirations of authorities and the activities of individuals and groups. these tactics , in their programmatic form , act directly and indirectly on the population ( and individuals who compose it ) to improve its conditions , welfare , wealth , longevity , health and so on ( foucault 1991 ) . these tactics become inseparable from a knowledge and understanding of processes related to population ( foucault 1991 ) . this form of governance is particularly concerned with processes that modulate health collectively rather than individually ; and this is why these processes have to be understood and managed using a broad range of tactics ( foucault 1991 ) . to govern , therefore , implies an ideological shift from the isolated person to the existing relations that are known to interfere with the production of a healthy population : relations between people and people , people and things , people and events , [ people and spaces] ( rose et al . these relations can then be administered directly or indirectly through tactics that make possible the optimization of health and so forth ( foucault 1991 ) . based on the work of foucault ( 1991 , 2003 ) , this article critically examines the use of community viral load as a new strategy of governmentality . also drawing on the work of miller and rose ( 2008 ) , this article further reconceptualizes specifically , it explores the deployment of community through the re - configuration of space , the problematization of viral concentrations in specific micro - locales , and the government ( in the foucauldian sense ) of specific bodies which are seen as it also examines community viral load as a necessary precondition forming the conditions of possibility for the recent shift to high impact prevention tactics that are being scaled up across north america . 2 ) describe community viral load as an aggregate biological measure of viral load for a particular geographic location for example the city of san francisco or a particular neighborhood and for a particular group of people who share socio - demographic characteristics. in order to determine the community viral load of any particular group or area of interest , one must take into account two distinct measures ; mean community viral load and total community viral load ( das et al . mean community viral load is the average of the most recent viral load of all reported hiv - positive persons in a particular target(ed ) population or geographical area ( das et al . total community viral load is the sum of the most recent viral loads of all reported hiv - positive persons in a particular target(ed ) population or geographical area ( das et al . these measurements make it possible to determine the mean average viral burden and the absolute level of the virus in any given population or geographical area . community viral load is a relatively new concept but has gained significant momentum in the field of hiv in recent years . this is due in part to the implementation of seek , test and treat ( now seek , test , treat and retain ) initiatives which rely on the premise that scaling up treatment collectively will result in a dramatic reduction of hiv transmission ( montaner et al . community viral load is considered by many researchers and clinicians to be a valuable marker of the direct relationship between hiv concentration ( in specific populations and geographical areas ) and overall hiv incidence ( wood et al . , community viral load has become an important concept under which to advocate for the expansion of testing and surveillance in the community and , most importantly , the reconceptualization of treatment as a prevention tool ( montaner et al . 2010 ) . recent studies in canada and the united states have demonstrated the value of measuring and mapping hiv viral load at the community level . in vancouver and across the province of british columbia ( canada ) , community viral load is increasingly being used as an epidemiological surveillance tool and an evaluation tool to monitor the outcomes of initiatives for the expansion of testing and treatment coverage ( montaner et al . 2010 ) . building on research findings , which suggest that community viral load is correlated with hiv incidence ( wood et al . 2009 ) , these initiatives were implemented across the province ( and more aggressively in the downtown eastside of vancouver ) and have led to a progressive decline in community viral load ( montaner et al . in some postal code areas of the province , however , the proportions of potentially infectious individuals ( as stated by the authors ) remain high regardless of the expansion of testing and treatment coverage ( lourenco et al . 2012 ) . similar findings have been reported in san francisco ( california , united states ) following the intensification of surveillance , the expansion of testing and earlier initiation of treatment ( das et al . 2011 ) . in addition to a documented decline in community viral load and new reported cases of hiv , the department of public health was also able to map areas with the highest concentration of viral load and further target them to produce improved outcomes ( das et al . areas such as tenderloin , south market , mission and castro were subsequently identified as hotspots ( as stated by the authors ) or areas with particularly high risk of hiv transmission due to the distribution of community viral load ( das et al . it was , therefore , recommended that more community - level interventions be implemented in these areas and that more aggressive prevention efforts ( including the use of treatment as a prevention tool ) be deployed to reduce community viral load . a similar mapping exercise was conducted in washington ( district of columbia , united states ) over a period of 4 years ( castel et al . ( 2012 ) included the most recent viral load data in the city , two indicators of socioeconomic status ( income and education ) , and race . their data were statistically analyzed and presented on various maps of the city of washington . these maps showed that the neighbourhoods with the worst socioeconomic indicators also had the highest hiv / aids prevalence rates ( castel et al . ( 2012 ) , can inform surveillance and the implementation of interventions that target populations with the highest viral load burden . it was determined that community viral load and its geospatial distribution are particularly useful to inform targeted public health interventions ( castel et al . similar arguments are presented by researchers who conducted studies on community viral load in new york city ( new york , united states ) from 2005 to 2009 ( laraque et al . based on their findings , they suggest that community viral load is valuable to public health agencies because it has the potential to identify high risk groups and target interventions to the groups whose viral control if achieved will most likely result in a rapid lowering of community viral load ( terzian et al . furthermore , it has the potential to identify groups at risk for sustained high viral load such as bronx residents who , according to terzian et al . ( 2012 ) , are more likely to have a detectable viral load and suffer from hiv - related disparities . identifying groups who are at risk or known to have sustained high viral load can thus provide an opportunity for the implementation of outreach initiatives in the community and the scale - up of efforts to educate , test , treat and engage members of the community in hiv care ( terzian et al . the inner workings of community viral load have not been critically examined in the hiv literature . the aim of this article is to interrogate the use of community viral load through a deductive process using theory to analyse empirics . theory makes it possible to engage in a critical analysis of a relatively new tool in the field of hiv and a limited body of empirical literature . it also allows us to disrupt normalizing discourses in the field of hiv and challenge the ways in which community viral load has been introduced as seemingly benign . this is particularly important considering that community viral load is now becoming normalized , standardized , and deployed in various public health agencies , without its assumptions and claims having been interrogated . in our view , it is imperative that researchers engage in discussions and debates around the use of community viral load and how it is intrinsically linked to the logics of governmentality . for this reason , we argue that a theory - based analysis of community viral load is of great value to these discussions and debates . community viral load links individual biomarkers and the concentration of the virus collectively through the production of particular kinds of spaces . these spaces are calculated , reconfigured , and imagined , with new levels of sophistication . first , community viral load signals an important shift in the way viral load is seen , imagined and represented both visually and spatially . specifically , we draw upon philo 's ( 2000 ) reading of foucault to expose three distinctive but interconnected representations of viral load . the first representation has to do with the tabulation of laboratory results that can attest to the progression of hiv and the response to treatment at the individual level . it draws a picture of how viral replication actually appears at every stage of hiv infection and what response is to be expected once treatment is initiated . here , tables and graphics of viral load patterns provide a reference point to assess individuals in the clinical setting and systematically position each of them accordingly in relation to an ideal patient with optimal viral suppression . it tracks viral replication within the body and penetrates its deepest recesses to expose the amount of copies of the virus in the blood , the breast milk , the sperm , the genital tract , the brain , the lymph nodes and so on . viral load is no longer seen as a laboratory value but rather as an evidence of viral activity in the body and a measure of infectiousness at the individual level . finally , the third representation allows for hiv to be located in space through mapping techniques . the location of the virus in space is compatible with foucault 's analysis of the shift from leper colony to plague city , wherein the risk of transmission is already present and requires the segmentation of space ( foucault 1990 ) . foucault explicitly linked mapping and government , and the need to have a rational plan to manage space ( crampton 2007 , p. 224 ) . in fact , he identified the ordering of space as central to the strategy for containment of biological risks and the government of processes that modulate health collectively rather than individually ( foucault 1990 , legg 2005 ) . these particular kinds of spaces take the form of specific neighbourhoods and locations where hiv is highly concentrated often located in geographical areas marked by high levels of marginalization , poverty , oppression and social exclusion . what is important here is not just that these spaces are rendered visible through viral load mapping and thus governable ( brown and knopp 2006 , 2010 ) , but the particular ways in which they are constructed based on the average viral burden of populations who occupy them and the absolute level of the virus that circulates among them . huxley ( 2006 ) explains that in order to better appreciate the articulation of spatial rationalities in the fabrication of governable spaces , we must pay close attention to the way authorities imagine these spaces and make them amenable to regulation . expanding on foucault 's work , huxley ( 2006 ) identifies three spatial rationalities : dispositional , generative and vitalist . for the purpose of this article , we will only refer to the dispositional and generative spatial rationalities . a dispositional rationality has to do with the production of boundaries and the spatial disposition of bodies ( huxley 2006 ) . it operates through the logics of quadrillage ( a term coined by foucault ) to prevent the spread of diseases and ensure a more effective regulation of processes that are responsible for epidemics ( huxley 2006 ) . in other words , it allows for the segmentation of space and the management of bodies to achieve a given end . entails systems or surveillance , as these are seen as essential to enforcing the ordering of spaces and bodies. generative rationality , on the other hand , concerns the effects of space on contagion , health , disease and death ( huxley 2006 ) . here , the problem is not so much the disposition of spaces but the concentration of diseased bodies in geographical areas ( huxley 2006 ) . these areas are seen in this imagination as particularly vulnerable and in need of attention ( brown and knopp 2010 , p. 394 ) . with this in mind , biomarkers become an important tool to circumscribe infectiousness and transmissibility ( or the risk thereof ) geographically . the emphasis is no longer on the disposition of diseased bodies in space but rather on areas where disease is highly concentrated and where the risk of transmission is intensified ( huxley 2006 ) . based on the work of huxley ( 2006 ) , we contend that the production of these spaces follows both dispositional and generational rationalities . by this , we mean that community viral load aims at drawing boundaries and mapping areas where the virus is unsuppressed areas that are then identified as vulnerable and in need of attention . we make a point here of referring to the virus because it is the real focus of this geographical ( and political ) undertaking even though we acknowledge that it reduces the bodies of people living with hiv to vectors of disease ( brown 1995 ) . community viral load , in fact , draws a rather partial and incomplete portrait of the hiv epidemic and continues to ignore the effect of context on hiv vulnerabilities -how the virus moves within a population and how it circulates across specific networks ( brown 1995 ) . what it does , however , is provide the necessary arguments to intensify surveillance , testing and prevention efforts in areas where we can find a number of people living with hiv whose viral load is unsuppressed. while our objective is not to portray people living with hiv as vectors of disease , we do have to stay true to the imaginations and rationales of those who see in community viral load a promising way to circumscribe infectiousness and transmissibility geographically . with this in mind , it is important to recognize that community viral load is increasingly being used as a tool to signal the existence of particular kinds of spaces , locate these spaces geographically , and allows for public health authorities to become more knowledgeable about the populations who occupy them . this knowledge generates new possibilities for the government of certain bodies which are seen as risky , dangerous , and in need of attention ( lupton 1995 ) . not surprisingly , it has led to the deployment of more aggressive interventions to lower the collective risk of acquiring or transmitting hiv , with little further consideration of the realities of people living with hiv on the ground , i.e. routine testing in institutions located in targeted areas , point of care testing in certain neighbourhoods , treatment as prevention initiatives in specific locations , and so forth . we shall now examine how community viral load cultivates specific ties between persons and communities through these new programmes of interventions . expanding on the work of michel foucault , miller and rose ( 2008 ) explain that the birth of the community marks a departure from governing a supposed collective social body to defining a new territory for the administration of collective life . this new territory has a number of significant features ( miller and rose 2008 ) . first , it calls on a particular re - configuration of space from a single , collective space to discrete communities which can be located geographically or constructed virtually ( i.e. lifestyle communities , moral communities , risk communities and so on ; miller and rose 2008 ) . second , it operates through the instrumentalization of personal allegiances and active responsibilities ( miller and rose 2008 , p. 90 ) . in other words , it makes use of allegiances and specific ties between individuals and communities to regulate , reform and mobilize ( miller and rose 2008 ) . from this perspective , members of particular communities are encouraged to practice active personal responsibility and conduct themselves accordingly ( miller and rose 2008 ) . as a sign of social citizenship , they must take responsibility , they must show themselves capable of calculated action and choice , they must shape their lives according to a moral code of individual responsibility and community obligation ( miller and rose 2008 , p. 105 ) . third , it concerns the identification of individuals as members of particular communities and the work required to make these individuals aware of their allegiances , for example , with the disability community , the gay community or the aids community . here , the sense of community is created and promoted through the work of educators , campaigns , activists , manipulators of symbols , narratives and identifications ( miller and rose 2008 , p. 92 ) . that is to say that these particular communities are created and marketed despite the fact that they may appear to be natural and may resonate with our own personal identity ( miller and rose 2008 ) . drawing on a number of examples , miller and rose ( 2008 ) explain that government through community works , even when it works upon pre - existing bonds of allegiance , transforms them , invests them with new values , affiliates them to expertise and reconfigures relations ( p. 93 ) in a productive way . miller and rose ( 2008 ) explain that the construction of community involves various strategies for making individuals aware of their personal allegiances . one of these strategies is to raise awareness ( i.e. awareness campaigns ) , educate ( i.e. community training sessions ) , communicate ( i.e. media and other communication tools ) , and make sure that individuals identify themselves as members of that community ( miller and rose 2008 ) . drawing on health promotion programmes in hiv and aids , miller and rose ( 2008 ) explain that government through community produces new personal allegiances and works upon pre - existing ones to make individuals aware of their collective affiliations . these affiliations , explain miller and rose ( 2008 ) , are to be celebrated , encouraged , nurtured , shaped and instrumentalized in the hope of producing consequences that are desirable for all and for each ( p. 93 ) . affiliations to particular communities , then , create new relations of identification and , incidentally , new relations of mutual obligation ( miller and rose 2008 , p. 88 ) . as such , they open up questions of personal responsibilities and obligations . these questions are particularly relevant to our analysis because community viral load is not just a means of identification and community affiliation . it is also used to govern individuals who are located here and not there , that can be expected to behave and engage in certain ways and not others , and whose distribution is spatially correlated with other characteristics that makes them part of risk communities ( viral load , serological status , gender , ethnicity , socio - economic status , sexuality , etc . ) ( brown and knopp 2010 ) . from this perspective , members of risk communities must practice active personal responsibility and are expected to shape their lives according to a moral code of individual responsibility and community obligation ( miller and rose 2008 , p. 105 ) . those who do not exercise caution and live according to this moral code , however , are generally found in the margins of communities and their particular difficulties thus need to be addressed through the activities of various specialists each of whom is an expert in a particular problem ( miller and rose 2008 , p. 104 ) . these relations of expertise , explain miller and rose ( 2008 ) , operate at the level of the community . professional gaze , where expertise now focuses on conduct itself and the cognitive and moral organization of perception , intention , action and evaluation ( miller and rose 2008 , p. 106 ) . in these relations , the subject of expertise is conceived as an individual who lacks cognitive , emotional , practical , and ethical skills to take personal responsibility for rational self - management and fulfill his moral obligations ( miller and rose 2008 , p. 106 ) . this form of governance relies on the deployment of expert knowledge from various fields ( i.e. nursing , social work , medicine , etc . ) and practices of empowerment . empowerment , then , is a matter of experts teaching , coaxing , requiring their clients to conduct themselves within their respective communities , according to certain prescribed codes of active personal responsibility and moral obligations ( miller and rose 2008 , p. 106 ) . seek , test and treat initiatives which have recently been implemented in both canada and the united states , including community as a unit of identity , responsibilization and autonomization of people who reside in certain neighbourhoods , empowerment of individuals located in the margins , and expert management and outreach in risk communities. despite a seemingly emancipatory or rights - based agenda and the will to empower individuals who have been traditionally labelled as hard to reach ( patton 2011 ) , the deployment of these initiatives is indicative of the very form of government that miller and rose ( 2008 ) describe : the subject constituted through techniques of empowerment is being shaped into a better functioning and more responsible neoliberal actor empowered to make better decisions , able to fulfill moral obligations , and capable of committing to the health of his community . the experts are working directly in the community , relaying messages to individuals who have to be made aware of their personal allegiances to particular the community , as imagined through community viral load and the distribution of bodies in space , is now something to be programmed , researched and managed . the use of the term community is a powerful rhetorical tool and appeals to a common sense of belonging indeed , do we not all belong to some community ? yet , public health is not interested in all communities equally . as mentioned earlier , community viral load is part of a larger public health programme to promote high impact prevention using combinations of scientifically proven , cost - effective , and scalable interventions targeted to the right populations in the right geographic areas ( centers for disease control and prevention 2011 , p. 1 ) ( italics added for emphasis ) . within this new prevention framework , public health agencies are asked to identify communities where concentrations of the virus overlap with concentrations of substance users , gay and other men of have sex with men , transgender persons and racialized persons . community viral load provides a new tool for public health authorities to imagine particular communities ( legg 2005 ) in ways that are flexible , fluid , and can be adapted to different needs ( brown and knopp 2006 ) . this is inclusive of virtual communities of individuals who share particular risk factors but are geographically dispersed to communities of individuals who share geographical coordinates and whose virus is unsuppressed. what is important to understand here is not just that community viral load allows for these communities to be imagined , but that it is part of a large - scale operation to seek , test and treat more aggressively in target areas with the highest community viral load . this is part of the inner workings of community viral load ; it generates new knowledge , maps the presence of the virus collectively , makes room for a new form of surveillance , and in the end allows for the creation of specific targets , which are clustered around predominantly impoverished and marginalized neighbourhoods . seek , test and treat initiatives are arguably the best examples of high impact prevention efforts that specifically target such neighbourhoods and use on - site outreach to implement more aggressive forms of testing , surveillance and treatment -including mass testing fares with incentives meant to draw in the most marginalized . the rationale for these initiatives is largely based on the need to know community viral load and its geographic distribution . these initiatives appeal to the discourse of community ( lynn 2006 ) , with its shared culture , needs , and responsibilities , and relations of expertise ( miller and rose 2008 ) . we are particularly interested here in how community viral load creates or builds on existing personal allegiances and specific ties between individuals and communities to support the implementation of seek , test and treat initiatives ( miller and rose 2008 ) . not only is community viral load a potent tool to re - configure space ( as previously discussed ) and justify the need for public health to intervene more aggressively in certain neighbourhoods and locations , but it is also used as a device of identification . on the one hand , it concerns the identification of individuals who reside in areas where hiv is most heavily concentrated as members of a community. on the other hand , it reinforces the personal allegiances that these individuals may have to this community based on their serological status , their viral load , or the fact that they share a common fate as residents in neighbourhoods and locations where hiv is these individuals may , in turn , become actively involved in the deployment of community - level initiatives and engaged in outreach efforts to scale up hiv prevention . they may also become increasingly politicized . while this phenomenon may be beneficial for particular communities , it also tends to extend and reinforce the instrumentalization of personal allegiances and active responsibilities ( miller and rose 2008 ) . seek , test and treat initiatives rely on the assumption that individuals who are made aware of their personal allegiances with a community will be more inclined to enrol , mobilize and act more responsibly ( miller and rose 2008 ) . one way to practice active personal responsibility is to take part in testing and link with prevention services . another way to practice active personal responsibility is to initiate antiretroviral treatment as soon as possible after diagnosis and demonstrate optimal treatment adherence . with this is mind , it is important to understand that seek , test and treat initiatives make room for new relations of expertise and new opportunities for experts to intervene directly in the community. in fact , these initiatives rely on a re - configuration of space and re - location of interventions traditionally done in clinical settings ; testing is now conducted in vans , shelters , community centres , gay bathhouses and drop - in services , results are communicated right away , and linkage to care is automatically provided , surveillance data are broadened and include geospatial analysis , individual viral load , census information and indicators of socioeconomic status , and treatment is initiated rapidly after diagnosis followed by ongoing follow - up and adherence counselling in the community . they also rely on the ongoing presence and visibility of experts in the community , working in neighbourhoods and locations that are most affected by the hiv / aids epidemic . again , this may be beneficial for some communities but it is important to acknowledge that the expansion of testing and treatment coverage are not substitute to the removal of vulnerabilities that place people at risk of infection in the first place ( which incidentally , overlap with vulnerabilities preventing access to [ hiv care and ] treatment ( nguyen et al . 2011 , p. 292 ) . while our goal is not refute the scientific evidence in support of high impact prevention , which is the guiding framework for seek , test and treat initiatives , we consider that these programmes require more reflection . in this article , we have acknowledged that this new framework is closely tied to the introduction of community viral and the need for more effective ways to govern bodies that are seen as contributing to the spread of hiv . in light of our analysis , we consider that this new framework has very little to do with the care of individuals who live with hiv/ aids and much more to do with the production of a healthy population and the control of certain bodies . it is concerned with matters of life and death , with health and illness , with infectiousness and transmissibility , and with the collective processes that contribute to the spread of hiv . it acts upon the health of the population as a whole by targeting geographical areas where hiv is highly concentrated , seeking and testing populations conceived as inherently risk - prone , vulnerable or unstable , and treating individuals who are found to be hiv positive in order to achieve individual viral suppression . therefore , in keeping with foucault 's ( 2003 ) work , we consider that community viral load has both individualizing and massifying effects . it was introduced to the field of hiv as a way to act upon communities who are located in specific geographical areas rather than individuals themselves . however , in order for it to be effective , it has to create specific ties between individuals who reside in areas where hiv is most heavily concentration and their respective risk communities. this has important implications for people living with hiv / aids , and may have broader implications for everyone who is being forced into these particular geographical areas these new ghettos that are marked by hiv and are under the radar of public health authorities . as such , we are concerned that community viral load has become a proxy for naming risk communities , but with new levels of sophistication . in particular , we are concerned with the potential to increase stigma directed at populations who occupy areas that are identified as highly virulent. this phenomenon has yet to be addressed in the literature and in research conducted on community viral load . community viral load links individual biomarkers and the concentration of the virus collectively through the production of particular kinds of spaces . these spaces are calculated , reconfigured , and imagined , with new levels of sophistication . first , community viral load signals an important shift in the way viral load is seen , imagined and represented both visually and spatially . specifically , we draw upon philo 's ( 2000 ) reading of foucault to expose three distinctive but interconnected representations of viral load . the first representation has to do with the tabulation of laboratory results that can attest to the progression of hiv and the response to treatment at the individual level . it draws a picture of how viral replication actually appears at every stage of hiv infection and what response is to be expected once treatment is initiated . here , tables and graphics of viral load patterns provide a reference point to assess individuals in the clinical setting and systematically position each of them accordingly in relation to an ideal patient with optimal viral suppression . it tracks viral replication within the body and penetrates its deepest recesses to expose the amount of copies of the virus in the blood , the breast milk , the sperm , the genital tract , the brain , the lymph nodes and so on . viral load is no longer seen as a laboratory value but rather as an evidence of viral activity in the body and a measure of infectiousness at the individual level . finally , the third representation allows for hiv to be located in space through mapping techniques . the location of the virus in space is compatible with foucault 's analysis of the shift from leper colony to plague city , wherein the risk of transmission is already present and requires the segmentation of space ( foucault 1990 ) . foucault explicitly linked mapping and government , and the need to have a rational plan to manage space ( crampton 2007 , p. 224 ) . in fact , he identified the ordering of space as central to the strategy for containment of biological risks and the government of processes that modulate health collectively rather than individually ( foucault 1990 , legg 2005 ) . these particular kinds of spaces take the form of specific neighbourhoods and locations where hiv is highly concentrated often located in geographical areas marked by high levels of marginalization , poverty , oppression and social exclusion . what is important here is not just that these spaces are rendered visible through viral load mapping and thus governable ( brown and knopp 2006 , 2010 ) , but the particular ways in which they are constructed based on the average viral burden of populations who occupy them and the absolute level of the virus that circulates among them . huxley ( 2006 ) explains that in order to better appreciate the articulation of spatial rationalities in the fabrication of governable spaces , we must pay close attention to the way authorities imagine these spaces and make them amenable to regulation . expanding on foucault 's work , huxley ( 2006 ) identifies three spatial rationalities : dispositional , generative and vitalist . for the purpose of this article a dispositional rationality has to do with the production of boundaries and the spatial disposition of bodies ( huxley 2006 ) . it operates through the logics of quadrillage ( a term coined by foucault ) to prevent the spread of diseases and ensure a more effective regulation of processes that are responsible for epidemics ( huxley 2006 ) . in other words , it allows for the segmentation of space and the management of bodies to achieve a given end . entails systems or surveillance , as these are seen as essential to enforcing the ordering of spaces and bodies. generative rationality , on the other hand , concerns the effects of space on contagion , health , disease and death ( huxley 2006 ) . here , the problem is not so much the disposition of spaces but the concentration of diseased bodies in geographical areas ( huxley 2006 ) . these areas are seen in this imagination as particularly vulnerable and in need of attention ( brown and knopp 2010 , p. 394 ) . with this in mind , biomarkers become an important tool to circumscribe infectiousness and transmissibility ( or the risk thereof ) geographically . the emphasis is no longer on the disposition of diseased bodies in space but rather on areas where disease is highly concentrated and where the risk of transmission is intensified ( huxley 2006 ) . based on the work of huxley ( 2006 ) , we contend that the production of these spaces follows both dispositional and generational rationalities . by this , we mean that community viral load aims at drawing boundaries and mapping areas where the virus is unsuppressed areas that are then identified as vulnerable and in need of attention . we make a point here of referring to the virus because it is the real focus of this geographical ( and political ) undertaking even though we acknowledge that it reduces the bodies of people living with hiv to vectors of disease ( brown 1995 ) . community viral load , in fact , draws a rather partial and incomplete portrait of the hiv epidemic and continues to ignore the effect of context on hiv vulnerabilities -how the virus moves within a population and how it circulates across specific networks ( brown 1995 ) . what it does , however , is provide the necessary arguments to intensify surveillance , testing and prevention efforts in areas where we can find a number of people living with hiv whose viral load is unsuppressed. while our objective is not to portray people living with hiv as vectors of disease , we do have to stay true to the imaginations and rationales of those who see in community viral load a promising way to circumscribe infectiousness and transmissibility geographically . with this in mind , it is important to recognize that community viral load is increasingly being used as a tool to signal the existence of particular kinds of spaces , locate these spaces geographically , and allows for public health authorities to become more knowledgeable about the populations who occupy them . this knowledge generates new possibilities for the government of certain bodies which are seen as not surprisingly , it has led to the deployment of more aggressive interventions to lower the collective risk of acquiring or transmitting hiv , with little further consideration of the realities of people living with hiv on the ground , i.e. routine testing in institutions located in targeted areas , point of care testing in certain neighbourhoods , treatment as prevention initiatives in specific locations , and so forth . we shall now examine how community viral load cultivates specific ties between persons and communities through these new programmes of interventions . expanding on the work of michel foucault , miller and rose ( 2008 ) explain that the birth of the community marks a departure from governing a supposed collective social body to defining a new territory for the administration of collective life . this new territory has a number of significant features ( miller and rose 2008 ) . first , it calls on a particular re - configuration of space from a single , collective space to discrete communities which can be located geographically or constructed virtually ( i.e. lifestyle communities , moral communities , risk communities and so on ; miller and rose 2008 ) . second , it operates through the instrumentalization of personal allegiances and active responsibilities ( miller and rose 2008 , p. 90 ) . in other words , it makes use of allegiances and specific ties between individuals and communities to regulate , reform and mobilize ( miller and rose 2008 ) . from this perspective , members of particular communities are encouraged to practice active personal responsibility and conduct themselves accordingly ( miller and rose 2008 ) . as a sign of social citizenship , they must take responsibility , they must show themselves capable of calculated action and choice , they must shape their lives according to a moral code of individual responsibility and community obligation ( miller and rose 2008 , p. 105 ) . third , it concerns the identification of individuals as members of particular communities and the work required to make these individuals aware of their allegiances , for example , with the disability community , the gay community or the aids community . here , the sense of community is created and promoted through the work of educators , campaigns , activists , manipulators of symbols , narratives and identifications ( miller and rose 2008 , p. 92 ) . that is to say that these particular communities are created and marketed despite the fact that they may appear to be natural and may resonate with our own personal identity ( miller and rose 2008 ) . drawing on a number of examples , miller and rose ( 2008 ) explain that government through community works , even when it works upon pre - existing bonds of allegiance , transforms them , invests them with new values , affiliates them to expertise and reconfigures relations ( p. 93 ) in a productive way . miller and rose ( 2008 ) explain that the construction of community involves various strategies for making individuals aware of their personal allegiances . one of these strategies is to raise awareness ( i.e. awareness campaigns ) , educate ( i.e. community training sessions ) , communicate ( i.e. media and other communication tools ) , and make sure that individuals identify themselves as members of that community ( miller and rose 2008 ) . drawing on health promotion programmes in hiv and aids , miller and rose ( 2008 ) explain that government through community produces new personal allegiances and works upon pre - existing ones to make individuals aware of their collective affiliations . these affiliations , explain miller and rose ( 2008 ) , are to be celebrated , encouraged , nurtured , shaped and instrumentalized in the hope of producing consequences that are desirable for all and for each ( p. 93 ) . affiliations to particular communities , then , create new relations of identification and , incidentally , new relations of mutual obligation ( miller and rose 2008 , p. 88 ) . these questions are particularly relevant to our analysis because community viral load is not just a means of identification and community affiliation . it is also used to govern individuals who are located here and not there , that can be expected to behave and engage in certain ways and not others , and whose distribution is spatially correlated with other characteristics that makes them part of risk communities ( viral load , serological status , gender , ethnicity , socio - economic status , sexuality , etc . ) ( brown and knopp 2010 ) . from this perspective , members of risk communities must practice active personal responsibility and are expected to shape their lives according to a moral code of individual responsibility and community obligation ( miller and rose 2008 , p. 105 ) . those who do not exercise caution and live according to this moral code , however , are generally found in the margins of communities and their particular difficulties thus need to be addressed through the activities of various specialists each of whom is an expert in a particular problem ( miller and rose 2008 , p. 104 ) . these relations of expertise , explain miller and rose ( 2008 ) , operate at the level of the community . professional gaze , where expertise now focuses on conduct itself and the cognitive and moral organization of perception , intention , action and evaluation ( miller and rose 2008 , p. 106 ) . in these relations , the subject of expertise is conceived as an individual who lacks cognitive , emotional , practical , and ethical skills to take personal responsibility for rational self - management and fulfill his moral obligations ( miller and rose 2008 , p. 106 ) . this form of governance relies on the deployment of expert knowledge from various fields ( i.e. nursing , social work , medicine , etc . ) and practices of empowerment . empowerment , then , is a matter of experts teaching , coaxing , requiring their clients to conduct themselves within their respective communities , according to certain prescribed codes of active personal responsibility and moral obligations ( miller and rose 2008 , p. 106 ) . seek , test and treat initiatives which have recently been implemented in both canada and the united states , including community as a unit of identity , responsibilization and autonomization of people who reside in certain neighbourhoods , empowerment of individuals located in the margins , and expert management and outreach in risk communities. despite a seemingly emancipatory or rights - based agenda and the will to empower individuals who have been traditionally labelled as hard to reach ( patton 2011 ) , the deployment of these initiatives is indicative of the very form of government that miller and rose ( 2008 ) describe : the subject constituted through techniques of empowerment is being shaped into a better functioning and more responsible neoliberal actor empowered to make better decisions , able to fulfill moral obligations , and capable of committing to the health of his community . the experts are working directly in the community , relaying messages to individuals who have to be made aware of their personal allegiances to particular the community , as imagined through community viral load and the distribution of bodies in space , is now something to be programmed , researched and managed . the use of the term community is a powerful rhetorical tool and appeals to a common sense of belonging indeed , do we not all belong to some community ? yet , public health is not interested in all communities equally . as mentioned earlier , community viral load is part of a larger public health programme to promote high impact prevention using combinations of scientifically proven , cost - effective , and scalable interventions targeted to the right populations in the right geographic areas ( centers for disease control and prevention 2011 , p. 1 ) ( italics added for emphasis ) . within this new prevention framework , public health agencies are asked to identify communities where concentrations of the virus overlap with concentrations of substance users , gay and other men of have sex with men , transgender persons and racialized persons . community viral load provides a new tool for public health authorities to imagine particular communities ( legg 2005 ) in ways that are flexible , fluid , and can be adapted to different needs ( brown and knopp 2006 ) . this is inclusive of virtual communities of individuals who share particular risk factors but are geographically dispersed to communities of individuals who share geographical coordinates and whose virus is unsuppressed. what is important to understand here is not just that community viral load allows for these communities to be imagined , but that it is part of a large - scale operation to seek , test and treat more aggressively in target areas with the highest community viral load . this is part of the inner workings of community viral load ; it generates new knowledge , maps the presence of the virus collectively , makes room for a new form of surveillance , and in the end allows for the creation of specific targets , which are clustered around predominantly impoverished and marginalized neighbourhoods . seek , test and treat initiatives are arguably the best examples of high impact prevention efforts that specifically target such neighbourhoods and use on - site outreach to implement more aggressive forms of testing , surveillance and treatment -including mass testing fares with incentives meant to draw in the most marginalized . the rationale for these initiatives is largely based on the need to know community viral load and its geographic distribution . these initiatives appeal to the discourse of community ( lynn 2006 ) , with its shared culture , needs , and responsibilities , and relations of expertise ( miller and rose 2008 ) . we are particularly interested here in how community viral load creates or builds on existing personal allegiances and specific ties between individuals and communities to support the implementation of seek , test and treat initiatives ( miller and rose 2008 ) . not only is community viral load a potent tool to re - configure space ( as previously discussed ) and justify the need for public health to intervene more aggressively in certain neighbourhoods and locations , but it is also used as a device of identification . on the one hand , it concerns the identification of individuals who reside in areas where hiv is most heavily concentrated as members of a community. on the other hand , it reinforces the personal allegiances that these individuals may have to this community based on their serological status , their viral load , or the fact that they share a common fate as residents in neighbourhoods and locations where hiv is these individuals may , in turn , become actively involved in the deployment of community - level initiatives and engaged in outreach efforts to scale up hiv prevention . they may also become increasingly politicized . while this phenomenon may be beneficial for particular communities , it also tends to extend and reinforce the instrumentalization of personal allegiances and active responsibilities ( miller and rose 2008 ) . seek , test and treat initiatives rely on the assumption that individuals who are made aware of their personal allegiances with a community will be more inclined to enrol , mobilize and act more responsibly ( miller and rose 2008 ) . one way to practice active personal responsibility is to take part in testing and link with prevention services . another way to practice active personal responsibility is to initiate antiretroviral treatment as soon as possible after diagnosis and demonstrate optimal treatment adherence . with this seek , test and treat initiatives make room for new relations of expertise and new opportunities for experts to intervene directly in the community. in fact , these initiatives rely on a re - configuration of space and re - location of interventions traditionally done in clinical settings ; testing is now conducted in vans , shelters , community centres , gay bathhouses and drop - in services , results are communicated right away , and linkage to care is automatically provided , surveillance data are broadened and include geospatial analysis , individual viral load , census information and indicators of socioeconomic status , and treatment is initiated rapidly after diagnosis followed by ongoing follow - up and adherence counselling in the community . they also rely on the ongoing presence and visibility of experts in the community , working in neighbourhoods and locations that are most affected by the hiv / aids epidemic . again , this may be beneficial for some communities but it is important to acknowledge that the expansion of testing and treatment coverage are not substitute to the removal of vulnerabilities that place people at risk of infection in the first place ( which incidentally , overlap with vulnerabilities preventing access to [ hiv care and ] treatment ( nguyen et al . 2011 , p. 292 ) . while our goal is not refute the scientific evidence in support of high impact prevention , which is the guiding framework for seek , test and treat initiatives , we consider that these programmes require more reflection . in this article , we have acknowledged that this new framework is closely tied to the introduction of community viral and the need for more effective ways to govern bodies that are seen as contributing to the spread of hiv . in light of our analysis , we consider that this new framework has very little to do with the care of individuals who live with hiv/ aids and much more to do with the production of a healthy population and the control of certain bodies . it is concerned with matters of life and death , with health and illness , with infectiousness and transmissibility , and with the collective processes that contribute to the spread of hiv . it acts upon the health of the population as a whole by targeting geographical areas where hiv is highly concentrated , seeking and testing populations conceived as inherently risk - prone , vulnerable or unstable , and treating individuals who are found to be hiv positive in order to achieve individual viral suppression . therefore , in keeping with foucault 's ( 2003 ) work , we consider that community viral load has both individualizing and massifying effects . it was introduced to the field of hiv as a way to act upon communities who are located in specific geographical areas rather than individuals themselves . however , in order for it to be effective , it has to create specific ties between individuals who reside in areas where hiv is most heavily concentration and their respective risk communities. this has important implications for people living with hiv / aids , and may have broader implications for everyone who is being forced into these particular geographical areas these new ghettos that are marked by hiv and are under the radar of public health authorities . as such , we are concerned that community viral load has become a proxy for naming risk communities , but with new levels of sophistication . in particular , we are concerned with the potential to increase stigma directed at populations who occupy areas that are identified as highly virulent. this phenomenon has yet to be addressed in the literature and in research conducted on community viral load . the objective of this article has been to formulate a critique and propose a novel way of theorizing community viral load through a foucauldian analytics of governmentality with an attention to the role of space and community . we have shown that particular kinds of spaces are being reconfigured through a combination of clinical measures and epidemiological techniques to map the distribution of infected and risky bodies . these spaces then become key targets of public health intervention in the form of high impact treatment and prevention technologies that necessitate an unprecedented investment in identifying individuals in need of treatment ( nguyen et al . once on treatment , individuals become part of the community viral load loop their individual viral load becomes used to assess their we have also shown that the term community and its uses represents a discursive terrain imbued with power and ideology and requires unpacking to surface how it is being used and to what ends ( lynn 2006 ) . in light of our analysis , we are struck by what appears to be a shift back to the beginnings of the aids epidemic when various groups were singled out as infectious ( novitsky and essex 2012 ) and characterized as dangerous [ for historical overview and critique of this , see epstein 1996 , patton 1996 ] . the concern became that identifying people in this way perpetuated stigma and could result in the potential for an even greater number of infections ( parker and aggleton 2003 , peretti - watel et al . , epidemiology moved to monitoring risk behaviours as a way of de - stigmatizing these groups . with the invention and deployment of community viral load , we see a return to this previous logic , but resting on the seeming neutrality of space as if all people freely inhabit spaces of their own choosing . the implications of this , some of which we have explored , are only speculative at this time as calculating community viral load in all jurisdictions has various logistical and policy challenges . there are however concerted efforts underway to reduce these remaining barriers as suggested by the most recent national hiv / aids strategy in the united states ( office of national aids policy 2010 ) and guidelines published by the centers for disease control and prevention ( 2012 ) on community viral load measures , definitions and methods for calculation . this return to previous ways of thinking about , and managing hiv , albeit through more sophisticated techniques than were available at the beginnings of the epidemic , raises important questions about how hiv is being seen by governments , policy makers and public health agencies . require testing and mandatory treatment on a scale seen only in dictatorships ( p. 263 ) . however , this bold statement fits within larger debates about hiv as a threat to inter / intra state security ( elbe 2005 , 2009 ) . how do the changing politics of hiv and the growing interest in the securitization of health and illness help inform our thinking about mapping community viral load what are the implications of these clinical advancements for other health issues , and the potential outcomes to those who reside in spaces characterized by high rates of hiv and other forms of chronic illness . we hope that by closing on such a note that others will be encouraged to revisit hiv , and these new treatment and new prevention technologies , as a site for critical debate .

hiv plasma viral load testing has become more than just a clinical tool to monitor treatment response at the individual level . increasingly , individual hiv plasma viral load testing is being reported to public health agencies and is used to inform epidemiological surveillance and monitor the presence of the virus collectively using techniques to measure community viral load. this article seeks to formulate a critique and propose a novel way of theorizing community viral load . based on the salient work of michel foucault , especially the governmentality literature , this article critically examines the use of community viral load as a new strategy of government . drawing also on the work of miller and rose , this article explores the deployment of community through the re - configuration of space , the problematization of viral concentrations in specific microlocales , and the government ( in the foucauldian sense ) of specific bodies which are seen as risky , dangerous and therefore , in need of attention . it also examines community viral load as a necessary precondition forming the conditions of possibility for the recent shift to high impact prevention tactics that are being scaled up across north america .

Introduction Defining community viral load Community viral load research in Vancouver, San Francisco, New York and Washington Mapping community viral load Governing through communities Beyond the rhetoric of community Final remarks

however , in recent years , plasma viral load testing has become more than just a clinical tool to monitor treatment response at the individual level . increasingly , individual plasma viral load testing is being reported to public health agencies and is used to inform epidemiological surveillance and monitor the presence of the virus collectively ( terzian et al . in particular need of critical appraisal is the development of techniques to measure , map and monitor community viral load. for the purpose of this article , we argue that community viral load should be examined in relation to the theoretical framework of foucault ( 1984 ) coined this concept to account for the ways through which capillary forms of power insert themselves into actions , attitudes , and discourses to produce a particular kind of person and population - productive , but docile and easily governed . miller and rose ( 2008 , p. 63 ) describe governmentality as a domain of strategies , techniques and procedures through which different forces seek to render programmes operable , and by means of which a multitude of connections are established between the aspirations of authorities and the activities of individuals and groups. based on the work of foucault ( 1991 , 2003 ) , this article critically examines the use of community viral load as a new strategy of governmentality . also drawing on the work of miller and rose ( 2008 ) , this article further reconceptualizes specifically , it explores the deployment of community through the re - configuration of space , the problematization of viral concentrations in specific micro - locales , and the government ( in the foucauldian sense ) of specific bodies which are seen as it also examines community viral load as a necessary precondition forming the conditions of possibility for the recent shift to high impact prevention tactics that are being scaled up across north america . 2 ) describe community viral load as an aggregate biological measure of viral load for a particular geographic location for example the city of san francisco or a particular neighborhood and for a particular group of people who share socio - demographic characteristics. community viral load is considered by many researchers and clinicians to be a valuable marker of the direct relationship between hiv concentration ( in specific populations and geographical areas ) and overall hiv incidence ( wood et al . , community viral load has become an important concept under which to advocate for the expansion of testing and surveillance in the community and , most importantly , the reconceptualization of treatment as a prevention tool ( montaner et al . in vancouver and across the province of british columbia ( canada ) , community viral load is increasingly being used as an epidemiological surveillance tool and an evaluation tool to monitor the outcomes of initiatives for the expansion of testing and treatment coverage ( montaner et al . in addition to a documented decline in community viral load and new reported cases of hiv , the department of public health was also able to map areas with the highest concentration of viral load and further target them to produce improved outcomes ( das et al . it was , therefore , recommended that more community - level interventions be implemented in these areas and that more aggressive prevention efforts ( including the use of treatment as a prevention tool ) be deployed to reduce community viral load . it was determined that community viral load and its geospatial distribution are particularly useful to inform targeted public health interventions ( castel et al . based on their findings , they suggest that community viral load is valuable to public health agencies because it has the potential to identify high risk groups and target interventions to the groups whose viral control if achieved will most likely result in a rapid lowering of community viral load ( terzian et al . identifying groups who are at risk or known to have sustained high viral load can thus provide an opportunity for the implementation of outreach initiatives in the community and the scale - up of efforts to educate , test , treat and engage members of the community in hiv care ( terzian et al . the inner workings of community viral load have not been critically examined in the hiv literature . the aim of this article is to interrogate the use of community viral load through a deductive process using theory to analyse empirics . it also allows us to disrupt normalizing discourses in the field of hiv and challenge the ways in which community viral load has been introduced as seemingly benign . this is particularly important considering that community viral load is now becoming normalized , standardized , and deployed in various public health agencies , without its assumptions and claims having been interrogated . in our view , it is imperative that researchers engage in discussions and debates around the use of community viral load and how it is intrinsically linked to the logics of governmentality . community viral load links individual biomarkers and the concentration of the virus collectively through the production of particular kinds of spaces . it tracks viral replication within the body and penetrates its deepest recesses to expose the amount of copies of the virus in the blood , the breast milk , the sperm , the genital tract , the brain , the lymph nodes and so on . viral load is no longer seen as a laboratory value but rather as an evidence of viral activity in the body and a measure of infectiousness at the individual level . what is important here is not just that these spaces are rendered visible through viral load mapping and thus governable ( brown and knopp 2006 , 2010 ) , but the particular ways in which they are constructed based on the average viral burden of populations who occupy them and the absolute level of the virus that circulates among them . these areas are seen in this imagination as particularly vulnerable and in need of attention ( brown and knopp 2010 , p. 394 ) . by this , we mean that community viral load aims at drawing boundaries and mapping areas where the virus is unsuppressed areas that are then identified as vulnerable and in need of attention . community viral load , in fact , draws a rather partial and incomplete portrait of the hiv epidemic and continues to ignore the effect of context on hiv vulnerabilities -how the virus moves within a population and how it circulates across specific networks ( brown 1995 ) . with this in mind , it is important to recognize that community viral load is increasingly being used as a tool to signal the existence of particular kinds of spaces , locate these spaces geographically , and allows for public health authorities to become more knowledgeable about the populations who occupy them . this knowledge generates new possibilities for the government of certain bodies which are seen as risky , dangerous , and in need of attention ( lupton 1995 ) . expanding on the work of michel foucault , miller and rose ( 2008 ) explain that the birth of the community marks a departure from governing a supposed collective social body to defining a new territory for the administration of collective life . here , the sense of community is created and promoted through the work of educators , campaigns , activists , manipulators of symbols , narratives and identifications ( miller and rose 2008 , p. 92 ) . these relations of expertise , explain miller and rose ( 2008 ) , operate at the level of the community . in these relations , the subject of expertise is conceived as an individual who lacks cognitive , emotional , practical , and ethical skills to take personal responsibility for rational self - management and fulfill his moral obligations ( miller and rose 2008 , p. 106 ) . seek , test and treat initiatives which have recently been implemented in both canada and the united states , including community as a unit of identity , responsibilization and autonomization of people who reside in certain neighbourhoods , empowerment of individuals located in the margins , and expert management and outreach in risk communities. despite a seemingly emancipatory or rights - based agenda and the will to empower individuals who have been traditionally labelled as hard to reach ( patton 2011 ) , the deployment of these initiatives is indicative of the very form of government that miller and rose ( 2008 ) describe : the subject constituted through techniques of empowerment is being shaped into a better functioning and more responsible neoliberal actor empowered to make better decisions , able to fulfill moral obligations , and capable of committing to the health of his community . the experts are working directly in the community , relaying messages to individuals who have to be made aware of their personal allegiances to particular the community , as imagined through community viral load and the distribution of bodies in space , is now something to be programmed , researched and managed . as mentioned earlier , community viral load is part of a larger public health programme to promote high impact prevention using combinations of scientifically proven , cost - effective , and scalable interventions targeted to the right populations in the right geographic areas ( centers for disease control and prevention 2011 , p. 1 ) ( italics added for emphasis ) . within this new prevention framework , public health agencies are asked to identify communities where concentrations of the virus overlap with concentrations of substance users , gay and other men of have sex with men , transgender persons and racialized persons . community viral load provides a new tool for public health authorities to imagine particular communities ( legg 2005 ) in ways that are flexible , fluid , and can be adapted to different needs ( brown and knopp 2006 ) . this is part of the inner workings of community viral load ; it generates new knowledge , maps the presence of the virus collectively , makes room for a new form of surveillance , and in the end allows for the creation of specific targets , which are clustered around predominantly impoverished and marginalized neighbourhoods . the rationale for these initiatives is largely based on the need to know community viral load and its geographic distribution . these initiatives appeal to the discourse of community ( lynn 2006 ) , with its shared culture , needs , and responsibilities , and relations of expertise ( miller and rose 2008 ) . not only is community viral load a potent tool to re - configure space ( as previously discussed ) and justify the need for public health to intervene more aggressively in certain neighbourhoods and locations , but it is also used as a device of identification . on the other hand , it reinforces the personal allegiances that these individuals may have to this community based on their serological status , their viral load , or the fact that they share a common fate as residents in neighbourhoods and locations where hiv is these individuals may , in turn , become actively involved in the deployment of community - level initiatives and engaged in outreach efforts to scale up hiv prevention . in fact , these initiatives rely on a re - configuration of space and re - location of interventions traditionally done in clinical settings ; testing is now conducted in vans , shelters , community centres , gay bathhouses and drop - in services , results are communicated right away , and linkage to care is automatically provided , surveillance data are broadened and include geospatial analysis , individual viral load , census information and indicators of socioeconomic status , and treatment is initiated rapidly after diagnosis followed by ongoing follow - up and adherence counselling in the community . in this article , we have acknowledged that this new framework is closely tied to the introduction of community viral and the need for more effective ways to govern bodies that are seen as contributing to the spread of hiv . this has important implications for people living with hiv / aids , and may have broader implications for everyone who is being forced into these particular geographical areas these new ghettos that are marked by hiv and are under the radar of public health authorities . community viral load links individual biomarkers and the concentration of the virus collectively through the production of particular kinds of spaces . it tracks viral replication within the body and penetrates its deepest recesses to expose the amount of copies of the virus in the blood , the breast milk , the sperm , the genital tract , the brain , the lymph nodes and so on . viral load is no longer seen as a laboratory value but rather as an evidence of viral activity in the body and a measure of infectiousness at the individual level . what is important here is not just that these spaces are rendered visible through viral load mapping and thus governable ( brown and knopp 2006 , 2010 ) , but the particular ways in which they are constructed based on the average viral burden of populations who occupy them and the absolute level of the virus that circulates among them . these areas are seen in this imagination as particularly vulnerable and in need of attention ( brown and knopp 2010 , p. 394 ) . based on the work of huxley ( 2006 ) , we contend that the production of these spaces follows both dispositional and generational rationalities . by this , we mean that community viral load aims at drawing boundaries and mapping areas where the virus is unsuppressed areas that are then identified as vulnerable and in need of attention . community viral load , in fact , draws a rather partial and incomplete portrait of the hiv epidemic and continues to ignore the effect of context on hiv vulnerabilities -how the virus moves within a population and how it circulates across specific networks ( brown 1995 ) . with this in mind , it is important to recognize that community viral load is increasingly being used as a tool to signal the existence of particular kinds of spaces , locate these spaces geographically , and allows for public health authorities to become more knowledgeable about the populations who occupy them . this knowledge generates new possibilities for the government of certain bodies which are seen as not surprisingly , it has led to the deployment of more aggressive interventions to lower the collective risk of acquiring or transmitting hiv , with little further consideration of the realities of people living with hiv on the ground , i.e. expanding on the work of michel foucault , miller and rose ( 2008 ) explain that the birth of the community marks a departure from governing a supposed collective social body to defining a new territory for the administration of collective life . first , it calls on a particular re - configuration of space from a single , collective space to discrete communities which can be located geographically or constructed virtually ( i.e. here , the sense of community is created and promoted through the work of educators , campaigns , activists , manipulators of symbols , narratives and identifications ( miller and rose 2008 , p. 92 ) . those who do not exercise caution and live according to this moral code , however , are generally found in the margins of communities and their particular difficulties thus need to be addressed through the activities of various specialists each of whom is an expert in a particular problem ( miller and rose 2008 , p. 104 ) . these relations of expertise , explain miller and rose ( 2008 ) , operate at the level of the community . seek , test and treat initiatives which have recently been implemented in both canada and the united states , including community as a unit of identity , responsibilization and autonomization of people who reside in certain neighbourhoods , empowerment of individuals located in the margins , and expert management and outreach in risk communities. despite a seemingly emancipatory or rights - based agenda and the will to empower individuals who have been traditionally labelled as hard to reach ( patton 2011 ) , the deployment of these initiatives is indicative of the very form of government that miller and rose ( 2008 ) describe : the subject constituted through techniques of empowerment is being shaped into a better functioning and more responsible neoliberal actor empowered to make better decisions , able to fulfill moral obligations , and capable of committing to the health of his community . the experts are working directly in the community , relaying messages to individuals who have to be made aware of their personal allegiances to particular the community , as imagined through community viral load and the distribution of bodies in space , is now something to be programmed , researched and managed . as mentioned earlier , community viral load is part of a larger public health programme to promote high impact prevention using combinations of scientifically proven , cost - effective , and scalable interventions targeted to the right populations in the right geographic areas ( centers for disease control and prevention 2011 , p. 1 ) ( italics added for emphasis ) . within this new prevention framework , public health agencies are asked to identify communities where concentrations of the virus overlap with concentrations of substance users , gay and other men of have sex with men , transgender persons and racialized persons . community viral load provides a new tool for public health authorities to imagine particular communities ( legg 2005 ) in ways that are flexible , fluid , and can be adapted to different needs ( brown and knopp 2006 ) . this is part of the inner workings of community viral load ; it generates new knowledge , maps the presence of the virus collectively , makes room for a new form of surveillance , and in the end allows for the creation of specific targets , which are clustered around predominantly impoverished and marginalized neighbourhoods . we are particularly interested here in how community viral load creates or builds on existing personal allegiances and specific ties between individuals and communities to support the implementation of seek , test and treat initiatives ( miller and rose 2008 ) . not only is community viral load a potent tool to re - configure space ( as previously discussed ) and justify the need for public health to intervene more aggressively in certain neighbourhoods and locations , but it is also used as a device of identification . on the other hand , it reinforces the personal allegiances that these individuals may have to this community based on their serological status , their viral load , or the fact that they share a common fate as residents in neighbourhoods and locations where hiv is these individuals may , in turn , become actively involved in the deployment of community - level initiatives and engaged in outreach efforts to scale up hiv prevention . in fact , these initiatives rely on a re - configuration of space and re - location of interventions traditionally done in clinical settings ; testing is now conducted in vans , shelters , community centres , gay bathhouses and drop - in services , results are communicated right away , and linkage to care is automatically provided , surveillance data are broadened and include geospatial analysis , individual viral load , census information and indicators of socioeconomic status , and treatment is initiated rapidly after diagnosis followed by ongoing follow - up and adherence counselling in the community . in this article , we have acknowledged that this new framework is closely tied to the introduction of community viral and the need for more effective ways to govern bodies that are seen as contributing to the spread of hiv . therefore , in keeping with foucault 's ( 2003 ) work , we consider that community viral load has both individualizing and massifying effects . this has important implications for people living with hiv / aids , and may have broader implications for everyone who is being forced into these particular geographical areas these new ghettos that are marked by hiv and are under the radar of public health authorities . the objective of this article has been to formulate a critique and propose a novel way of theorizing community viral load through a foucauldian analytics of governmentality with an attention to the role of space and community . these spaces then become key targets of public health intervention in the form of high impact treatment and prevention technologies that necessitate an unprecedented investment in identifying individuals in need of treatment ( nguyen et al . once on treatment , individuals become part of the community viral load loop their individual viral load becomes used to assess their we have also shown that the term community and its uses represents a discursive terrain imbued with power and ideology and requires unpacking to surface how it is being used and to what ends ( lynn 2006 ) . with the invention and deployment of community viral load , we see a return to this previous logic , but resting on the seeming neutrality of space as if all people freely inhabit spaces of their own choosing . this return to previous ways of thinking about , and managing hiv , albeit through more sophisticated techniques than were available at the beginnings of the epidemic , raises important questions about how hiv is being seen by governments , policy makers and public health agencies . how do the changing politics of hiv and the growing interest in the securitization of health and illness help inform our thinking about mapping community viral load what are the implications of these clinical advancements for other health issues , and the potential outcomes to those who reside in spaces characterized by high rates of hiv and other forms of chronic illness .

the poor access to water supply is a prevalent issue in over 850 million people worldwide with over 2.5 billion limited by access to sanitation facilities . the global burden of disease and mortality rates could be reduced by about 9.1% and 6.3% , respectively , if rapid success is attained in facilitating access to water , sanitation , and hygiene facilities . a large proportion of these diseases are related to diarrhea incidences which contribute to the mortality rate of about 1.9 million and new diarrhea cases estimated at 4 billion annually especially among children under five years old . the world health statistics review done in 2009 showed that the highest case fatality rates due to diarrheal incidences occurred in india with over 386,000 diarrheal deaths . high mortality rates of about 13.9% are still attributed to diarrheal deaths in egypt among children less than five years old irrespective of the recent reduction in child mortality rates . the leading cause of infant mortality and health - related expenditures has been attributed to diarrheal incidences among children in indonesia . diarrheal diseases are also the third cause responsible for increased morbidity rates in all age groups in indonesia . previous literature has shown considerable studies regarding the effects of lack of appropriate water facilities , hand washing , and hygiene practices on child health outcomes . impaired cognitive learning and learning performance are long - term outcomes of the negative effects of infections such as diarrhea , worm infestations , and dehydrations which are largely attributed to poor water , sanitation , and hygiene conditions . diarrheal incidences in children during their first few years of life have been shown to limit their growth by about 8 cm and cause an iq point reduction when they progress to about 7 or 8 years of age . information regarding absenteeism from middle and higher income countries has shown that poor academic and social development , high dropout rates , and reduced learning performance are attributed to school absence in children [ 811 ] . absenteeism due to illness has been shown to be reduced by implementation of mandatory hand hygiene and sanitary procedures based on the results of previous interventions . the availability and utilization of alcohol - based sanitizers in schools have also been shown to reduce absenteeism by about 2050% [ 1417 ] . a hand hygiene intervention in two public elementary schools in chicago involving instructions in hand hygiene practices and provision of hand hygiene facilities significantly reduced absenteeism among students in prekindergarten to the eighth grade ( ages 414 ) . there have been considerable studies that have examined the effect of water treatment , hygiene , and sanitary practices on reducing absenteeism , diarrhea prevalence , and acute respiratory infections in school - age children . however , limited research has been done to evaluate the effectiveness of water , sanitation , and hygiene practices through randomized controlled clinical trials to gauge the long - term impact of these interventions on improving child health outcomes . the objective of the study is to examine and describe the gaps in the existing water , sanitation , and hygiene interventions to improve child health outcomes such as acute respiratory infections , diarrheal episodes , and absenteeism in various settings . a search was conducted in january 2013 using the scientific databases pubmed and google scholar for studies published between 2009 and 2012 and focusing on the effects of access to safe water , hand washing facilities , and hygiene education among school - age children . water access and waterborne illnesses , school enrollment and hygiene education , hand washing facilities and absenteeism , and hygiene education and children . studies included were those that documented the provision of water and sanitation in schools for children less than 18 years of age , interventions which assessed the impact of wash practices , and english - language , full - text peer reviewed papers . studies which did not have a school - based component in assessing wash practices were excluded . a secondary search was also done to review the references of the articles included in the final analysis . this was reduced to 15 studies after the exclusion criteria were applied and duplicates removed ( figure 1 ) . 40% ( n = 6 ) of the studies were published in 2011 and 33% ( n = 5 ) in 2012 . 73% ( n = 11 ) of the studies were conducted in developing countries including india , kenya , and egypt and were rural based ( 53% , n = 8) . 60% ( n = 9 ) of the studies incorporated an educational component in the interventions assessing the impact of water treatment and sanitation hygiene and this was shown to facilitate the success of those studies . the outcomes assessed were reducing illness - related absenteeism , gastro - intestinal , and respiratory infections and adoption of point - of - use water treatment in children . hygiene and sanitation interventions have had considerable impact on reducing diarrhea and absenteeism rates in school - age children . age / grade level : comparisons of the effectiveness of wash interventions were reported for some of the studies stratified by various age categories and measured in years.gender : the gender of the children in the various studies was noted to observe if there were any differences in the effectiveness of the interventions.socioeconomic status / household income : information on socioeconomic status was abstracted from the articles to observe its significance towards promoting access to water facilities and improving hygiene practices in children.parental literacy : it was assessed to monitor the effect of role - modeling behaviors in children towards imbibing knowledge , attitudes , and practices of sanitation and hygiene . age / grade level : comparisons of the effectiveness of wash interventions were reported for some of the studies stratified by various age categories and measured in years . gender : the gender of the children in the various studies was noted to observe if there were any differences in the effectiveness of the interventions . socioeconomic status / household income : information on socioeconomic status was abstracted from the articles to observe its significance towards promoting access to water facilities and improving hygiene practices in children . parental literacy : it was assessed to monitor the effect of role - modeling behaviors in children towards imbibing knowledge , attitudes , and practices of sanitation and hygiene . study design : information was gathered to determine the types of studies that were performed including randomized controlled trials , cross - sectional studies , cohort studies , and case series.study location : information was gathered about the locations where the studies were conducted.study duration : the period of the study was also recorded.sample size of the populations : it was also recorded . study design : information was gathered to determine the types of studies that were performed including randomized controlled trials , cross - sectional studies , cohort studies , and case series . knowledge : knowledge , as an educational component of the various studies reviewed , was assessed to observe their effects on sustained impact of the interventions . comparisons were also made to interventions that did not utilize this component to weigh its effectiveness . knowledge : knowledge , as an educational component of the various studies reviewed , was assessed to observe their effects on sustained impact of the interventions . comparisons were also made to interventions that did not utilize this component to weigh its effectiveness . the commonly studied age groups of children studied fell between 5 years and 16 years ( 53% , n = 8) . 13% ( n = 2 ) of the studies focused on children less than 5 years while 26% ( n = 4 ) of the studies did not specify the age groups of the children . 40% ( n = 2 ) of the studies were conducted in africa ( figure 2 ) . others were conducted in asia ( 33% , n = 5 ) , north america ( 20% , n = 3 ) , and europe ( 6% , n = 1 ) . more than half ( 53% , n = 8) of the studies were randomized clinical trials while 47% ( n = 7 ) were cross - sectional studies . 100% ( n = 15 ) of the studies reviewed were school based , conducted in primary / middle / elementary schools , with one study incorporating a community component . more than half of the studies were done in rural settings ( 53% , n = 8) . 33.3% ( n = 2 ) of the studies done in africa were urban based , 80% ( n = 4 ) of the studies done in asia were rural based , and 100% ( n = 3 ) of the studies done in north america were urban based . 53% ( n = 8) of the studies had sample sizes ranging between 500 and 1000 . the data collection process was carried out using surveys at several levels including students , parents , teachers , health workers , social workers , or a combination of them ( figure 3 ) . several variable categories were gathered based on the literature review of the articles included in the final analysis . 80% ( n = 12 ) of the studies reviewed assessed the ages of the children in relation to the outcomes . 53% ( n = 8) of the studies included children with age groups between 5 years and 16 years . 13% ( n = 2 ) of the studies reviewed involved children less than 5 years of age , while 26.6% ( n = 4 ) of the studies did not specify the ages of the children . 27% ( n = 3 ) of the studies which assessed age found a significant association with the outcomes . results from a study that examined the prevalence of helminthic infections in children showed that the age of children was a significant factor in the risk of helminthic and protozoan infections . older children aged 8 and 10 years were less prone to infections by helminthes and intestinal protozoa , unlike the younger children between 7 and 8 years who had very high infection rates . however , in another study , the infection rates were significantly associated with older children between 810 years old . the ages of the children were also significantly associated with latrine use especially in the 810 age groups . 86% ( n = 13 ) of the studies assessed the school grade levels of the children . majority of the children studied fell between grades ranging from pre - kindergarten to the eighth grade ( 86% , n = 13 ) . 13.3% ( n = 2 ) of the studies did not specify the grades of the children . the association between school grades and the outcomes was significant in 23% ( n = 3 ) of the studies . the child health outcomes reviewed were more significantly associated with children in higher grade levels . the gender of the participating children was described in majority of the studies reviewed ( n = 12 , 80% ) . 20% ( n = 3 ) of the studies found a significant association between gender and the outcomes . two of those studies were randomized control trials assessing the impact of water treatment and sanitation hygiene ( wash ) on reducing absenteeism and diarrhea / ari prevalence and 1 was a cross - sectional study . in the randomized controlled trials , absenteeism reduction was significant in girls [ 1 , 20 ] , but in the cross - sectional study , the impact was associated with the male gender . 66% of the studies assessed parental literacy ( n = 10 ) and 50% of those collected data specifically on maternal education ( n = 5 ) . maternal literacy was found to be insignificant in one of the studies involving the factors associated with the use of improved water sources and sanitation among children in cambodia and its effect was not specified in others studies [ 18 , 2124 ] . the significance of parental literacy on the outcomes assessed was not stated in 5 of the studies reviewed [ 18 , 20 , 21 , 24 , 25 ] . the socioeconomic index was described in the studies reviewed to include wealth index , wealth , household income , household assets , and housing type . 60% of the studies analyzed the association between socioeconomic index and the outcomes ( n = 9 ) , but its significance was only stated in 3 studies [ 18 , 20 , 21 ] . higher socioeconomic status ( defined by housing type ) was a key independent factor associated with the use of improved water sources and sanitation . results from a study showed that children belonging to households of the middle 40% which were on a higher scale had a reduced risk of being infected compared to their poorer counterparts ( or = 0.58 , 95% ci : 0.380.90 ) . individuals with higher socioeconomic index generally reflected the positive outcomes of the health interventions compared to those with a lower index . the socioeconomic index was described in the studies reviewed to include wealth index , wealth , household income , household assets , and housing type . 60% of the studies analyzed the association between socioeconomic index and the outcomes ( n = 9 ) , but its significance was only stated in 3 studies [ 18 , 20 , 21 ] . higher socioeconomic status ( defined by housing type ) was a key independent factor associated with the use of improved water sources and sanitation . results from a study showed that children belonging to households of the middle 40% which were on a higher scale had a reduced risk of being infected compared to their poorer counterparts ( or = 0.58 , 95% ci : 0.380.90 ) . individuals with higher socioeconomic index generally reflected the positive outcomes of the health interventions compared to those with a lower index . n = 4 ) and the number of female - headed households ( 20% , n = 3 ) were the most common household variables assessed . 13.3% ( n = 2 ) of the articles showed a significant association between female - headed households and the outcomes while the remaining article did not specify its significance . 25% ( n = 1 ) of the studies that assessed family size showed a significant association with the outcomes . the other variables assessed included distance to school from home , distance to water source , climatic season , number of people sharing bedroom / toilet with child , pupil to latrine ratio , water quantity used , and crowding index . data on other household variables than the listed above were rarely collected in all the studies reviewed . n = 4 ) and the number of female - headed households ( 20% , n = 3 ) were the most common household variables assessed . 13.3% ( n = 2 ) of the articles showed a significant association between female - headed households and the outcomes while the remaining article did not specify its significance . 25% ( n = 1 ) of the studies that assessed family size showed a significant association with the outcomes . the other variables assessed included distance to school from home , distance to water source , climatic season , number of people sharing bedroom / toilet with child , pupil to latrine ratio , water quantity used , and crowding index . data on other household variables than the listed above were rarely collected in all the studies reviewed . latrine coverage in schools was the most common variable assessed ( 60% , n = 9 ) followed by school area / location and water source at school ( 13.3% , n = 2 ) each . information gathered on school characteristics included school area / location , latrine coverage , pupil - latrine ratio , water source at school , hand washing soap on basin , means of waste disposal , and washup point after toilet . 44.4% ( n = 4 ) of the studies that assessed latrine coverage showed a significant association with the outcomes while 50% ( n = 2 ) of the studies that assessed school area ( highlands versus lowlands ) and water sources found a significant association with the outcomes . the prevalence of diarrheal infections was lowest in children from households located in mountainous regions . in rural cambodian school children , diarrheal incidences were largely reduced in households which utilized improved water sources and sanitation facilities . latrine coverage in schools was the most common variable assessed ( 60% , n = 9 ) followed by school area / location and water source at school ( 13.3% , n information gathered on school characteristics included school area / location , latrine coverage , pupil - latrine ratio , water source at school , hand washing soap on basin , means of waste disposal , and washup point after toilet . 44.4% ( n = 4 ) of the studies that assessed latrine coverage showed a significant association with the outcomes while 50% ( n = 2 ) of the studies that assessed school area ( highlands versus lowlands ) and water sources found a significant association with the outcomes . the prevalence of diarrheal infections was lowest in children from households located in mountainous regions . in rural cambodian school children , diarrheal incidences were largely reduced in households which utilized improved water sources and sanitation facilities . the major data on environmental characteristics collected in most studies were those regarding drinking water sources in households ( 46.6% , n = 7 ) and obtaining water samples ( 26.6% , n = 4 ) . 42.9% ( n = 3 ) of the studies that assessed drinking water sources found a significant association with the outcome . improved water sources including public wells and stand pipes were significantly associated with positive health outcomes . chlorine treatment was not significantly associated with improved child health outcomes in a randomized controlled trial . other data assessed included water source contamination / water quality and water access ( availability ) . the major data on environmental characteristics collected in most studies were those regarding drinking water sources in households ( 46.6% , n = 7 ) and obtaining water samples ( 26.6% , n = 4 ) . 42.9% ( n = 3 ) of the studies that assessed drinking water sources found a significant association with the outcome . improved water sources including public wells and stand pipes were significantly associated with positive health outcomes . chlorine treatment was not significantly associated with improved child health outcomes in a randomized controlled trial . other data assessed included water source contamination / water quality and water access ( availability ) . 20% ( n = 3 ) of the studies assessed the association between nutrition practices and the outcomes . the variables collected included : food handling food preparation , food consumption , acute malnutrition , breast feeding , and meat consumption . 20% ( n = 3 ) of the studies assessed the association between nutrition practices and the outcomes . the variables collected included : food handling food preparation , food consumption , acute malnutrition , breast feeding , and meat consumption . other data collected in the studies reviewed included nasal swab samples ( for influenza testing ) , age of respondents , crowding index , sewage spillage , and contaminated fomites . the most commonly assessed variables in the studies reviewed include hand washing ( 66.6% , n = 10 ) , latrine coverage ( 60% , n = 9 ) , sanitation practices ( 53.3% , n = 8) , prior knowledge , and use of soap ( 46.6% , n = 7 ) . majority of the studies showed a significant association between these variables and the outcomes ( see table 1 ) . a reduced risk of parasitic infections was observed in children with substantial knowledge regarding hygiene and sanitation practices ( aor 0.78 , ci 0.561.09 ) . their knowledge was also reflected in their clean clothing ( aor 1.62 , ci 1.142.29 ) . there was only one study that examined the impact of hygiene practices on absenteeism due to infectious illness among pupils in elementary schools . the students at the intervention school were required to wash their hands before the first lesson , before lunch , and before going home while those at the control school continued their usual hand washing practices . the rates of absenteeism for the students in the intervention school were significantly reduced compared with those in the control school ( p = 0.002 ) . the effects of reduced absenteeism were more prominent on female students ( 1.05 , 95% ci 0.901.22 ) compared to their male counterparts ( 0.87 , 95% ci = 0.721.05 ) . there were 4 studies that examined the impact of the combination of hand hygiene instructions and provision of hygiene facilities . they included both us- and non - us - based studies conducted in texas , chicago , egypt , and pittsburgh . the study conducted in north texas was geared towards controlling a shigella outbreak in an elementary and middle school . installing liquid soap in dispensers in student restrooms followed by sustained instruction in hand washing and monitoring of hygiene practices among students the results showed that appropriate soap supplies and repeated instruction in hand washing and its monitoring were needed to control the shigella outbreak . the study conducted in chicago assessed the role of hand hygiene instruction in decreasing illness - related absenteeism in two public elementary schools during the peak flu season . students in the intervention group also received short repetitive instruction in hand hygiene every two months . the results of the study showed higher rates of attendance among students that received hand hygiene instruction during the flu season ( p = 0.002 , p < 0.001 , resp . ) . the study conducted in egypt assessed the effects of hand hygiene campaigns on the incidence of laboratory - confirmed influenza and absenteeism in school children . children in the intervention schools were required to wash hands twice daily and health messages were provided through entertainment activities . an intensive campaign to promote hand hygiene was launched in the intervention schools to raise the awareness of students , teachers , nurses , and parents ; it required students to wash their hands at least twice during the school day for 45 seconds , followed by proper rinsing and drying with a clean cloth towel . the results showed a significant reduction in illness - related absenteeism , infections rates including diarrhea , conjunctivitis , and influenza by the following frequencies : 40% , 30% , 67% , and 50% , respectively ( p < 0.0001 ) . in another randomized controlled trial , children in 5 intervention schools received training about hand and respiratory hygiene and were provided and encouraged to use hand sanitizer regularly while the children in the other 5 schools acted as controls . prior to implementation of the interventions , there was a 45-minute presentation at intervention schools regarding influenza and proper hand washing technique and sanitizer use . hand sanitizer dispensers with 62% alcohol - based hand sanitizer were installed in each classroom and all major common areas of intervention schools . the children were required to utilize it several times daily including upon arrival , before and after lunch , and prior to departure was taught to students . they were also encouraged to wash hands or use additional doses of hand sanitizer as needed . the total absence episodes were significantly reduced among children in the intervention group compared to the controls , adjusted irr 0.74 ( 95% ci : 0.56 , 0.97 ) . there were 3 randomized controlled trials that utilized a combination of water treatment and hygiene practices and education ( kenya , n = 2 ; india , n = 1 ) [ 21 , 28 , 29 ] . one of the prior studies done in kenya assessed the impact of a hygiene curriculum and the installation of hand washing and drinking stations towards reducing diarrhea and acute respiratory infections in students . hygiene education was provided through the provision of instructional materials and training for teachers and students while water stations installed were strategically located near latrines for hand washing to improve hygiene practices . the results showed a decrease in the median percentage of students with acute respiratory illness but no decrease in diarrhea among students . in another study conducted in kenya , the role of school children in the promotion of point - of - use water treatment and hand washing in schools and their impact on reducing pupil absenteeism rates water stations which utilized a flocculent / disinfectant called pur were installed and teachers and students received training on hygiene as well as instructional books . student absenteeism rates decreased after implementation by 26% . in another study conducted in india , the impact of a school - based safe water intervention on household adoption of point - of - use water treatment practices was assessed . the intervention consisted of providing classrooms of 200 schools with a commercial water purifier , training of students and teachers , and provision of basic hygiene and water treatment information . there was no evidence that the intervention was effective in improving uptake of water treatment practices . only one study was conducted in kenya which utilized multiple intervention components involving water treatment , hygiene promotion , and sanitation to assess their impact on pupil absence . schools were randomized to one of three study arms including water treatment and hygiene promotion ( wt and hp ) , water treatment , hygiene promotion , and sanitation ( wt , hp , and sanitation ) , and a control group . the intervention provided water guard ( a 1.2% chlorine based point - of - use water disinfectant for water treatment ) . hygiene promotion incorporated an education component which involved a 3-day training of teachers as well as provision of hand - washing and drinking water containers while the sanitation component involved the provision of latrine facilities to the students . the addition of a sanitation component as an intervention arm resulted in a marginally significant reduction in absenteeism among the students ( or 0.47 , 0.211.05 ) . the studies reviewed assessed 4 major outcomes of the effect of water and sanitation hygiene practices in children . these included absenteeism , infections ( diarrhea / acute respiratory ) , knowledge / attitudes / practices , and adoption of point of use water treatment ( table 2 ) . information regarding absenteeism was defined and collected differently in the various studies reviewed [ 13 , 19 , 22 , 27 , 30 ] . absence periods were defined as number of days absent due to a single cause , with at most 2 days of attendance or a weekend between events and was recorded based on the number of absences due to infectious causes as reported by pupils , teachers and parents , or number of absences caused by illness per 100 student - weeks and was recorded based on the number of absences due to illness as reported by parents and school records , or measured using number of absences due to illness and non - illness from both parents and teachers . one of the studies did not define absence days but absence records were ascertained from schools . illness - related absenteeism children constitutes about 75% of all school absences and is largely attributed to respiratory and gastro - intestinal infections . incorporating an educational component in the interventions access to hand hygiene instruction and hygiene facilities improved attendance at public elementary schools during the flu season . of all the studies that were geared towards reducing absenteeism , gender was significant in 33% ( n = 2 ) of the studies . the benefits of hand washing were more pronounced in females with the highest rates of absenteeism [ 10 , 13 ] . some of the limitations of the existing studies were self - reporting with regard to illness incidences and compliance . others include small sample sizes , and loss to followup . the variables assessed regarding knowledge , attitudes , and practices included knowledge of prior water treatment , use of soap / sanitizer ( 46% , n = 7 ) , latrine coverage in household and community ( 60% , n = 9 ) , hand washing frequency ( 80% , n = 12 ) , sanitation practice ( latrine use , 53% , n = 8) , household water treatment , water storage practices ( 20% , n = 3 ) , and prior knowledge of hygiene practices ( 20% , n = 3 ) . only 5 studies gathered information about the knowledge , attitudes , and practices of school children in relation to infections ( diarrhea , intestinal protozoa , schistosomiasis ) and the use of improved water sources . data regarding drying material availability , hygiene practices ( taking bath , brushing teeth , and washing hair and feet ) , sewage spillage , and vaccination were sparsely collected in the studied reviewed . 46% ( n = 7 ) of the studies in this review assessed water access , sanitation , and hygiene practices in relation to diarrhea prevalence and other infectious disease incidences among school - age children . the risk factors for diarrhea identified included ; a lack of education on hygiene practices , age of child , area of residence , maternal education , and source of water , toilet facility , disposal of waste and multiple children aged less than 5 yrs residing together . 40% ( n = 6 ) of the studies utilized education as a key component of the interventions and it was found to be significant in 4 of those studies . a key factor which determined the child 's access to safe water sources and improved sanitation and hygiene infrastructure was socioeconomic status . individuals with higher socioeconomic index especially in the middle 40% generally reflected the positive outcomes of the health interventions compared to those with a lower index . the incidence of respiratory infections was also significantly associated with independent variables including child 's age , gender , and family wealth . the studies were conducted in india , ( n = 1 ) and kenya , ( n = 1 ) . the study conducted in india assessed the impact of a school - based safe water intervention on household adoption of point - of - use water treatment practices while the study conducted in kenya evaluated the role of school children in the promotion of point - of - use water treatment and hand washing in households as well as its effect on students absence . they were both randomized controlled trials and utilized combined interventions including water treatment , hygiene practices , and hygiene education . the study conducted in india did not show statistically significant results while that conducted in kenya showed improved knowledge of water treatment ( 4991% , p < 0.0001 ) and this effect was retained even after a followup period of 13 months ( p = 0.53 ) . results of our systematic review yielded fifteen studies which assessed the impact of wash practices to improve the knowledge , attitudes , practices , and child health outcomes . these health outcomes include absenteeism , diarrhea , acute respiratory infections , and adoption of point of use water treatment . more than half of the studies ( 53% , n = 8) were randomized controlled trials , followed by 40% ( n = 6 ) cross - sectional studies and 6% ( n = 1 ) being a case series study . 60% ( n = 9 ) of the studies were interventions that involved water treatment , hygiene practices , hygiene education , and sanitation in single form or in combination . various data collected included socio - demographics , household and school characteristics , and environmental factors . higher rates of infection by helminthes and protozoa were more prevalent in the younger age group consisting of children aged 7 - 8 years old compared to the older children aged 8 to 10 years . negative child health outcomes were more common in children in lower grade levels , specifically grade 3 . the impact of the wash interventions differed with respect to gender and socioeconomic indices with children in higher socioeconomic levels showing better outcomes . 44.4% ( n = 4 ) of the studies that assessed latrine coverage showed significant association with the outcomes while 50% ( n = 2 ) of the studies that assessed school area ( highlands versus lowlands ) and water sources found a significant association with the outcomes . the structure , cleanliness , and general outlook of latrines were implicated in the rate of utilization of sanitary facilities by the school children , with children being more likely to utilize well - kept sanitary facilities or outside sources . the most commonly assessed variables with highest significance were latrine coverage and hand washing practices while the least commonly assessed were school characteristics including pupil - latrine ratio , availability of soaps in toilets , water source contamination , water availability , drying material availability , and several household variables including number of people sharing bedroom or toilet with child . this review showed that several independent variables were implicated in the adoption of wash practices and found to be significantly associated with the outcomes . they include the age of the child , gender , grade level , socioeconomic index , access to hygiene and sanitary facilities , and prior knowledge of hygiene practices . all the studies done in developing countries reported a positive effect of sanitation practices on reducing diarrhea prevalence [ 19 , 22 , 28 ] . most of the studies involved children within grade level 3 , who were at high risk for gastrointestinal and acute respiratory infections which could have been a source of bias . studies have shown that children in grade 3 are at high risk of being infected with schistosomiasis in communities with high prevalence of this disease . some of the limitations observed in the various studies in this group included self - reporting of illness incidences leading to misclassification , underrepresented sample sizes , recall bias by parents in diarrheal incidences of their children , restricting studies to a particular age group , gender restriction , and the use of cross - sectional study designs . in the study done in pittsburg , reason for absence in students could only be ascertained in 34% of absences ; this is largely attributed to the inability to contact the parent or guardian . other limitations included a confounding of the impact of interventions due to similar ongoing interventions making it difficult to evaluate the outcomes , restriction of the study to a particular gender , and study sample restriction to only children present at the start of the school term leading to a loss of generalizability [ 13 , 21 , 27 , 30 ] . this review identified a gap in assessment of nutrition practices which is a key factor related to the various outcomes studied especially diarrheal infections and should therefore be given more attention in future research . the studies assessed the health and educational effects of wash practices in schools on reducing absenteeism and diarrhea prevalence / infections among school - age children on a short term . however , there have been little or no empirical studies which examined the long term impact of wash interventions on child health outcomes , and therefore limited data to support future interventions . this was noted as a limitation in various studies showing a high loss to followup , where followup was present . the positive effect of an education component in the intervention on the uptake and adherence to hygiene practices should be noted in future research . knowledge was implicated in several studies in this review as a facilitator in the uptake of hygiene practices and interventions [ 5 , 21 ] . several key independent variables including age of the child , gender , grade level , socioeconomic index , access to hygiene and sanitary facilities use and prior knowledge of hygiene practices which were significantly associated with child health outcomes should be noted and controlled for in future interventions . the review concluded that the importance of access to safe water , hand washing facilities , and hygiene education can not be underscored in abating water - borne illnesses , malnutrition , school absenteeism , and generally improving the quality of life and learning performance in children .

purpose . this review was done to explore the impact of water treatment , hygiene , and sanitary interventions on improving child health outcomes such as absenteeism , infections , knowledge , attitudes , and practices and adoption of point - of - use water treatment . methods . a literature search was conducted using the databases pubmed and google scholar for studies published between 2009 and 2012 and focusing on the effects of access to safe water , hand washing facilities , and hygiene education among school - age children . studies included were those that documented the provision of water and sanitation in schools for children less than 18 years of age , interventions which assessed wash practices , and english - language , full - text peer reviewed papers . results . fifteen studies were included in the final analysis . 73% ( n = 11 ) of the studies were conducted in developing countries and were rural based ( 53% , n = 8) . the child 's age , gender , grade level , socioeconomic index , access to hygiene and sanitary facilities , and prior knowledge of hygiene practices were significantly associated with the outcomes . nutrition practices which are key factors associated with the outcomes were rarely assessed . conclusion . further research is required to assess the long - term impact of such interventions in different settings .

1. Introduction 2. Methods 3. Results 4. Variable Extraction 5. Interventions 6. Results 7. Discussion 8. Conclusion

the global burden of disease and mortality rates could be reduced by about 9.1% and 6.3% , respectively , if rapid success is attained in facilitating access to water , sanitation , and hygiene facilities . previous literature has shown considerable studies regarding the effects of lack of appropriate water facilities , hand washing , and hygiene practices on child health outcomes . impaired cognitive learning and learning performance are long - term outcomes of the negative effects of infections such as diarrhea , worm infestations , and dehydrations which are largely attributed to poor water , sanitation , and hygiene conditions . there have been considerable studies that have examined the effect of water treatment , hygiene , and sanitary practices on reducing absenteeism , diarrhea prevalence , and acute respiratory infections in school - age children . however , limited research has been done to evaluate the effectiveness of water , sanitation , and hygiene practices through randomized controlled clinical trials to gauge the long - term impact of these interventions on improving child health outcomes . the objective of the study is to examine and describe the gaps in the existing water , sanitation , and hygiene interventions to improve child health outcomes such as acute respiratory infections , diarrheal episodes , and absenteeism in various settings . a search was conducted in january 2013 using the scientific databases pubmed and google scholar for studies published between 2009 and 2012 and focusing on the effects of access to safe water , hand washing facilities , and hygiene education among school - age children . water access and waterborne illnesses , school enrollment and hygiene education , hand washing facilities and absenteeism , and hygiene education and children . studies included were those that documented the provision of water and sanitation in schools for children less than 18 years of age , interventions which assessed the impact of wash practices , and english - language , full - text peer reviewed papers . a secondary search was also done to review the references of the articles included in the final analysis . 40% ( n = 6 ) of the studies were published in 2011 and 33% ( n = 5 ) in 2012 . 73% ( n = 11 ) of the studies were conducted in developing countries including india , kenya , and egypt and were rural based ( 53% , n = 8) . 60% ( n = 9 ) of the studies incorporated an educational component in the interventions assessing the impact of water treatment and sanitation hygiene and this was shown to facilitate the success of those studies . the outcomes assessed were reducing illness - related absenteeism , gastro - intestinal , and respiratory infections and adoption of point - of - use water treatment in children . hygiene and sanitation interventions have had considerable impact on reducing diarrhea and absenteeism rates in school - age children . age / grade level : comparisons of the effectiveness of wash interventions were reported for some of the studies stratified by various age categories and measured in years.gender : the gender of the children in the various studies was noted to observe if there were any differences in the effectiveness of the interventions.socioeconomic status / household income : information on socioeconomic status was abstracted from the articles to observe its significance towards promoting access to water facilities and improving hygiene practices in children.parental literacy : it was assessed to monitor the effect of role - modeling behaviors in children towards imbibing knowledge , attitudes , and practices of sanitation and hygiene . parental literacy : it was assessed to monitor the effect of role - modeling behaviors in children towards imbibing knowledge , attitudes , and practices of sanitation and hygiene . study design : information was gathered to determine the types of studies that were performed including randomized controlled trials , cross - sectional studies , cohort studies , and case series.study location : information was gathered about the locations where the studies were conducted.study duration : the period of the study was also recorded.sample size of the populations : it was also recorded . the commonly studied age groups of children studied fell between 5 years and 16 years ( 53% , n = 8) . 13% ( n = 2 ) of the studies focused on children less than 5 years while 26% ( n = 4 ) of the studies did not specify the age groups of the children . 40% ( n = 2 ) of the studies were conducted in africa ( figure 2 ) . others were conducted in asia ( 33% , n = 5 ) , north america ( 20% , n = 3 ) , and europe ( 6% , n = 1 ) . more than half ( 53% , n = 8) of the studies were randomized clinical trials while 47% ( n = 7 ) were cross - sectional studies . 100% ( n = 15 ) of the studies reviewed were school based , conducted in primary / middle / elementary schools , with one study incorporating a community component . more than half of the studies were done in rural settings ( 53% , n = 8) . 33.3% ( n = 2 ) of the studies done in africa were urban based , 80% ( n = 4 ) of the studies done in asia were rural based , and 100% ( n = 3 ) of the studies done in north america were urban based . 53% ( n = 8) of the studies had sample sizes ranging between 500 and 1000 . several variable categories were gathered based on the literature review of the articles included in the final analysis . 80% ( n = 12 ) of the studies reviewed assessed the ages of the children in relation to the outcomes . 53% ( n = 8) of the studies included children with age groups between 5 years and 16 years . 13% ( n = 2 ) of the studies reviewed involved children less than 5 years of age , while 26.6% ( n = 4 ) of the studies did not specify the ages of the children . 27% ( n = 3 ) of the studies which assessed age found a significant association with the outcomes . however , in another study , the infection rates were significantly associated with older children between 810 years old . the ages of the children were also significantly associated with latrine use especially in the 810 age groups . 86% ( n = 13 ) of the studies assessed the school grade levels of the children . 13.3% ( n = 2 ) of the studies did not specify the grades of the children . the association between school grades and the outcomes was significant in 23% ( n = 3 ) of the studies . the child health outcomes reviewed were more significantly associated with children in higher grade levels . the gender of the participating children was described in majority of the studies reviewed ( n = 12 , 80% ) . 20% ( n = 3 ) of the studies found a significant association between gender and the outcomes . two of those studies were randomized control trials assessing the impact of water treatment and sanitation hygiene ( wash ) on reducing absenteeism and diarrhea / ari prevalence and 1 was a cross - sectional study . in the randomized controlled trials , absenteeism reduction was significant in girls [ 1 , 20 ] , but in the cross - sectional study , the impact was associated with the male gender . 66% of the studies assessed parental literacy ( n = 10 ) and 50% of those collected data specifically on maternal education ( n = 5 ) . maternal literacy was found to be insignificant in one of the studies involving the factors associated with the use of improved water sources and sanitation among children in cambodia and its effect was not specified in others studies [ 18 , 2124 ] . the significance of parental literacy on the outcomes assessed was not stated in 5 of the studies reviewed [ 18 , 20 , 21 , 24 , 25 ] . the socioeconomic index was described in the studies reviewed to include wealth index , wealth , household income , household assets , and housing type . 60% of the studies analyzed the association between socioeconomic index and the outcomes ( n = 9 ) , but its significance was only stated in 3 studies [ 18 , 20 , 21 ] . higher socioeconomic status ( defined by housing type ) was a key independent factor associated with the use of improved water sources and sanitation . the socioeconomic index was described in the studies reviewed to include wealth index , wealth , household income , household assets , and housing type . 60% of the studies analyzed the association between socioeconomic index and the outcomes ( n = 9 ) , but its significance was only stated in 3 studies [ 18 , 20 , 21 ] . 13.3% ( n = 2 ) of the articles showed a significant association between female - headed households and the outcomes while the remaining article did not specify its significance . 25% ( n = 1 ) of the studies that assessed family size showed a significant association with the outcomes . 13.3% ( n = 2 ) of the articles showed a significant association between female - headed households and the outcomes while the remaining article did not specify its significance . 25% ( n = 1 ) of the studies that assessed family size showed a significant association with the outcomes . 44.4% ( n = 4 ) of the studies that assessed latrine coverage showed a significant association with the outcomes while 50% ( n = 2 ) of the studies that assessed school area ( highlands versus lowlands ) and water sources found a significant association with the outcomes . latrine coverage in schools was the most common variable assessed ( 60% , n = 9 ) followed by school area / location and water source at school ( 13.3% , n information gathered on school characteristics included school area / location , latrine coverage , pupil - latrine ratio , water source at school , hand washing soap on basin , means of waste disposal , and washup point after toilet . 44.4% ( n = 4 ) of the studies that assessed latrine coverage showed a significant association with the outcomes while 50% ( n = 2 ) of the studies that assessed school area ( highlands versus lowlands ) and water sources found a significant association with the outcomes . the major data on environmental characteristics collected in most studies were those regarding drinking water sources in households ( 46.6% , n = 7 ) and obtaining water samples ( 26.6% , n = 4 ) . 42.9% ( n = 3 ) of the studies that assessed drinking water sources found a significant association with the outcome . improved water sources including public wells and stand pipes were significantly associated with positive health outcomes . chlorine treatment was not significantly associated with improved child health outcomes in a randomized controlled trial . the major data on environmental characteristics collected in most studies were those regarding drinking water sources in households ( 46.6% , n = 7 ) and obtaining water samples ( 26.6% , n = 4 ) . 42.9% ( n = 3 ) of the studies that assessed drinking water sources found a significant association with the outcome . improved water sources including public wells and stand pipes were significantly associated with positive health outcomes . chlorine treatment was not significantly associated with improved child health outcomes in a randomized controlled trial . 20% ( n = 3 ) of the studies assessed the association between nutrition practices and the outcomes . 20% ( n = 3 ) of the studies assessed the association between nutrition practices and the outcomes . other data collected in the studies reviewed included nasal swab samples ( for influenza testing ) , age of respondents , crowding index , sewage spillage , and contaminated fomites . the most commonly assessed variables in the studies reviewed include hand washing ( 66.6% , n = 10 ) , latrine coverage ( 60% , n = 9 ) , sanitation practices ( 53.3% , n = 8) , prior knowledge , and use of soap ( 46.6% , n = 7 ) . majority of the studies showed a significant association between these variables and the outcomes ( see table 1 ) . there was only one study that examined the impact of hygiene practices on absenteeism due to infectious illness among pupils in elementary schools . there were 4 studies that examined the impact of the combination of hand hygiene instructions and provision of hygiene facilities . installing liquid soap in dispensers in student restrooms followed by sustained instruction in hand washing and monitoring of hygiene practices among students the results showed that appropriate soap supplies and repeated instruction in hand washing and its monitoring were needed to control the shigella outbreak . the study conducted in egypt assessed the effects of hand hygiene campaigns on the incidence of laboratory - confirmed influenza and absenteeism in school children . in another randomized controlled trial , children in 5 intervention schools received training about hand and respiratory hygiene and were provided and encouraged to use hand sanitizer regularly while the children in the other 5 schools acted as controls . there were 3 randomized controlled trials that utilized a combination of water treatment and hygiene practices and education ( kenya , n = 2 ; india , n = 1 ) [ 21 , 28 , 29 ] . one of the prior studies done in kenya assessed the impact of a hygiene curriculum and the installation of hand washing and drinking stations towards reducing diarrhea and acute respiratory infections in students . hygiene education was provided through the provision of instructional materials and training for teachers and students while water stations installed were strategically located near latrines for hand washing to improve hygiene practices . in another study conducted in kenya , the role of school children in the promotion of point - of - use water treatment and hand washing in schools and their impact on reducing pupil absenteeism rates water stations which utilized a flocculent / disinfectant called pur were installed and teachers and students received training on hygiene as well as instructional books . in another study conducted in india , the impact of a school - based safe water intervention on household adoption of point - of - use water treatment practices was assessed . the intervention consisted of providing classrooms of 200 schools with a commercial water purifier , training of students and teachers , and provision of basic hygiene and water treatment information . only one study was conducted in kenya which utilized multiple intervention components involving water treatment , hygiene promotion , and sanitation to assess their impact on pupil absence . schools were randomized to one of three study arms including water treatment and hygiene promotion ( wt and hp ) , water treatment , hygiene promotion , and sanitation ( wt , hp , and sanitation ) , and a control group . the intervention provided water guard ( a 1.2% chlorine based point - of - use water disinfectant for water treatment ) . the studies reviewed assessed 4 major outcomes of the effect of water and sanitation hygiene practices in children . these included absenteeism , infections ( diarrhea / acute respiratory ) , knowledge / attitudes / practices , and adoption of point of use water treatment ( table 2 ) . of all the studies that were geared towards reducing absenteeism , gender was significant in 33% ( n = 2 ) of the studies . the variables assessed regarding knowledge , attitudes , and practices included knowledge of prior water treatment , use of soap / sanitizer ( 46% , n = 7 ) , latrine coverage in household and community ( 60% , n = 9 ) , hand washing frequency ( 80% , n = 12 ) , sanitation practice ( latrine use , 53% , n = 8) , household water treatment , water storage practices ( 20% , n = 3 ) , and prior knowledge of hygiene practices ( 20% , n = 3 ) . only 5 studies gathered information about the knowledge , attitudes , and practices of school children in relation to infections ( diarrhea , intestinal protozoa , schistosomiasis ) and the use of improved water sources . data regarding drying material availability , hygiene practices ( taking bath , brushing teeth , and washing hair and feet ) , sewage spillage , and vaccination were sparsely collected in the studied reviewed . 46% ( n = 7 ) of the studies in this review assessed water access , sanitation , and hygiene practices in relation to diarrhea prevalence and other infectious disease incidences among school - age children . the risk factors for diarrhea identified included ; a lack of education on hygiene practices , age of child , area of residence , maternal education , and source of water , toilet facility , disposal of waste and multiple children aged less than 5 yrs residing together . 40% ( n = 6 ) of the studies utilized education as a key component of the interventions and it was found to be significant in 4 of those studies . a key factor which determined the child 's access to safe water sources and improved sanitation and hygiene infrastructure was socioeconomic status . individuals with higher socioeconomic index especially in the middle 40% generally reflected the positive outcomes of the health interventions compared to those with a lower index . the incidence of respiratory infections was also significantly associated with independent variables including child 's age , gender , and family wealth . the studies were conducted in india , ( n = 1 ) and kenya , ( n = 1 ) . the study conducted in india assessed the impact of a school - based safe water intervention on household adoption of point - of - use water treatment practices while the study conducted in kenya evaluated the role of school children in the promotion of point - of - use water treatment and hand washing in households as well as its effect on students absence . they were both randomized controlled trials and utilized combined interventions including water treatment , hygiene practices , and hygiene education . the study conducted in india did not show statistically significant results while that conducted in kenya showed improved knowledge of water treatment ( 4991% , p < 0.0001 ) and this effect was retained even after a followup period of 13 months ( p = 0.53 ) . results of our systematic review yielded fifteen studies which assessed the impact of wash practices to improve the knowledge , attitudes , practices , and child health outcomes . these health outcomes include absenteeism , diarrhea , acute respiratory infections , and adoption of point of use water treatment . more than half of the studies ( 53% , n = 8) were randomized controlled trials , followed by 40% ( n = 6 ) cross - sectional studies and 6% ( n = 1 ) being a case series study . 60% ( n = 9 ) of the studies were interventions that involved water treatment , hygiene practices , hygiene education , and sanitation in single form or in combination . 44.4% ( n = 4 ) of the studies that assessed latrine coverage showed significant association with the outcomes while 50% ( n = 2 ) of the studies that assessed school area ( highlands versus lowlands ) and water sources found a significant association with the outcomes . this review showed that several independent variables were implicated in the adoption of wash practices and found to be significantly associated with the outcomes . they include the age of the child , gender , grade level , socioeconomic index , access to hygiene and sanitary facilities , and prior knowledge of hygiene practices . some of the limitations observed in the various studies in this group included self - reporting of illness incidences leading to misclassification , underrepresented sample sizes , recall bias by parents in diarrheal incidences of their children , restricting studies to a particular age group , gender restriction , and the use of cross - sectional study designs . other limitations included a confounding of the impact of interventions due to similar ongoing interventions making it difficult to evaluate the outcomes , restriction of the study to a particular gender , and study sample restriction to only children present at the start of the school term leading to a loss of generalizability [ 13 , 21 , 27 , 30 ] . the studies assessed the health and educational effects of wash practices in schools on reducing absenteeism and diarrhea prevalence / infections among school - age children on a short term . however , there have been little or no empirical studies which examined the long term impact of wash interventions on child health outcomes , and therefore limited data to support future interventions . the positive effect of an education component in the intervention on the uptake and adherence to hygiene practices should be noted in future research . knowledge was implicated in several studies in this review as a facilitator in the uptake of hygiene practices and interventions [ 5 , 21 ] . several key independent variables including age of the child , gender , grade level , socioeconomic index , access to hygiene and sanitary facilities use and prior knowledge of hygiene practices which were significantly associated with child health outcomes should be noted and controlled for in future interventions . the review concluded that the importance of access to safe water , hand washing facilities , and hygiene education can not be underscored in abating water - borne illnesses , malnutrition , school absenteeism , and generally improving the quality of life and learning performance in children .

we conducted this population - based cohort study using data from medical registries in northern denmark over the period from 1998 to 2010 . data were obtained from the clinical laboratory information system research database ( labka ) ( 20 ) , the aarhus university prescription database ( aupd ) ( 21 ) , the danish cancer registry ( dcr ) ( 22 ) , and the danish national registry of patients ( dnrp ) ( 23 ) . the danish civil registration system , established in 1968 , assigns a civil registration number to all residents , allowing for unequivocal individual - level data linkage among all danish registries ( 24 ) . the labka database ( 20 ) contains all test results from routine tests performed in hospital laboratories in northern denmark , which has a total population of 2.2 million inhabitants . results of more than 1700 different types of analyses are included in the database . for each analysis , the database stores the test result ( or indicates that it is missing ) , civil registration number , date , and the international nomenclature , properties and units code . for some analyses , we identified all patients in the labka database with a plasma cbl measurement of greater than 200 pmol / l [ lower reference limit in northern denmark = 271 pg / ml ( 27 ) ] recorded from 1998 through 2009 . if a patient had more than one cbl test result , only the first test was included in our analysis . ( see supplementary table 1 , available online , for all codes used in this study . ) the aupd ( 21 ) collects data on all prescriptions reimbursed to patients in the study area . most prescription medications in denmark are eligible for full or partial tax - paid reimbursement . only over - the - counter drugs and a few prescription medications , such as oral contraceptives and sedatives , are normally not eligible for reimbursement , although reimbursement can be granted on a case - by - case basis . all records in the aupd include the date of dispensing , the patient s civil registration number , codes for the prescribing physician / clinic / hospital department , the anatomical therapeutic chemical code , and the medication name , pack size , dose units , and manufacturer . patients were classified as having received cbl therapy if they had one or more relevant prescriptions recorded in the aupd up to 2 years before measurement of their plasma cbl level . these patients were excluded from the analysis . in denmark , cbl therapy in pharmacological doses is available only by prescription . the dcr ( 22 ) includes all cancer diagnoses at the individual level in denmark since 1943 , coded according to the international classification of diseases , 10th revision ( icd-10 ) . the dcr was used to identify subsequent diagnoses of cancer from 1998 to 2010 among patients with a cbl test result in the labka database . patients were excluded if they had a cancer diagnosis before the date of the plasma cbl measurement . the dnrp ( 23 ) , established in 1977 , contains information on all inpatient hospitalizations and , since 1995 , all hospital outpatient clinic visits . the dnrp includes the patient s civil registration number , hospital admission and discharge date , date of hospital outpatient visit , and up to 20 diagnoses coded by physicians , including a primary diagnosis representing the main reason for the inpatient or outpatient hospital contact . all diagnoses are coded according to the international classification of diseases , 8th revision during the period from 1977 to 1993 and icd-10 since 1994 . to assess possible confounding from underlying diseases , we obtained data on all diagnoses before cbl measurement from the dnrp and grouped them according to icd-10 codes . patients were classified as inpatients or outpatients if they were admitted and/or had a hospital outpatient clinic visit up to 30 days before or 7 days after cbl measurement . if no hospital contact was recorded during the defined period , patients were classified as outpatients . we calculated the expected number of cancers , based on national incidence rates by age , sex , and year of diagnosis in 1-year intervals ( 25 ) . the number of cancers to be expected if the patients had the same risk as the general population was calculated by multiplying person - years of follow - up by the population - based cancer incidence rates . the risk of cancer in the study cohort was calculated as the standardized incidence ratio ( sir ; ie , the ratio of observed cancers to expected cancers ) . the 95% confidence intervals ( cis ) for the sir estimates were calculated assuming a poisson distribution of the observed number of specific cancers in the follow - up period . byar s approximation was used unless the observed number was less than 10 , in which case , exact 95% confidence intervals were calculated ( 26 ) . in the analyses , we first excluded patients receiving cbl therapy . a priori , we grouped incident cancers through 2010 into four different categories : smoking and alcohol - related cancers , hematological cancers , immune - related cancers , and hormone - related cancers ( see table 1 ) . we divided patients into three groups according to plasma cbl level : 200 to 600 pmol / l ( regional reference interval ( 27 ) = 271813 pg / ml ) ; 601 to 800 pmol / l ( regional reference interval = 8141084 pg / ml ) , and greater than 800 pmol / l ( regional reference level = > 1084 pg / ml ) . patients were also stratified according to age ( 050 years , 51 years ) , sex , length of follow - up ( 1 , > 1 year , and > 5 years ) , and receipt of inpatient or outpatient care . standardized incidence ratios were assessed in 100 to 200 pmol / l cbl level intervals to examine a possible cbl cutoff level for high cancer risk . because high plasma cbl levels also have been associated with other conditions ( 19 ) , we assessed cancer risk in relation to selected morbidities , categorized according to icd-10 codes ( see supplementary table 1 , available online ) . all statistical analyses were performed using sas version 9.2 ( sas institute inc , cary , nc ) . the study was approved by the danish data protection agency ( record number : 2009 - 41 - 3866 ) . we conducted this population - based cohort study using data from medical registries in northern denmark over the period from 1998 to 2010 . data were obtained from the clinical laboratory information system research database ( labka ) ( 20 ) , the aarhus university prescription database ( aupd ) ( 21 ) , the danish cancer registry ( dcr ) ( 22 ) , and the danish national registry of patients ( dnrp ) ( 23 ) . the danish civil registration system , established in 1968 , assigns a civil registration number to all residents , allowing for unequivocal individual - level data linkage among all danish registries ( 24 ) . the labka database ( 20 ) contains all test results from routine tests performed in hospital laboratories in northern denmark , which has a total population of 2.2 million inhabitants . results of more than 1700 different types of analyses are included in the database . for each analysis , the database stores the test result ( or indicates that it is missing ) , civil registration number , date , and the international nomenclature , properties and units code . for some analyses , we identified all patients in the labka database with a plasma cbl measurement of greater than 200 pmol / l [ lower reference limit in northern denmark = 271 pg / ml ( 27 ) ] recorded from 1998 through 2009 . if a patient had more than one cbl test result , only the first test was included in our analysis . ( see supplementary table 1 , available online , for all codes used in this study . ) the aupd ( 21 ) collects data on all prescriptions reimbursed to patients in the study area . most prescription medications in denmark are eligible for full or partial tax - paid reimbursement . only over - the - counter drugs and a few prescription medications , such as oral contraceptives and sedatives , are normally not eligible for reimbursement , although reimbursement can be granted on a case - by - case basis . all records in the aupd include the date of dispensing , the patient s civil registration number , codes for the prescribing physician / clinic / hospital department , the anatomical therapeutic chemical code , and the medication name , pack size , dose units , and manufacturer . patients were classified as having received cbl therapy if they had one or more relevant prescriptions recorded in the aupd up to 2 years before measurement of their plasma cbl level . these patients were excluded from the analysis . in denmark , cbl therapy in pharmacological doses is available only by prescription . the dcr ( 22 ) includes all cancer diagnoses at the individual level in denmark since 1943 , coded according to the international classification of diseases , 10th revision ( icd-10 ) . the dcr was used to identify subsequent diagnoses of cancer from 1998 to 2010 among patients with a cbl test result in the labka database . patients were excluded if they had a cancer diagnosis before the date of the plasma cbl measurement . the dnrp ( 23 ) , established in 1977 , contains information on all inpatient hospitalizations and , since 1995 , all hospital outpatient clinic visits . the dnrp includes the patient s civil registration number , hospital admission and discharge date , date of hospital outpatient visit , and up to 20 diagnoses coded by physicians , including a primary diagnosis representing the main reason for the inpatient or outpatient hospital contact . all diagnoses are coded according to the international classification of diseases , 8th revision during the period from 1977 to 1993 and icd-10 since 1994 . to assess possible confounding from underlying diseases , we obtained data on all diagnoses before cbl measurement from the dnrp and grouped them according to icd-10 codes . patients were classified as inpatients or outpatients if they were admitted and/or had a hospital outpatient clinic visit up to 30 days before or 7 days after cbl measurement . if no hospital contact was recorded during the defined period , patients were classified as outpatients . we calculated the expected number of cancers , based on national incidence rates by age , sex , and year of diagnosis in 1-year intervals ( 25 ) . the number of cancers to be expected if the patients had the same risk as the general population was calculated by multiplying person - years of follow - up by the population - based cancer incidence rates . the risk of cancer in the study cohort was calculated as the standardized incidence ratio ( sir ; ie , the ratio of observed cancers to expected cancers ) . the 95% confidence intervals ( cis ) for the sir estimates were calculated assuming a poisson distribution of the observed number of specific cancers in the follow - up period . byar s approximation was used unless the observed number was less than 10 , in which case , exact 95% confidence intervals were calculated ( 26 ) . in the analyses , we first excluded patients receiving cbl therapy . a priori , we grouped incident cancers through 2010 into four different categories : smoking and alcohol - related cancers , hematological cancers , immune - related cancers , and hormone - related cancers ( see table 1 ) . we divided patients into three groups according to plasma cbl level : 200 to 600 pmol / l ( regional reference interval ( 27 ) = 271813 pg / ml ) ; 601 to 800 pmol / l ( regional reference interval = 8141084 pg / ml ) , and greater than 800 pmol / l ( regional reference level = > 1084 pg / ml ) . patients were also stratified according to age ( 050 years , 51 years ) , sex , length of follow - up ( 1 , > 1 year , and > 5 years ) , and receipt of inpatient or outpatient care . standardized incidence ratios were assessed in 100 to 200 pmol / l cbl level intervals to examine a possible cbl cutoff level for high cancer risk . because high plasma cbl levels also have been associated with other conditions ( 19 ) , we assessed cancer risk in relation to selected morbidities , categorized according to icd-10 codes ( see supplementary table 1 , available online ) . all statistical analyses were performed using sas version 9.2 ( sas institute inc , cary , nc ) . the study was approved by the danish data protection agency ( record number : 2009 - 41 - 3866 ) . we identified 352831 persons without prevalent cancer who had plasma cbl levels 200 pmol / l , of which 19164 patients were receiving cbl therapy at the time of plasma cbl measurement and were therefore excluded . of the patients included in the study , 19,665 ( 6% ) had levels greater than the upper reference limit ( 601 pmol / l ) . the total number of person - years of follow - up was 1421512 and median follow - up time was 3.5 years ( interquartile range [ iqr]= 1.96.2 years ) . the distribution of outpatients and inpatients was 276 229 ( 83% ) and 57 438 ( 17% ) , respectively . a total of 22 652 ( 7% ) patients in the study cohort subsequently developed cancer during the period from 1998 to 2010 . the overall standardized incidence ratio was 1.26 ( 95% ci = 1.24 to 1.28 ; p < .001 ) ( table 2 ) . the overall cancer risk increased with higher cbl levels and with a peak during the first year of follow - up ( cbl 601800 pmol / l : sir = 3.44 , 95% ci = 3.14 to 3.76 , p < 0.001 ; cbl > 800 pmol / l : sir = 6.27 , 95% ci = 5.70 to 6.88 , p < 0.001 ) . for all cancer groups , the first year risk increased with higher cbl levels , most substantially for cbl levels greater than 800 pmol / l . after the first year , the risk remained elevated for patients with cbl levels greater than 800 pmol / l for smoking and alcohol - related cancers and for hematological cancers . the standardized incidence ratios also remained elevated after the first year in patients with cbl levels in the 601 to 800 pmol / l range for smoking- and alcohol - related cancers ( table 2 ) . for hematological cancers and smoking- and alcohol - related cancers , the standardized incidence ratios remained elevated for more than 5 years of follow - up , although for hematological cancers , this was the case only for patients with cbl levels greater than 800 the numbers and percentages of patients diagnosed with cancer according to plasma cbl levels and length of follow - up are shown in table 3 and are as follows : overall : 200 to 600 pmol / l : 6.7% , 601 to 800 pmol / l : 11.0% ; first year : 200 to 600 pmol / l : 2.3% , 601 to 800 pmol / l : 6.6% ; after the first year : 200 to 600 pmol / l : 4.4% , 601 to 800 pmol cancer risk diagnosed after a plasma cobalamin ( cbl ) measurement according to sex , follow - up interval , age , cancer group , and plasma cbl levels * * all statistical tests were two - sided . percentages and numbers of patients diagnosed with cancer during the study period disaggregated according to plasma cobalamin levels * * percentages are the fraction of patients diagnosed with cancer in each cobalamin ( cbl ) level group , presented as overall percentages and according to follow - up interval . figure 1 presents the first year standardized incidence ratios in detailed cbl - level intervals . the figure indicates that only risks of smoking- and alcohol - related cancers and hematological cancers substantially increased with higher cbl levels . also , it shows that a specific cbl level cutoff for high cancer risk could not be established . the standardized incidence ratios were elevated fourfold to 37-fold for hematological cancers , twofold to 10-fold for smoking- and alcohol - related cancers , and twofold to threefold for hormone - related cancers ; they were not elevated for immune - related cancers . one - year risk of cancer in groups according to plasma cobalamin ( cbl ) levels in 100 to 200 pmol / l intervals . the figure shows the 1-year standardized incidence ratios ( sirs ) with corresponding 95% confidence intervals ( cis ; vertical bars ) disaggregated according to cbl levels for hematological cancers ( solid line ) , smoking- and alcohol - related cancers ( dashed line ) , immune - related cancers ( dotted / dashed line ) , and hormone - related cancers ( dotted line ) . note the logarithmic scale for standardized incidence ratio on the y - axis . the standardized incidence ratios for inpatients and outpatients showed similar associations , both overall and by cancer group . the highest risks again were found for smoking- and alcohol - related cancers and for hematological cancers . the estimates were highest for inpatients in all cancer groups , although cancer risk was elevated also for outpatients with high cbl levels ( data not shown ) . when we examined the association between high cbl levels and specific cancer types within the first year of follow - up ( figure 2 ) ( sirs and 95% cis for cbl levels > 800 pmol / l ) , we found associations with gastric ( sir = 13.24 ; 95% ci = 7.23 to 22.21 ; p < .001 ) , colorectal ( sir = 5.48 ; 95% ci = 4.01 to 7.31 ; p < .001 ) , liver ( sir = 40.70 ; 95% ci = 25.50 to 61.62 ; p < .001 ) , pancreatic ( sir = 15.57 ; 95% ci = 10.34 to 22.50 ; p < .001 ) , lung ( sir = 9.27 ; 95% ci = 7.24 to 11.69 ; p < .001 ) , renal ( sir = 9.56 ; 95% ci = 4.58 to 17.58 ; p < .001 ) , urinary bladder ( sir = 5.34 ; 95% ci = 3.11 to 8.55 ; p < .001 ) , lymphatic leukemia ( sir = 8.88 ; 95% ci = 3.56 to 18.29 ; p < .001 ) , myeloid malignancies ( sir = 105.73 ; 95% ci = 81.24 to 135.28 ; p < .001 ) , non - hodgkin lymphoma ( sir = 6.64 ; 95% ci = 3.31 to 11.89 ; p < .001 ) , and multiple myeloma ( sir = 16.08 ; 95% ci = 7.70 to 29.58 ; p < .001 ) . the associations increased with higher cbl levels and were greatest among patients with the highest cbl levels ( > 800 pmol / l ) . the cancer risk remained elevated after the first year of follow - up for liver , pancreatic , lung cancer , and myeloid malignancies , with highest standardized incidence ratios observed for patients with cbl levels greater than 800 pmol / l ( supplementary table 3 , available online ) . risk of specific cancer types within the first year after plasma cobalamin ( cbl ) measurement . the figure shows the 1-year standardized incidence ratios ( sirs ) with corresponding 95% confidence intervals ( cis ) disaggregated according to cbl levels : x : 200 to 600 pmol / l ; o : 601 to 800 pmol / l ; and : greater than 800 the associations between cancer risk and high cbl levels remained robust in patients with selected morbidities , with no differences among specific morbidities ( data not shown ) . we identified 352831 persons without prevalent cancer who had plasma cbl levels 200 pmol / l , of which 19164 patients were receiving cbl therapy at the time of plasma cbl measurement and were therefore excluded . of the patients included in the study , 19,665 ( 6% ) had levels greater than the upper reference limit ( 601 pmol / l ) . the total number of person - years of follow - up was 1421512 and median follow - up time was 3.5 years ( interquartile range [ iqr]= 1.96.2 years ) . the distribution of outpatients and inpatients was 276 229 ( 83% ) and 57 438 ( 17% ) , respectively . a total of 22 652 ( 7% ) patients in the study cohort subsequently developed cancer during the period from 1998 to 2010 . the overall standardized incidence ratio was 1.26 ( 95% ci = 1.24 to 1.28 ; p < .001 ) ( table 2 ) . the overall cancer risk increased with higher cbl levels and with a peak during the first year of follow - up ( cbl 601800 pmol / l : sir = 3.44 , 95% ci = 3.14 to 3.76 , p < 0.001 ; cbl > 800 pmol / l : sir = 6.27 , 95% ci = 5.70 to 6.88 , p < 0.001 ) . for all cancer groups , the first year risk increased with higher cbl levels , most substantially for cbl levels greater than 800 pmol / l . after the first year , the risk remained elevated for patients with cbl levels greater than 800 pmol / l for smoking and alcohol - related cancers and for hematological cancers . the standardized incidence ratios also remained elevated after the first year in patients with cbl levels in the 601 to 800 pmol / l range for smoking- and alcohol - related cancers ( table 2 ) . for hematological cancers and smoking- and alcohol - related cancers , the standardized incidence ratios remained elevated for more than 5 years of follow - up , although for hematological cancers , this was the case only for patients with cbl levels greater than 800 the numbers and percentages of patients diagnosed with cancer according to plasma cbl levels and length of follow - up are shown in table 3 and are as follows : overall : 200 to 600 pmol / l : 6.7% , 601 to 800 pmol / l : 11.0% ; first year : 200 to 600 pmol / l : 2.3% , 601 to 800 pmol / l : 3.7% , and greater than 800 pmol / l : 6.6% ; after the first year : 200 to 600 pmol / l : 4.4% , 601 to 800 pmol cancer risk diagnosed after a plasma cobalamin ( cbl ) measurement according to sex , follow - up interval , age , cancer group , and plasma cbl levels * * all statistical tests were two - sided . percentages and numbers of patients diagnosed with cancer during the study period disaggregated according to plasma cobalamin levels * * percentages are the fraction of patients diagnosed with cancer in each cobalamin ( cbl ) level group , presented as overall percentages and according to follow - up interval . figure 1 presents the first year standardized incidence ratios in detailed cbl - level intervals . the figure indicates that only risks of smoking- and alcohol - related cancers and hematological cancers substantially increased with higher cbl levels . also , it shows that a specific cbl level cutoff for high cancer risk could not be established . the standardized incidence ratios were elevated fourfold to 37-fold for hematological cancers , twofold to 10-fold for smoking- and alcohol - related cancers , and twofold to threefold for hormone - related cancers ; they were not elevated for immune - related cancers . one - year risk of cancer in groups according to plasma cobalamin ( cbl ) levels in 100 to 200 pmol / l intervals . the figure shows the 1-year standardized incidence ratios ( sirs ) with corresponding 95% confidence intervals ( cis ; vertical bars ) disaggregated according to cbl levels for hematological cancers ( solid line ) , smoking- and alcohol - related cancers ( dashed line ) , immune - related cancers ( dotted / dashed line ) , and hormone - related cancers ( dotted line ) . the standardized incidence ratios for inpatients and outpatients showed similar associations , both overall and by cancer group . the highest risks again were found for smoking- and alcohol - related cancers and for hematological cancers . the estimates were highest for inpatients in all cancer groups , although cancer risk was elevated also for outpatients with high cbl levels ( data not shown ) . when we examined the association between high cbl levels and specific cancer types within the first year of follow - up ( figure 2 ) ( sirs and 95% cis for cbl levels > 800 pmol / l ) , we found associations with gastric ( sir = 13.24 ; 95% ci = 7.23 to 22.21 ; p < .001 ) , colorectal ( sir = 5.48 ; 95% ci = 4.01 to 7.31 ; p < .001 ) , liver ( sir = 40.70 ; 95% ci = 25.50 to 61.62 ; p < .001 ) , pancreatic ( sir = 15.57 ; 95% ci = 10.34 to 22.50 ; p < .001 ) , lung ( sir = 9.27 ; 95% ci = 7.24 to 11.69 ; p < .001 ) , renal ( sir = 9.56 ; 95% ci = 4.58 to 17.58 ; p < .001 ) , urinary bladder ( sir = 5.34 ; 95% ci = 3.11 to 8.55 ; p < .001 ) , lymphatic leukemia ( sir = 8.88 ; 95% ci = 3.56 to 18.29 ; p < .001 ) , myeloid malignancies ( sir = 105.73 ; 95% ci = 81.24 to 135.28 ; p < .001 ) , non - hodgkin lymphoma ( sir = 6.64 ; 95% ci = 3.31 to 11.89 ; p < .001 ) , and multiple myeloma ( sir = 16.08 ; 95% ci = 7.70 to 29.58 ; p < .001 ) . the associations increased with higher cbl levels and were greatest among patients with the highest cbl levels ( > 800 pmol / l ) . the cancer risk remained elevated after the first year of follow - up for liver , pancreatic , lung cancer , and myeloid malignancies , with highest standardized incidence ratios observed for patients with cbl levels greater than 800 pmol / l ( supplementary table 3 , available online ) . risk of specific cancer types within the first year after plasma cobalamin ( cbl ) measurement . the figure shows the 1-year standardized incidence ratios ( sirs ) with corresponding 95% confidence intervals ( cis ) disaggregated according to cbl levels : x : 200 to 600 pmol / l ; o : 601 to 800 pmol / l ; and : greater than 800 pmol / l . the associations between cancer risk and high cbl levels remained robust in patients with selected morbidities , with no differences among specific morbidities ( data not shown ) . our study shows that elevated plasma cbl levels are markers for various types of cancers , most notably hematological cancers within the first year after cbl measurement . our results extend those of earlier research ( 3,511 ) by showing a strong association between elevated cbl levels and cancer in a large study with a longitudinal design . the majority of these previous studies have been negative ( 1216 ) , except for lung and prostate cancer ( 17,18 ) . we focused on high cbl levels and thereby avoided the limitations of these earlier studies . our study was based on a large cohort of patients referred for plasma cbl measurement rather than patients with a particular type of cancer or persons from the general population . second , unlike the earlier studies , we assessed risk among patients with cbl levels above a given reference range . because we specifically focused on high cbl levels , we were able to demonstrate associations with cancer , including types not previously associated with high cbl levels , such as cancers of the pancreas and urinary bladder and multiple myeloma . we showed that approximately 15% of the population in northern denmark has had a plasma cbl measurement during the study period and that these measurements show cbl levels within or above the reference range . this suggests that plasma cbl measurements are used routinely and frequently to screen for cbl deficiency in the presence of symptoms or other risks . although our study can not directly assess use of high cbl levels as a nonspecific marker of cancer , our findings may be relevant to the clinical interpretation of such high levels . a number of different diseases have been suggested as causing high cbl levels ( 19 ) , but common to most of them is that the pathogenesis involving elevated cbl levels is not fully understood . in principle , some of these diseases might be responsible for the long - term associations observed in our study , thereby possibly confounding the associations between high cbl levels and cancer . for example , the association between liver cancer and high cbl levels could , in theory , be caused by nonmalignant liver disease underlying the liver cancer . however , when we stratified the analyses according to prior diseases , the results remained robust . this indicates that high cbl levels could be a marker for cancer both in the short- and long - term , exceeding 5 years for hematological cancers ( supplementary table 2 , available online ) . such persistent long - term associations are more likely to be affected by unrecorded confounders , such as lifestyle factors . although smoking is not associated with disruptions in cbl status ( 28 ) , alcoholism and alcohol - related liver disease have previously been linked to high cbl levels ( 3,29 ) . this could influence the long - term risk that we observed for some cancers , such as liver cancer , whereas the long - term association with hematological cancers is more peculiar . despite its large size and the use of registries to ensure complete follow - up , however , danish medical registries have proven to be of high validity and completeness ( 2023,30 ) , and any potential misclassification of information is likely to be minor and nondifferential ( ie , not associated with cbl levels ) . another concern is that we could not directly assess the clinical criteria for measuring plasma cbl levels because we did not have access to medical files and there are currently no specific guidelines for requesting plasma cbl measurement . mainly speculative , unspecific symptoms ( eg , anemia , fatigue , weight loss ) could be the reason for requesting plasma cbl measurement , and the same symptoms may also be related to the suspicion of cancer . this may explain why we found elevated standardized incidence ratios also for patients within normal cbl levels . however , we find it unlikely that the finding of elevated cbl levels in itself led to increased surveillance for cancer , although this statement can not be further assessed . hence , we find it reasonable to believe that the risk of confounding by indication is minimal . this may explain the decrease in cancer risk after the first year that we found for immune - related and hormone - related cancers , a compensatory deficit . finally , we chose to classify cancers a priori to analysis according to type ( hematological ) , life - style factors ( smoking- and alcohol - related ) , and two groups based on potential pathogenesis ( hormone related and immune related ) . to circumvent that some specific cancer types could fall in to more than one of the predefined groups , we also analyzed risk estimates for all specific cancers to obtain more detailed information within the predefined groups . the underlying pathogenesis leading to high cbl levels is poorly elucidated , with a few exceptions ( 6,10,11 ) . it is not thought to involve increased cbl intake because intestinal absorption capacity is saturable ( 31 ) and high physiological consumption does not increase plasma cbl levels substantially . only cbl therapy in the form of injections or extremely high oral doses can produce high circulating levels , and in this study , patients treated with cbl were excluded . we therefore conclude that the mechanisms resulting in high cbl levels may be related to malignant pathogenesis . our recent study showed that levels of the circulating cbl binding protein haptocorrin were high in patients with high plasma cbl levels ( 3 ) . this protein originates from a variety of tissues , but its physiological function remains unknown ( 32 ) . it is elevated in patients with some cancer types ( 6,10,11 ) and has been suggested as a marker for disease progression ( 6,10 ) . thus , haptocorrin may be a candidate factor to include in future studies of the possible pathogenic mechanisms leading to high cbl levels in cancer patients , in particular for the novel associations demonstrated in this study . in conclusion , our study showed that high plasma cbl levels increased the risk of subsequently diagnosed cancer , mostly within the first year of follow - up . although our results may have clinical implications for interpreting high cbl levels , further studies are warranted to examine the possible diagnostic value of high plasma cbl levels . this work was funded by the faculty of health sciences , aarhus university , aarhus , denmark ( to jfba , lp , en , hts ) ; aarhus university hospital , aarhus , denmark ( to lp , en , hts ) ; frits , georg , and marie cecilie gluds foundation and else og mogens wedell - wedellborgs foundation ( to jfba ) ; and the danish cancer society ( grant no . the department of clinical epidemiology , aarhus university hospital , receives funding for other studies from companies in the form of research grants to ( and administered by ) aarhus university .

backgrounda substantial proportion of patients referred for plasma vitamin b12 ( cobalamin [ cbl ] ) measurement present with high cbl levels , which have been reported in patients with different cancer types . however , the cancer risk among patients with newly diagnosed high cbl levels has not been adequately examined.methodswe conducted this cohort study using population - based danish medical registries . patients referred for cbl measurement with levels greater than the lower reference limit ( 200 pmol / l ) were identified from the population of northern denmark during the period of 1998 to 2009 using a database of laboratory test results covering the entire population . data on cancer incidence ( follow - up 19982010 ) , cbl treatment , and prior diagnoses were obtained from medical registries . patients receiving cbl treatment were excluded . cancer risks were calculated as standardized incidence ratios ( sirs ) with 95% confidence intervals ( cis ) , stratified by plasma cbl levels . all statistical tests were two-sided.resultswe identified 333 667 persons without prevalent cancer and not receiving cbl treatment . six percent had cbl levels greater than the upper reference limit ( 601 pmol / l ) . cancer risk increased with higher cbl levels and was highest during the first year of follow - up ( cbl 601800 pmol / l : sir = 3.44 , 95% ci = 3.14 to 3.76 ; cbl > 800 pmol / l : sir = 6.27 , 95% ci = 5.70 to 6.88 ; both p < .001 ) . the risks were particularly elevated for hematological and smoking- and alcohol - related cancers for persons with high cbl levels.conclusionshigh cbl levels were associated with the risk of subsequently diagnosed cancer , mostly within the first year of follow - up . this may have clinical implications for the interpretation of high cbl levels .

Methods Design, Study Cohort, and Data Sources Statistical Analyses Results Study Cohort Cancer Risks Patient and Cancer Subtypes Discussion Funding Supplementary Material

we conducted this population - based cohort study using data from medical registries in northern denmark over the period from 1998 to 2010 . data were obtained from the clinical laboratory information system research database ( labka ) ( 20 ) , the aarhus university prescription database ( aupd ) ( 21 ) , the danish cancer registry ( dcr ) ( 22 ) , and the danish national registry of patients ( dnrp ) ( 23 ) . for some analyses , we identified all patients in the labka database with a plasma cbl measurement of greater than 200 pmol / l [ lower reference limit in northern denmark = 271 pg / ml ( 27 ) ] recorded from 1998 through 2009 . the number of cancers to be expected if the patients had the same risk as the general population was calculated by multiplying person - years of follow - up by the population - based cancer incidence rates . the risk of cancer in the study cohort was calculated as the standardized incidence ratio ( sir ; ie , the ratio of observed cancers to expected cancers ) . the 95% confidence intervals ( cis ) for the sir estimates were calculated assuming a poisson distribution of the observed number of specific cancers in the follow - up period . a priori , we grouped incident cancers through 2010 into four different categories : smoking and alcohol - related cancers , hematological cancers , immune - related cancers , and hormone - related cancers ( see table 1 ) . we divided patients into three groups according to plasma cbl level : 200 to 600 pmol / l ( regional reference interval ( 27 ) = 271813 pg / ml ) ; 601 to 800 pmol / l ( regional reference interval = 8141084 pg / ml ) , and greater than 800 pmol / l ( regional reference level = > 1084 pg / ml ) . patients were also stratified according to age ( 050 years , 51 years ) , sex , length of follow - up ( 1 , > 1 year , and > 5 years ) , and receipt of inpatient or outpatient care . standardized incidence ratios were assessed in 100 to 200 pmol / l cbl level intervals to examine a possible cbl cutoff level for high cancer risk . because high plasma cbl levels also have been associated with other conditions ( 19 ) , we assessed cancer risk in relation to selected morbidities , categorized according to icd-10 codes ( see supplementary table 1 , available online ) . we conducted this population - based cohort study using data from medical registries in northern denmark over the period from 1998 to 2010 . data were obtained from the clinical laboratory information system research database ( labka ) ( 20 ) , the aarhus university prescription database ( aupd ) ( 21 ) , the danish cancer registry ( dcr ) ( 22 ) , and the danish national registry of patients ( dnrp ) ( 23 ) . for some analyses , we identified all patients in the labka database with a plasma cbl measurement of greater than 200 pmol / l [ lower reference limit in northern denmark = 271 pg / ml ( 27 ) ] recorded from 1998 through 2009 . the number of cancers to be expected if the patients had the same risk as the general population was calculated by multiplying person - years of follow - up by the population - based cancer incidence rates . the risk of cancer in the study cohort was calculated as the standardized incidence ratio ( sir ; ie , the ratio of observed cancers to expected cancers ) . the 95% confidence intervals ( cis ) for the sir estimates were calculated assuming a poisson distribution of the observed number of specific cancers in the follow - up period . a priori , we grouped incident cancers through 2010 into four different categories : smoking and alcohol - related cancers , hematological cancers , immune - related cancers , and hormone - related cancers ( see table 1 ) . we divided patients into three groups according to plasma cbl level : 200 to 600 pmol / l ( regional reference interval ( 27 ) = 271813 pg / ml ) ; 601 to 800 pmol / l ( regional reference interval = 8141084 pg / ml ) , and greater than 800 pmol / l ( regional reference level = > 1084 pg / ml ) . patients were also stratified according to age ( 050 years , 51 years ) , sex , length of follow - up ( 1 , > 1 year , and > 5 years ) , and receipt of inpatient or outpatient care . standardized incidence ratios were assessed in 100 to 200 pmol / l cbl level intervals to examine a possible cbl cutoff level for high cancer risk . because high plasma cbl levels also have been associated with other conditions ( 19 ) , we assessed cancer risk in relation to selected morbidities , categorized according to icd-10 codes ( see supplementary table 1 , available online ) . we identified 352831 persons without prevalent cancer who had plasma cbl levels 200 pmol / l , of which 19164 patients were receiving cbl therapy at the time of plasma cbl measurement and were therefore excluded . of the patients included in the study , 19,665 ( 6% ) had levels greater than the upper reference limit ( 601 pmol / l ) . the overall standardized incidence ratio was 1.26 ( 95% ci = 1.24 to 1.28 ; p < .001 ) ( table 2 ) . the overall cancer risk increased with higher cbl levels and with a peak during the first year of follow - up ( cbl 601800 pmol / l : sir = 3.44 , 95% ci = 3.14 to 3.76 , p < 0.001 ; cbl > 800 pmol / l : sir = 6.27 , 95% ci = 5.70 to 6.88 , p < 0.001 ) . for all cancer groups , the first year risk increased with higher cbl levels , most substantially for cbl levels greater than 800 pmol / l . after the first year , the risk remained elevated for patients with cbl levels greater than 800 pmol / l for smoking and alcohol - related cancers and for hematological cancers . the standardized incidence ratios also remained elevated after the first year in patients with cbl levels in the 601 to 800 pmol / l range for smoking- and alcohol - related cancers ( table 2 ) . for hematological cancers and smoking- and alcohol - related cancers , the standardized incidence ratios remained elevated for more than 5 years of follow - up , although for hematological cancers , this was the case only for patients with cbl levels greater than 800 the numbers and percentages of patients diagnosed with cancer according to plasma cbl levels and length of follow - up are shown in table 3 and are as follows : overall : 200 to 600 pmol / l : 6.7% , 601 to 800 pmol / l : 11.0% ; first year : 200 to 600 pmol / l : 2.3% , 601 to 800 pmol / l : 6.6% ; after the first year : 200 to 600 pmol / l : 4.4% , 601 to 800 pmol cancer risk diagnosed after a plasma cobalamin ( cbl ) measurement according to sex , follow - up interval , age , cancer group , and plasma cbl levels * * all statistical tests were two - sided . percentages and numbers of patients diagnosed with cancer during the study period disaggregated according to plasma cobalamin levels * * percentages are the fraction of patients diagnosed with cancer in each cobalamin ( cbl ) level group , presented as overall percentages and according to follow - up interval . the figure indicates that only risks of smoking- and alcohol - related cancers and hematological cancers substantially increased with higher cbl levels . the standardized incidence ratios were elevated fourfold to 37-fold for hematological cancers , twofold to 10-fold for smoking- and alcohol - related cancers , and twofold to threefold for hormone - related cancers ; they were not elevated for immune - related cancers . one - year risk of cancer in groups according to plasma cobalamin ( cbl ) levels in 100 to 200 pmol / l intervals . the figure shows the 1-year standardized incidence ratios ( sirs ) with corresponding 95% confidence intervals ( cis ; vertical bars ) disaggregated according to cbl levels for hematological cancers ( solid line ) , smoking- and alcohol - related cancers ( dashed line ) , immune - related cancers ( dotted / dashed line ) , and hormone - related cancers ( dotted line ) . the highest risks again were found for smoking- and alcohol - related cancers and for hematological cancers . when we examined the association between high cbl levels and specific cancer types within the first year of follow - up ( figure 2 ) ( sirs and 95% cis for cbl levels > 800 pmol / l ) , we found associations with gastric ( sir = 13.24 ; 95% ci = 7.23 to 22.21 ; p < .001 ) , colorectal ( sir = 5.48 ; 95% ci = 4.01 to 7.31 ; p < .001 ) , liver ( sir = 40.70 ; 95% ci = 25.50 to 61.62 ; p < .001 ) , pancreatic ( sir = 15.57 ; 95% ci = 10.34 to 22.50 ; p < .001 ) , lung ( sir = 9.27 ; 95% ci = 7.24 to 11.69 ; p < .001 ) , renal ( sir = 9.56 ; 95% ci = 4.58 to 17.58 ; p < .001 ) , urinary bladder ( sir = 5.34 ; 95% ci = 3.11 to 8.55 ; p < .001 ) , lymphatic leukemia ( sir = 8.88 ; 95% ci = 3.56 to 18.29 ; p < .001 ) , myeloid malignancies ( sir = 105.73 ; 95% ci = 81.24 to 135.28 ; p < .001 ) , non - hodgkin lymphoma ( sir = 6.64 ; 95% ci = 3.31 to 11.89 ; p < .001 ) , and multiple myeloma ( sir = 16.08 ; 95% ci = 7.70 to 29.58 ; p < .001 ) . the associations increased with higher cbl levels and were greatest among patients with the highest cbl levels ( > 800 pmol / l ) . the cancer risk remained elevated after the first year of follow - up for liver , pancreatic , lung cancer , and myeloid malignancies , with highest standardized incidence ratios observed for patients with cbl levels greater than 800 pmol / l ( supplementary table 3 , available online ) . risk of specific cancer types within the first year after plasma cobalamin ( cbl ) measurement . the figure shows the 1-year standardized incidence ratios ( sirs ) with corresponding 95% confidence intervals ( cis ) disaggregated according to cbl levels : x : 200 to 600 pmol / l ; o : 601 to 800 pmol / l ; and : greater than 800 the associations between cancer risk and high cbl levels remained robust in patients with selected morbidities , with no differences among specific morbidities ( data not shown ) . we identified 352831 persons without prevalent cancer who had plasma cbl levels 200 pmol / l , of which 19164 patients were receiving cbl therapy at the time of plasma cbl measurement and were therefore excluded . of the patients included in the study , 19,665 ( 6% ) had levels greater than the upper reference limit ( 601 pmol / l ) . the overall standardized incidence ratio was 1.26 ( 95% ci = 1.24 to 1.28 ; p < .001 ) ( table 2 ) . the overall cancer risk increased with higher cbl levels and with a peak during the first year of follow - up ( cbl 601800 pmol / l : sir = 3.44 , 95% ci = 3.14 to 3.76 , p < 0.001 ; cbl > 800 pmol / l : sir = 6.27 , 95% ci = 5.70 to 6.88 , p < 0.001 ) . for all cancer groups , the first year risk increased with higher cbl levels , most substantially for cbl levels greater than 800 pmol / l . after the first year , the risk remained elevated for patients with cbl levels greater than 800 pmol / l for smoking and alcohol - related cancers and for hematological cancers . the standardized incidence ratios also remained elevated after the first year in patients with cbl levels in the 601 to 800 pmol / l range for smoking- and alcohol - related cancers ( table 2 ) . for hematological cancers and smoking- and alcohol - related cancers , the standardized incidence ratios remained elevated for more than 5 years of follow - up , although for hematological cancers , this was the case only for patients with cbl levels greater than 800 the numbers and percentages of patients diagnosed with cancer according to plasma cbl levels and length of follow - up are shown in table 3 and are as follows : overall : 200 to 600 pmol / l : 6.7% , 601 to 800 pmol / l : 11.0% ; first year : 200 to 600 pmol / l : 2.3% , 601 to 800 pmol / l : 3.7% , and greater than 800 pmol / l : 6.6% ; after the first year : 200 to 600 pmol / l : 4.4% , 601 to 800 pmol cancer risk diagnosed after a plasma cobalamin ( cbl ) measurement according to sex , follow - up interval , age , cancer group , and plasma cbl levels * * all statistical tests were two - sided . percentages and numbers of patients diagnosed with cancer during the study period disaggregated according to plasma cobalamin levels * * percentages are the fraction of patients diagnosed with cancer in each cobalamin ( cbl ) level group , presented as overall percentages and according to follow - up interval . the figure indicates that only risks of smoking- and alcohol - related cancers and hematological cancers substantially increased with higher cbl levels . the standardized incidence ratios were elevated fourfold to 37-fold for hematological cancers , twofold to 10-fold for smoking- and alcohol - related cancers , and twofold to threefold for hormone - related cancers ; they were not elevated for immune - related cancers . the figure shows the 1-year standardized incidence ratios ( sirs ) with corresponding 95% confidence intervals ( cis ; vertical bars ) disaggregated according to cbl levels for hematological cancers ( solid line ) , smoking- and alcohol - related cancers ( dashed line ) , immune - related cancers ( dotted / dashed line ) , and hormone - related cancers ( dotted line ) . the highest risks again were found for smoking- and alcohol - related cancers and for hematological cancers . when we examined the association between high cbl levels and specific cancer types within the first year of follow - up ( figure 2 ) ( sirs and 95% cis for cbl levels > 800 pmol / l ) , we found associations with gastric ( sir = 13.24 ; 95% ci = 7.23 to 22.21 ; p < .001 ) , colorectal ( sir = 5.48 ; 95% ci = 4.01 to 7.31 ; p < .001 ) , liver ( sir = 40.70 ; 95% ci = 25.50 to 61.62 ; p < .001 ) , pancreatic ( sir = 15.57 ; 95% ci = 10.34 to 22.50 ; p < .001 ) , lung ( sir = 9.27 ; 95% ci = 7.24 to 11.69 ; p < .001 ) , renal ( sir = 9.56 ; 95% ci = 4.58 to 17.58 ; p < .001 ) , urinary bladder ( sir = 5.34 ; 95% ci = 3.11 to 8.55 ; p < .001 ) , lymphatic leukemia ( sir = 8.88 ; 95% ci = 3.56 to 18.29 ; p < .001 ) , myeloid malignancies ( sir = 105.73 ; 95% ci = 81.24 to 135.28 ; p < .001 ) , non - hodgkin lymphoma ( sir = 6.64 ; 95% ci = 3.31 to 11.89 ; p < .001 ) , and multiple myeloma ( sir = 16.08 ; 95% ci = 7.70 to 29.58 ; p < .001 ) . the associations increased with higher cbl levels and were greatest among patients with the highest cbl levels ( > 800 pmol / l ) . the cancer risk remained elevated after the first year of follow - up for liver , pancreatic , lung cancer , and myeloid malignancies , with highest standardized incidence ratios observed for patients with cbl levels greater than 800 pmol / l ( supplementary table 3 , available online ) . risk of specific cancer types within the first year after plasma cobalamin ( cbl ) measurement . the figure shows the 1-year standardized incidence ratios ( sirs ) with corresponding 95% confidence intervals ( cis ) disaggregated according to cbl levels : x : 200 to 600 pmol / l ; o : 601 to 800 pmol / l ; and : greater than 800 pmol / l . our study shows that elevated plasma cbl levels are markers for various types of cancers , most notably hematological cancers within the first year after cbl measurement . our study was based on a large cohort of patients referred for plasma cbl measurement rather than patients with a particular type of cancer or persons from the general population . because we specifically focused on high cbl levels , we were able to demonstrate associations with cancer , including types not previously associated with high cbl levels , such as cancers of the pancreas and urinary bladder and multiple myeloma . we showed that approximately 15% of the population in northern denmark has had a plasma cbl measurement during the study period and that these measurements show cbl levels within or above the reference range . although our study can not directly assess use of high cbl levels as a nonspecific marker of cancer , our findings may be relevant to the clinical interpretation of such high levels . although smoking is not associated with disruptions in cbl status ( 28 ) , alcoholism and alcohol - related liver disease have previously been linked to high cbl levels ( 3,29 ) . despite its large size and the use of registries to ensure complete follow - up , however , danish medical registries have proven to be of high validity and completeness ( 2023,30 ) , and any potential misclassification of information is likely to be minor and nondifferential ( ie , not associated with cbl levels ) . this may explain the decrease in cancer risk after the first year that we found for immune - related and hormone - related cancers , a compensatory deficit . finally , we chose to classify cancers a priori to analysis according to type ( hematological ) , life - style factors ( smoking- and alcohol - related ) , and two groups based on potential pathogenesis ( hormone related and immune related ) . our recent study showed that levels of the circulating cbl binding protein haptocorrin were high in patients with high plasma cbl levels ( 3 ) . in conclusion , our study showed that high plasma cbl levels increased the risk of subsequently diagnosed cancer , mostly within the first year of follow - up . although our results may have clinical implications for interpreting high cbl levels , further studies are warranted to examine the possible diagnostic value of high plasma cbl levels .

this was a multinational , randomized , double - blind , parallel - group , 54-week study . the study included a 1-week screening period , a diet / exercise and oral antihyperglycemic agent ( aha ) wash - off period of up to 14 weeks , a 2-week , single - blind placebo run - in period , and a 54-week , double - blind treatment period . at screening , patients not taking ahas for 12 weeks with an a1c of 79% directly entered the single - blind placebo run - in period and those with an a1c > 9% entered a 6-week diet and exercise period . patients taking oral ahas with an a1c of 79% entered an 8-week drug wash - off and diet and exercise period ( those taking thiazolidinediones underwent a 10-week wash - off period ) , and those with an a1c of 6.5 to < 7% entered an 812-week drug wash - off and diet and exercise period ( those on thiazolidinediones underwent a 1014-week wash - off period ) . patients received diet and exercise counseling throughout the study , consistent with american diabetes association recommendations and appropriate for their renal insufficiency status . following the placebo run - in , eligible patients were randomized ( 1:1 ) using a computer - generated randomization schedule to receive sitagliptin or glipizide . randomization was stratified based on : 1 ) renal insufficiency status ( moderate or severe ) , 2 ) history of cardiovascular disease ( yes or no ) , and 3 ) history of heart failure ( yes or no ) . patients with moderate renal insufficiency received 50 mg / day of sitagliptin ( two 25-mg tablets ) or matching placebo . patients with severe renal insufficiency received 25 mg / day of sitagliptin ( one 25-mg tablet ) or matching placebo . the dose of sitagliptin was reduced from 50 mg / day to 25 mg / day for patients whose renal status changed from moderate to severe during the study . glipizide was administered at a starting dose of 2.5 mg / day , prior to the morning meal , and electively titrated to a maximum of 20 mg / day as considered appropriate by the investigator based on the patient s glycemic control ; the dose of glipizide could also be reduced or interrupted to prevent hypoglycemia . after maximally tolerated uptitration of glipizide or matching placebo , patients had insulin rescue therapy initiated , with the regimen and dose determined by investigator , if they met the following criteria : confirmed fpg > 240 mg / dl any time from randomization to week 6 ; confirmed fpg > 220 mg / dl from week 6 to 12 ; confirmed fpg > 200 mg / dl from week 12 to 24 ; and confirmed a1c > 8% after week 24 . once insulin rescue therapy was initiated , patients continued to take blinded sitagliptin or matching placebo , but discontinued blinded glipizide or matching placebo . patients with t2 dm could participate if they had moderate to severe chronic renal insufficiency ( egfr < 50 ml / min/1.73 m using the modification of diet in renal disease equation ) , were not on dialysis and unlikely to require dialysis for the duration of the study , had an a1c 7.0 and 9.0% , and were 30 years of age at the screening visit . patients taking insulin within 12 weeks of the screening visit , patients with type 1 diabetes , history of ketoacidosis , acute renal disease , history of renal transplant , liver disease , a recent ( within 3 months ) cardiovascular event , hepatic transaminase levels two or more times the upper limit of normal , thyroid stimulating hormone outside the reference range , or triglycerides > 600 mg / dl were excluded from participation . patients were also excluded if they met one of the following prespecified glycemic criteria : visit 2 , fpg > 260 mg / dl , unlikely to improve with diet / exercise ; visit 3 , fpg > 250 mg / dl consistently ( i.e. , measurement repeated and confirmed within 7 days ) ; visit 4 , fpg > 240 mg / dl consistently ; and visit 5 , finger - stick glucose > 240 or < 120 mg / dl . the study was performed in accordance with good clinical practice standards and the ethical principles that have their origin in the declaration of helsinki . the primary efficacy end point was the change from baseline in a1c at week 54 . other efficacy assessments included fpg , fasting serum insulin and proinsulin , and plasma lipid profiles ( total cholesterol , ldl cholesterol [ ldl - c ] , hdl cholesterol [ hdl - c ] , non hdl - c , and triglycerides ) . homeostasis model assessment-cell function ( homa- ) , homa - insulin resistance ( homa - ir ) , and proinsulin / insulin ratio were calculated from fasting measurements of fpg , insulin , and proinsulin ( 16,17 ) . the proportion of individuals whose a1c values met glycemic goals ( < 7.0% as primary ; < 6.5% as secondary ) at week 54 was analyzed . a prespecified analysis evaluated the consistency of the a1c - lowering effects of sitagliptin versus glipizide across predefined subgroups based on baseline characteristics a1c ( < and 8% ) , age ( < and 65 years ) , severity of ckd ( egfr 30 to < 50 [ moderate ] and < 30 [ severe ] ml / min/1.73 m ) , bmi ( < and median ) , duration of t2 dm ( < and median ) , ethnicity ( hispanic or latino and non - hispanic or non - latino ) , sex , prior antihyperglycemic therapy status ( yes or no ) , and race ( asian , black , white , or other ) . a post hoc analysis evaluated the effect of sitagliptin versus glipizide on a composite end point consisting of glycemic control ( reduction in a1c > 0.5% ) , no body weight gain , and no hypoglycemia . safety measurements included evaluation of adverse events ( aes ) , physical examination and vital signs , and electrocardiograms . serious aes consistent with vascular events ( cardiovascular , cerebrovascular , and peripheral vascular events ) and heart failure , revascularization procedures , and all deaths , regardless of cause , were adjudicated by an expert committee external to merck sharp & dohme corp . patients were instructed to monitor and record their glucose concentrations and counseled regarding the symptoms of hypoglycemia , as previously described ( 16 ) . events deemed by the investigator as hypoglycemia were reported as an ae of symptomatic hypoglycemia and did not require documentation of a glucose measurement . events of hypoglycemia requiring ( nonmedical ) assistance of others , requiring medical intervention , or exhibiting markedly depressed level of consciousness , loss of consciousness , or seizure were considered severe . other parameters of special interest included change in body weight and gastrointestinal aes ( nausea , vomiting , diarrhea , and abdominal pain ) . patients were instructed to fast overnight for 10 h prior to collection of blood for laboratory assessment . blood was collected at baseline ( predose ) and at various time points throughout the treatment period of 54 weeks for efficacy and safety measurements . all laboratory measurements were performed by a central laboratory ( ppd global central laboratories , llc , highland heights , ky , and zaventem , belgium ) . the primary study end points were change from baseline in a1c , the incidence of hypoglycemia events , and overall safety and tolerability . the primary efficacy analysis population was the per - protocol ( pp ) population , which included all randomized patients who had measurements of the respective end point both at baseline and week 54 and did not have any major protocol violations . the ancova model included terms for treatment , renal insufficiency stratum at visit 4/week 2 ( moderate or severe ) , prior diabetes pharmacotherapy ( on or not on aha ) , and a covariate for baseline a1c . sitagliptin was to be declared noninferior to glipizide in lowering a1c at week 54 if the upper bound of the 95% ci around the between - group difference was less than the noninferiority margin of 0.4% . the changes ( or percent changes ) from baseline in all other continuous efficacy end points at week 54 , except for serum triglycerides and homa- , were analyzed using the ancova model described for a1c at week 54 , substituting the relevant baseline efficacy measurement as a covariate . the analysis of triglycerides used a nonparametric approach based on ranks . due to the presence of outliers , homa- was analyzed using a robust m - estimation approach that minimized the influence of outliers ( 18 ) . the proportion of individuals whose a1c values met predefined glycemic goals was based on the miettinen and nurminen ( m&n ) method ( 19 ) . for each subgroup factor , the ancova model included the following terms : treatment , renal insufficiency stratum , prior diabetes pharmacotherapy , baseline value , subgroup , and treatment - by - subgroup interaction . due to enrollment challenges , the total sample size was revised from 500 ( original design ) to 430 . approximately 324 patients ( 162 patients per group ) were expected to be available for the pp analysis . using an sd of 1.1% for a1c change from baseline at week 54 , the study had 90% power to declare noninferiority for a margin of 0.4% at an level of 0.05 , assuming the true mean difference was 0% . a composite end point of a1c reduction from baseline of > 0.5% , no body weight gain , and no hypoglycemia was evaluated post hoc . the 95% ci for proportion difference was calculated using the m&n method ( 19 ) , stratified by renal insufficiency stratum , prior diabetes pharmacotherapy ( on or not on oral ahas ) , and baseline a1c ( < 8 or 8% ) . the odds ratio and 95% ci of achieving the composite end point for sitagliptin versus glipizide were provided using logistic regression , adjusting for treatment , renal insufficiency stratum , prior diabetes pharmacotherapy , and baseline a1c . all patients who had measurements at baseline and week 54 for both a1c and body weight were included in the analysis . safety analyses included all randomized patients who received at least one dose of study drug and included data through 28 days following the last day of study medication . additional analyses of cardiovascular events included data through 28 days following week 54 . in these additional analyses , events that occurred up to 54 weeks postrandomization in patients who discontinued prior to week 54 for all ae end points , the proportions of patients with one or more events were analyzed using the m&n method ( 19 ) . analysis of continuous safety end points required measurements at baseline and at one or more postbaseline time points . change from baseline in body weight at week 54 was analyzed using an ancova model similar to that used for the efficacy analyses , based on all patients with measurements both at baseline and week 54 . between - group differences and 95% cis for the exposure - adjusted incidence rates ( i.e. , number of patients with 1 event per 100 patient - years ) were provided [ using the m&n method ( 19 ) , stratified as per the study randomization ] for confirmed , adjudicated cardiovascular aes . except for end points related to confirmed , adjudicated heart failure and cardiovascular aes , analyses of both efficacy and safety excluded data following initiation of insulin rescue therapy . this was a multinational , randomized , double - blind , parallel - group , 54-week study . the study included a 1-week screening period , a diet / exercise and oral antihyperglycemic agent ( aha ) wash - off period of up to 14 weeks , a 2-week , single - blind placebo run - in period , and a 54-week , double - blind treatment period . at screening , patients not taking ahas for 12 weeks with an a1c of 79% directly entered the single - blind placebo run - in period and those with an a1c > 9% entered a 6-week diet and exercise period . patients taking oral ahas with an a1c of 79% entered an 8-week drug wash - off and diet and exercise period ( those taking thiazolidinediones underwent a 10-week wash - off period ) , and those with an a1c of 6.5 to < 7% entered an 812-week drug wash - off and diet and exercise period ( those on thiazolidinediones underwent a 1014-week wash - off period ) . patients received diet and exercise counseling throughout the study , consistent with american diabetes association recommendations and appropriate for their renal insufficiency status . following the placebo run - in , eligible patients were randomized ( 1:1 ) using a computer - generated randomization schedule to receive sitagliptin or glipizide . randomization was stratified based on : 1 ) renal insufficiency status ( moderate or severe ) , 2 ) history of cardiovascular disease ( yes or no ) , and 3 ) history of heart failure ( yes or no ) . patients with moderate renal insufficiency received 50 mg / day of sitagliptin ( two 25-mg tablets ) or matching placebo . patients with severe renal insufficiency received 25 mg / day of sitagliptin ( one 25-mg tablet ) or matching placebo . the dose of sitagliptin was reduced from 50 mg / day to 25 mg / day for patients whose renal status changed from moderate to severe during the study . glipizide was administered at a starting dose of 2.5 mg / day , prior to the morning meal , and electively titrated to a maximum of 20 mg / day as considered appropriate by the investigator based on the patient s glycemic control ; the dose of glipizide could also be reduced or interrupted to prevent hypoglycemia . after maximally tolerated uptitration of glipizide or matching placebo , patients had insulin rescue therapy initiated , with the regimen and dose determined by investigator , if they met the following criteria : confirmed fpg > 240 mg / dl any time from randomization to week 6 ; confirmed fpg > 220 mg / dl from week 6 to 12 ; confirmed fpg > 200 mg / dl from week 12 to 24 ; and confirmed a1c > 8% after week 24 . once insulin rescue therapy was initiated , patients continued to take blinded sitagliptin or matching placebo , but discontinued blinded glipizide or matching placebo . patients with t2 dm could participate if they had moderate to severe chronic renal insufficiency ( egfr < 50 ml / min/1.73 m using the modification of diet in renal disease equation ) , were not on dialysis and unlikely to require dialysis for the duration of the study , had an a1c 7.0 and 9.0% , and were 30 years of age at the screening visit . patients taking insulin within 12 weeks of the screening visit , patients with type 1 diabetes , history of ketoacidosis , acute renal disease , history of renal transplant , liver disease , a recent ( within 3 months ) cardiovascular event , hepatic transaminase levels two or more times the upper limit of normal , thyroid stimulating hormone outside the reference range , or triglycerides > 600 mg / dl were excluded from participation . patients were also excluded if they met one of the following prespecified glycemic criteria : visit 2 , fpg > 260 mg / dl , unlikely to improve with diet / exercise ; visit 3 , fpg > 250 mg / dl consistently ( i.e. , measurement repeated and confirmed within 7 days ) ; visit 4 , fpg > 240 mg / dl consistently ; and visit 5 , finger - stick glucose > 240 or < 120 mg / dl . the study was performed in accordance with good clinical practice standards and the ethical principles that have their origin in the declaration of helsinki . the primary efficacy end point was the change from baseline in a1c at week 54 . other efficacy assessments included fpg , fasting serum insulin and proinsulin , and plasma lipid profiles ( total cholesterol , ldl cholesterol [ ldl - c ] , hdl cholesterol [ hdl - c ] , non hdl - c , and triglycerides ) . homeostasis model assessment-cell function ( homa- ) , homa - insulin resistance ( homa - ir ) , and proinsulin / insulin ratio were calculated from fasting measurements of fpg , insulin , and proinsulin ( 16,17 ) . the proportion of individuals whose a1c values met glycemic goals ( < 7.0% as primary ; < 6.5% as secondary ) at week 54 was analyzed . a prespecified analysis evaluated the consistency of the a1c - lowering effects of sitagliptin versus glipizide across predefined subgroups based on baseline characteristics a1c ( < and 8% ) , age ( < and 65 years ) , severity of ckd ( egfr 30 to < 50 [ moderate ] and < 30 [ severe ] ml / min/1.73 m ) , bmi ( < and median ) , duration of t2 dm ( < and median ) , ethnicity ( hispanic or latino and non - hispanic or non - latino ) , sex , prior antihyperglycemic therapy status ( yes or no ) , and race ( asian , black , white , or other ) . a post hoc analysis evaluated the effect of sitagliptin versus glipizide on a composite end point consisting of glycemic control ( reduction in a1c > 0.5% ) , no body weight gain , and no hypoglycemia . safety measurements included evaluation of adverse events ( aes ) , physical examination and vital signs , and electrocardiograms . serious aes consistent with vascular events ( cardiovascular , cerebrovascular , and peripheral vascular events ) and heart failure , revascularization procedures , and all deaths , regardless of cause , were adjudicated by an expert committee external to merck sharp & dohme corp . patients were instructed to monitor and record their glucose concentrations and counseled regarding the symptoms of hypoglycemia , as previously described ( 16 ) . events deemed by the investigator as hypoglycemia were reported as an ae of symptomatic hypoglycemia and did not require documentation of a glucose measurement . events of hypoglycemia requiring ( nonmedical ) assistance of others , requiring medical intervention , or exhibiting markedly depressed level of consciousness , loss of consciousness , or seizure were considered severe . other parameters of special interest included change in body weight and gastrointestinal aes ( nausea , vomiting , diarrhea , and abdominal pain ) . patients were instructed to fast overnight for 10 h prior to collection of blood for laboratory assessment . blood was collected at baseline ( predose ) and at various time points throughout the treatment period of 54 weeks for efficacy and safety measurements . all laboratory measurements were performed by a central laboratory ( ppd global central laboratories , llc , highland heights , ky , and zaventem , belgium ) . the primary study end points were change from baseline in a1c , the incidence of hypoglycemia events , and overall safety and tolerability . the primary efficacy analysis population was the per - protocol ( pp ) population , which included all randomized patients who had measurements of the respective end point both at baseline and week 54 and did not have any major protocol violations . the ancova model included terms for treatment , renal insufficiency stratum at visit 4/week 2 ( moderate or severe ) , prior diabetes pharmacotherapy ( on or not on aha ) , and a covariate for baseline a1c . sitagliptin was to be declared noninferior to glipizide in lowering a1c at week 54 if the upper bound of the 95% ci around the between - group difference was less than the noninferiority margin of 0.4% . the changes ( or percent changes ) from baseline in all other continuous efficacy end points at week 54 , except for serum triglycerides and homa- , were analyzed using the ancova model described for a1c at week 54 , substituting the relevant baseline efficacy measurement as a covariate . the analysis of triglycerides used a nonparametric approach based on ranks . due to the presence of outliers , homa- was analyzed using a robust m - estimation approach that minimized the influence of outliers ( 18 ) . the proportion of individuals whose a1c values met predefined glycemic goals was based on the miettinen and nurminen ( m&n ) method ( 19 ) . the ancova model included the following terms : treatment , renal insufficiency stratum , prior diabetes pharmacotherapy , baseline value , subgroup , and treatment - by - subgroup interaction . due to enrollment challenges , approximately 324 patients ( 162 patients per group ) were expected to be available for the pp analysis . using an sd of 1.1% for a1c change from baseline at week 54 , the study had 90% power to declare noninferiority for a margin of 0.4% at an level of 0.05 , assuming the true mean difference was 0% . a composite end point of a1c reduction from baseline of > 0.5% , no body weight gain , and no hypoglycemia was evaluated post hoc . the 95% ci for proportion difference was calculated using the m&n method ( 19 ) , stratified by renal insufficiency stratum , prior diabetes pharmacotherapy ( on or not on oral ahas ) , and baseline a1c ( < 8 or 8% ) . the odds ratio and 95% ci of achieving the composite end point for sitagliptin versus glipizide were provided using logistic regression , adjusting for treatment , renal insufficiency stratum , prior diabetes pharmacotherapy , and baseline a1c . all patients who had measurements at baseline and week 54 for both a1c and body weight were included in the analysis . safety analyses included all randomized patients who received at least one dose of study drug and included data through 28 days following the last day of study medication . additional analyses of cardiovascular events included data through 28 days following week 54 . in these additional analyses , events that occurred up to 54 weeks postrandomization in patients who discontinued prior to week 54 the proportions of patients with one or more events were analyzed using the m&n method ( 19 ) . analysis of continuous safety end points required measurements at baseline and at one or more postbaseline time points . change from baseline in body weight at week 54 was analyzed using an ancova model similar to that used for the efficacy analyses , based on all patients with measurements both at baseline and week 54 . between - group differences and 95% cis for the exposure - adjusted incidence rates ( i.e. , number of patients with 1 event per 100 patient - years ) were provided [ using the m&n method ( 19 ) , stratified as per the study randomization ] for confirmed , adjudicated cardiovascular aes . except for end points related to confirmed , adjudicated heart failure and cardiovascular aes , analyses of both efficacy and safety excluded data following initiation of insulin rescue therapy . data from one study site ( three patients ) were considered potentially unreliable due to lack of compliance with good clinical practice and excluded from all analyses . of the remaining 423 randomized patients ( sitagliptin , n = 211 ; glipizide , n = 212 ) , 77.7% in the sitagliptin group and 80.2% in the glipizide group completed the 54-week study . the primary reasons for discontinuation in both treatment groups were aes and withdrawal of informed consent ( supplementary fig . demographic and anthropometric traits were generally similar for both treatment groups ( supplementary table 1 ) . the mean dose of glipizide was 7.7 mg / day for the pp population . for the overall cohort of patients , the mean duration of exposure to study drug was 312 days in the sitagliptin group and 318 days in the glipizide group . at week 54 , similar reductions from baseline ( mean of 7.8% in both groups ) in a1c were observed in both treatment groups ( table 1 ) . the difference in least squares ( ls ) mean change for sitagliptin versus glipizide ( 0.11% [ 95% ci 0.29 to 0.06 ] ) met the criterion for noninferiority because the upper bound of the 95% ci was less than the prespecified noninferiority margin ( 0.4% ) . , 47.4% of patients in the sitagliptin group versus 41.5% in the glipizide group met the a1c goal of < 7% ( difference 5.6% [ 95% ci 5.9 to 16.9 ] ) and 17.8% in the sitagliptin group versus 14.8% in the glipizide group met the a1c goal of < 6.5% ( difference 3.3% [ 95% ci 5.7 to 12.1 ] ) . however , greater changes from baseline in a1c were observed in patients with higher a1c at baseline ( baseline a1c 8% ; ls mean change from baseline 1.25% [ 95% ci 1.48 to 1.02 ] in the sitagliptin group and 1.10% [ 1.32 to 0.87 ] in the glipizide group ) relative to those with lower a1c at baseline ( baseline a1c < 8% ; ls mean change from baseline 0.46% [ 95% ci 0.63 to 0.28 ] in the sitagliptin group and 0.38% [ 0.55 to 0.21 ] in the glipizide group ) for both treatment groups . the post hoc composite end point ( reduction in a1c > 0.5% , no body weight gain , and no hypoglycemia ) was achieved in a significantly greater proportion of patients with sitagliptin versus glipizide ( 35.7 vs. 14.2% , respectively ) . the odds ratio for achieving the composite end point with sitagliptin compared with glipizide was 3.4 ( 95% ci 1.96.2 ) . change from baseline in efficacy end points in the pp population at week 54 a : change in a1c over time in pp population . reductions from baseline in fpg at week 54 were observed in both treatment groups ( table 1 ) . the profiles of mean change from baseline in fpg over time showed similar trends in both groups , beginning with a decrease in the first 12 weeks , followed by generally stable levels for the remainder of the study ( fig . proinsulin / insulin ratio , and homa - ir were similar for both treatment groups ( table 1 ) . significant increases from baseline in homa- were observed in both treatment groups , with a greater increase in the glipizide group ( table 1 ) . through the 54-week treatment period , 19 of 211 ( 9.0% ) patients in the sitagliptin group and 23 of 212 ( 10.8% ) in the glipizide group decreases relative to baseline for total cholesterol , ldl - c , non hdl - c , and triglycerides and an increase relative to baseline for hdl - c were observed ( table 1 ) . in the glipizide group , increases relative to baseline were observed for total cholesterol , hdl - c , ldl - c , and non hdl - c , and a decrease relative to baseline was observed for triglycerides ( table 1 ) . between - group comparisons showed significantly greater reductions in the sitagliptin group compared with the glipizide group for total cholesterol , ldl - c , and non hdl - c ( table 1 ) . the incidences of overall aes and discontinuation due to aes were similar between groups ( table 2 ) . a total of 10 patient deaths were reported during the study : 3 in the sitagliptin group and 7 in the glipizide group ( supplementary table 2 ) . aes in the neoplasms , benign , malignant , and unspecified ( including cysts and polyps ) system organ class were reported for six patients ( 2.9% ) in the sitagliptin group and none in the glipizide group ( supplementary table 3 ) . of the six events reported in the sitagliptin group , three were confirmed malignant disease , and three were either nonmalignant or not histologically confirmed . each event comprised a different type of lesion ( breast cancer , chronic myeloid leukemia , lung cancer , pancreatic head mass , polycythemia vera , and thyroid nodule ) , all were first identified within 6 months of initiation of therapy , and all were considered as not related to the study drug by the investigator . the proportion of patients reporting aes of symptomatic hypoglycemia was significantly lower ( p = 0.001 ) in the sitagliptin group ( 6.2% ) compared with the glipizide group ( 17.0% ) . overall , 1.4% of patients in the sitagliptin group were reported with a severe episode of hypoglycemia compared with 2.8% in the glipizide group ( between - group difference 1.4 [ 95% ci 4.8 to 1.5 ] ) . for gastrointestinal aes , there were no significant differences between groups in the incidences of abdominal pain , diarrhea , and vomiting and a significantly lower incidence of nausea ( p = 0.025 ) with sitagliptin versus glipizide . eight patients experienced vascular events in the sitagliptin group and 11 in the glipizide group . in the prespecified analysis adjusting for exposure to treatment , the incidence rate of vascular events was 3.5 incident events per 100 patient - years with sitagliptin compared with 4.8 incident events per 100 patient - years with glipizide . no patients in the sitagliptin group and four patients in the glipizide group experienced an ae of heart failure . over the study period , body weight decreased in the sitagliptin group ( 0.6 kg ) and increased in the glipizide group ( 1.2 kg ) , resulting in a statistically significant ( p < 0.001 ) between - group difference of 1.8 kg ( fig . 2 ) . change in body weight over time . in both treatment groups , similar decreases from baseline in egfr were observed at week 54 ( sitagliptin , 3.9 ml / min/1.73 m ; glipizide , 3.3 ml / min/1.73 m ; supplementary fig . of the randomized patients with moderate renal insufficiency at baseline , 28 of 149 ( 18.8% ) in the sitagliptin group and 17 of 154 ( 11.0% ) in the glipizide group transitioned to severe renal insufficiency status during the study . the change from baseline in urine albumin / creatinine ratio at week 54 is provided in supplementary fig . 2b ; the 95% ci around the between - group difference for this analysis included 0 . no clinically meaningful between - group differences were noted in the proportions of patients with values meeting predefined limits of change criteria for any of the measured chemistry and hematology parameters or in blood pressure or other vital signs . data from one study site ( three patients ) were considered potentially unreliable due to lack of compliance with good clinical practice and excluded from all analyses . of the remaining 423 randomized patients ( sitagliptin , n = 211 ; glipizide , n = 212 ) , 77.7% in the sitagliptin group and 80.2% in the glipizide group completed the 54-week study . the primary reasons for discontinuation in both treatment groups were aes and withdrawal of informed consent ( supplementary fig . demographic and anthropometric traits were generally similar for both treatment groups ( supplementary table 1 ) . the mean dose of glipizide was 7.7 mg / day for the pp population . for the overall cohort of patients , the mean duration of exposure to study drug was 312 days in the sitagliptin group and 318 days in the glipizide group . at week 54 , similar reductions from baseline ( mean of 7.8% in both groups ) in a1c were observed in both treatment groups ( table 1 ) . the difference in least squares ( ls ) mean change for sitagliptin versus glipizide ( 0.11% [ 95% ci 0.29 to 0.06 ] ) met the criterion for noninferiority because the upper bound of the 95% ci was less than the prespecified noninferiority margin ( 0.4% ) . change from baseline in a1c over time is presented in fig . , 47.4% of patients in the sitagliptin group versus 41.5% in the glipizide group met the a1c goal of < 7% ( difference 5.6% [ 95% ci 5.9 to 16.9 ] ) and 17.8% in the sitagliptin group versus 14.8% in the glipizide group met the a1c goal of < 6.5% ( difference 3.3% [ 95% ci 5.7 to 12.1 ] ) . however , greater changes from baseline in a1c were observed in patients with higher a1c at baseline ( baseline a1c 8% ; ls mean change from baseline 1.25% [ 95% ci 1.48 to 1.02 ] in the sitagliptin group and 1.10% [ 1.32 to 0.87 ] in the glipizide group ) relative to those with lower a1c at baseline ( baseline a1c < 8% ; ls mean change from baseline 0.46% [ 95% ci 0.63 to 0.28 ] in the sitagliptin group and 0.38% [ 0.55 to 0.21 ] in the glipizide group ) for both treatment groups . the post hoc composite end point ( reduction in a1c > 0.5% , no body weight gain , and no hypoglycemia ) was achieved in a significantly greater proportion of patients with sitagliptin versus glipizide ( 35.7 vs. 14.2% , respectively ) . the odds ratio for achieving the composite end point with sitagliptin compared with glipizide was 3.4 ( 95% ci 1.96.2 ) . change from baseline in efficacy end points in the pp population at week 54 a : change in a1c over time in pp population . reductions from baseline in fpg at week 54 were observed in both treatment groups ( table 1 ) . the profiles of mean change from baseline in fpg over time showed similar trends in both groups , beginning with a decrease in the first 12 weeks , followed by generally stable levels for the remainder of the study ( fig . proinsulin / insulin ratio , and homa - ir were similar for both treatment groups ( table 1 ) . significant increases from baseline in homa- were observed in both treatment groups , with a greater increase in the glipizide group ( table 1 ) . through the 54-week treatment period , 19 of 211 ( 9.0% ) patients in the sitagliptin group and 23 of 212 ( 10.8% ) in the glipizide group were started on glycemic rescue therapy with insulin . in the sitagliptin group , decreases relative to baseline for total cholesterol , ldl - c , non hdl - c , and triglycerides and an increase relative to baseline for hdl - c were observed ( table 1 ) . in the glipizide group , increases relative to baseline were observed for total cholesterol , hdl - c , ldl - c , and non hdl - c , and a decrease relative to baseline was observed for triglycerides ( table 1 ) . between - group comparisons showed significantly greater reductions in the sitagliptin group compared with the glipizide group for total cholesterol , ldl - c , and non hdl - c ( table 1 ) . the incidences of overall aes and discontinuation due to aes were similar between groups ( table 2 ) . a total of 10 patient deaths were reported during the study : 3 in the sitagliptin group and 7 in the glipizide group ( supplementary table 2 ) . aes in the neoplasms , benign , malignant , and unspecified ( including cysts and polyps ) system organ class were reported for six patients ( 2.9% ) in the sitagliptin group and none in the glipizide group ( supplementary table 3 ) . of the six events reported in the sitagliptin group , three were confirmed malignant disease , and three were either nonmalignant or not histologically confirmed . each event comprised a different type of lesion ( breast cancer , chronic myeloid leukemia , lung cancer , pancreatic head mass , polycythemia vera , and thyroid nodule ) , all were first identified within 6 months of initiation of therapy , and all were considered as not related to the study drug by the investigator . the proportion of patients reporting aes of symptomatic hypoglycemia was significantly lower ( p = 0.001 ) in the sitagliptin group ( 6.2% ) compared with the glipizide group ( 17.0% ) . overall , 1.4% of patients in the sitagliptin group were reported with a severe episode of hypoglycemia compared with 2.8% in the glipizide group ( between - group difference 1.4 [ 95% ci 4.8 to 1.5 ] ) . for gastrointestinal aes , there were no significant differences between groups in the incidences of abdominal pain , diarrhea , and vomiting and a significantly lower incidence of nausea ( p = 0.025 ) with sitagliptin versus glipizide . eight patients experienced vascular events in the sitagliptin group and 11 in the glipizide group . in the prespecified analysis adjusting for exposure to treatment , the incidence rate of vascular events was 3.5 incident events per 100 patient - years with sitagliptin compared with 4.8 incident events per 100 patient - years with glipizide . no patients in the sitagliptin group and four patients in the glipizide group experienced an ae of heart failure . over the study period , body weight decreased in the sitagliptin group ( 0.6 kg ) and increased in the glipizide group ( 1.2 kg ) , resulting in a statistically significant ( p < 0.001 ) between - group difference of 1.8 kg ( fig . similar decreases from baseline in egfr were observed at week 54 ( sitagliptin , 3.9 ml / min/1.73 m ; glipizide , 3.3 ml / min/1.73 m ; supplementary fig . 2a ) . of the randomized patients with moderate renal insufficiency at baseline , 28 of 149 ( 18.8% ) in the sitagliptin group and 17 of 154 ( 11.0% ) in the glipizide group the change from baseline in urine albumin / creatinine ratio at week 54 is provided in supplementary fig . 2b ; the 95% ci around the between - group difference for this analysis included 0 . no clinically meaningful between - group differences were noted in the proportions of patients with values meeting predefined limits of change criteria for any of the measured chemistry and hematology parameters or in blood pressure or other vital signs . in patients with t2 dm and chronic renal insufficiency , sitagliptin and glipizide provided similar a1c - lowering efficacy after 54 weeks of treatment , confirming noninferiority of sitagliptin relative to glipizide . in the sitagliptin group , the maximal a1c reduction occurred at week 42 , with a subsequent small increase at week 54 . in the glipizide group , the maximal a1c reduction occurred at week 18 , which remained generally stable for the duration of the study . the stable response with glipizide was likely due to dose uptitration , which was permitted throughout the study to maintain adequate glycemic control . in contrast , the sitagliptin dose was to remain unchanged throughout the study , except for dose reduction as required if a patient s renal insufficiency status changed from moderate to severe . the effects on a1c were generally consistent across prespecified demographic and disease - related subgroups . in both treatment groups , greater a1c reductions were observed in patients with baseline a1c > 8% relative to patients with a1c 8% . similar to the reductions from baseline in a1c , fpg decreased to a similar extent in the two treatment groups over 54 weeks . results from assessment of additional efficacy end points were generally similar with sitagliptin and glipizide . no significant within - group changes from baseline in fasting insulin , proinsulin , or the proinsulin / insulin ratio were observed with either treatment . these results are consistent with the findings of a prior study ( 12 ) . greater reductions from baseline in total cholesterol , ldl - c , and non hdl - c and increased hdl - c were observed with sitagliptin compared with glipizide . the results from this study support the favorable safety and tolerability profile of sitagliptin in patients with moderate or severe renal insufficiency . the incidences of aes , including those considered drug - related by the investigator , serious aes , as well as most other ae summary measures , tended to be lower for the sitagliptin group relative to the glipizide group . the percent of patients with events of symptomatic hypoglycemia in the glipizide group was almost three times higher and the number of events of hypoglycemia was more than four times higher with glipizide than with sitagliptin , despite the similar a1c reductions in both groups . in addition , a higher incidence of aes of decreased blood glucose was reported for patients in the glipizide group compared with those in the sitagliptin group . these results are consistent with those from another study comparing sitagliptin and glipizide ( 12 ) . in this study , neoplasms were reported for six patients ( 2.9% ) in the sitagliptin group and none in the glipizide group . none of the events was considered by the investigator as related to the study drug , the neoplasms reported comprised diverse types and are those generally observed in older patients , and all were first identified within 6 months of initiation of therapy . neither the overall neoplasm incidence nor the characteristics of these events are suggestive of an underlying relationship between the events and treatment with sitagliptin . assessment of vascular events was of particular interest , given the high frequency of such events in patients with t2 dm and chronic renal insufficiency . overall , three ( 1.4% ) patients in the sitagliptin group died , compared with seven ( 3.3% ) patients in the glipizide group . the overall mortality observed was consistent with the expected rate for a patient population with long - standing diabetes ( mean of 10 years ) and multiple comorbidities , including hypertension , that typically accompany chronic renal insufficiency . a thorough assessment of the effects of study therapy on renal function showed no meaningful between - group differences in changes from baseline in egfr , serum creatinine , urine albumin / creatinine ratio , or uric acid or in the number of patients with laboratory values meeting the predefined limits of change for serum creatinine or uric acid . change in body weight was notably different between groups . over the 54-week treatment period , treatment with glipizide resulted in weight gain , whereas treatment with sitagliptin resulted in weight loss , resulting in a statistically significant and clinically meaningful between - group difference of 1.8 kg . these effects on body weight are consistent with previous reports ( 20,21 ) . a significantly greater proportion of patients in the sitagliptin group achieved the composite end point of reduction in a1c > 0.5% , no body weight gain , and no hypoglycemia compared with those in the glipizide group . in summary , sitagliptin provided glycemic control that was noninferior to that observed with glipizide in patients with t2 dm and chronic renal insufficiency who had inadequate glycemic control . the percentage of patients with reported hypoglycemia and the number of events of hypoglycemia were substantially and clinically meaningfully lower with sitagliptin compared with glipizide . treatment with sitagliptin led to a small reduction in body weight , and treatment with glipizide led to weight gain . other than the differences in hypoglycemia and body weight , both agents had similar safety profiles , with a low and similar rate of serious aes and discontinuations due to aes .

objectivepatients with type 2 diabetes mellitus ( t2 dm ) and chronic kidney disease have an increased risk of micro- and macrovascular disease , but limited options for antihyperglycemic therapy . we compared the efficacy and safety of sitagliptin with glipizide in patients with t2 dm and moderate - to - severe chronic renal insufficiency and inadequate glycemic control.research design and methodspatients ( n = 426 ) were randomized 1:1 to sitagliptin ( 50 mg every day [ q.d . ] for moderate renal insufficiency and 25 mg q.d . for severe renal insufficiency ) or glipizide ( 2.5 mg q.d . , adjusted based on glycemic control to a 10-mg twice a day maximum dose ) . randomization was stratified by : 1 ) renal status ( moderate or severe renal insufficiency ) ; 2 ) history of cardiovascular disease ; and 3 ) history of heart failure.resultsat week 54 , treatment with sitagliptin was noninferior to treatment with glipizide in a1c change from baseline ( 0.8 vs. 0.6% ; between - group difference 0.11% ; 95% ci 0.29 to 0.06 ) because the upper bound of the 95% ci was less than the prespecified noninferiority margin of 0.4% . there was a lower incidence of symptomatic hypoglycemia adverse events ( aes ) with sitagliptin versus glipizide ( 6.2 and 17.0% , respectively ; p = 0.001 ) and a decrease in body weight with sitagliptin ( 0.6 kg ) versus an increase ( 1.2 kg ) with glipizide ( difference , 1.8 kg ; p < 0.001 ) . the incidence of gastrointestinal aes was low with both treatments.conclusionsin patients with t2 dm and chronic renal insufficiency , sitagliptin and glipizide provided similar a1c - lowering efficacy . sitagliptin was generally well - tolerated , with a lower risk of hypoglycemia and weight loss versus weight gain , relative to glipizide .

RESEARCH DESIGN AND METHODS Study design Patients Efficacy assessments Safety and tolerability assessments Blood collection and assays Statistical analysis RESULTS Patient characteristics and disposition Efficacy Safety CONCLUSIONS

randomization was stratified based on : 1 ) renal insufficiency status ( moderate or severe ) , 2 ) history of cardiovascular disease ( yes or no ) , and 3 ) history of heart failure ( yes or no ) . patients with moderate renal insufficiency received 50 mg / day of sitagliptin ( two 25-mg tablets ) or matching placebo . patients with severe renal insufficiency received 25 mg / day of sitagliptin ( one 25-mg tablet ) or matching placebo . the dose of sitagliptin was reduced from 50 mg / day to 25 mg / day for patients whose renal status changed from moderate to severe during the study . glipizide was administered at a starting dose of 2.5 mg / day , prior to the morning meal , and electively titrated to a maximum of 20 mg / day as considered appropriate by the investigator based on the patient s glycemic control ; the dose of glipizide could also be reduced or interrupted to prevent hypoglycemia . patients with t2 dm could participate if they had moderate to severe chronic renal insufficiency ( egfr < 50 ml / min/1.73 m using the modification of diet in renal disease equation ) , were not on dialysis and unlikely to require dialysis for the duration of the study , had an a1c 7.0 and 9.0% , and were 30 years of age at the screening visit . patients taking insulin within 12 weeks of the screening visit , patients with type 1 diabetes , history of ketoacidosis , acute renal disease , history of renal transplant , liver disease , a recent ( within 3 months ) cardiovascular event , hepatic transaminase levels two or more times the upper limit of normal , thyroid stimulating hormone outside the reference range , or triglycerides > 600 mg / dl were excluded from participation . a prespecified analysis evaluated the consistency of the a1c - lowering effects of sitagliptin versus glipizide across predefined subgroups based on baseline characteristics a1c ( < and 8% ) , age ( < and 65 years ) , severity of ckd ( egfr 30 to < 50 [ moderate ] and < 30 [ severe ] ml / min/1.73 m ) , bmi ( < and median ) , duration of t2 dm ( < and median ) , ethnicity ( hispanic or latino and non - hispanic or non - latino ) , sex , prior antihyperglycemic therapy status ( yes or no ) , and race ( asian , black , white , or other ) . a post hoc analysis evaluated the effect of sitagliptin versus glipizide on a composite end point consisting of glycemic control ( reduction in a1c > 0.5% ) , no body weight gain , and no hypoglycemia . the primary study end points were change from baseline in a1c , the incidence of hypoglycemia events , and overall safety and tolerability . the ancova model included terms for treatment , renal insufficiency stratum at visit 4/week 2 ( moderate or severe ) , prior diabetes pharmacotherapy ( on or not on aha ) , and a covariate for baseline a1c . sitagliptin was to be declared noninferior to glipizide in lowering a1c at week 54 if the upper bound of the 95% ci around the between - group difference was less than the noninferiority margin of 0.4% . using an sd of 1.1% for a1c change from baseline at week 54 , the study had 90% power to declare noninferiority for a margin of 0.4% at an level of 0.05 , assuming the true mean difference was 0% . a composite end point of a1c reduction from baseline of > 0.5% , no body weight gain , and no hypoglycemia was evaluated post hoc . the 95% ci for proportion difference was calculated using the m&n method ( 19 ) , stratified by renal insufficiency stratum , prior diabetes pharmacotherapy ( on or not on oral ahas ) , and baseline a1c ( < 8 or 8% ) . change from baseline in body weight at week 54 was analyzed using an ancova model similar to that used for the efficacy analyses , based on all patients with measurements both at baseline and week 54 . randomization was stratified based on : 1 ) renal insufficiency status ( moderate or severe ) , 2 ) history of cardiovascular disease ( yes or no ) , and 3 ) history of heart failure ( yes or no ) . patients with moderate renal insufficiency received 50 mg / day of sitagliptin ( two 25-mg tablets ) or matching placebo . patients with severe renal insufficiency received 25 mg / day of sitagliptin ( one 25-mg tablet ) or matching placebo . the dose of sitagliptin was reduced from 50 mg / day to 25 mg / day for patients whose renal status changed from moderate to severe during the study . glipizide was administered at a starting dose of 2.5 mg / day , prior to the morning meal , and electively titrated to a maximum of 20 mg / day as considered appropriate by the investigator based on the patient s glycemic control ; the dose of glipizide could also be reduced or interrupted to prevent hypoglycemia . patients with t2 dm could participate if they had moderate to severe chronic renal insufficiency ( egfr < 50 ml / min/1.73 m using the modification of diet in renal disease equation ) , were not on dialysis and unlikely to require dialysis for the duration of the study , had an a1c 7.0 and 9.0% , and were 30 years of age at the screening visit . patients taking insulin within 12 weeks of the screening visit , patients with type 1 diabetes , history of ketoacidosis , acute renal disease , history of renal transplant , liver disease , a recent ( within 3 months ) cardiovascular event , hepatic transaminase levels two or more times the upper limit of normal , thyroid stimulating hormone outside the reference range , or triglycerides > 600 mg / dl were excluded from participation . a prespecified analysis evaluated the consistency of the a1c - lowering effects of sitagliptin versus glipizide across predefined subgroups based on baseline characteristics a1c ( < and 8% ) , age ( < and 65 years ) , severity of ckd ( egfr 30 to < 50 [ moderate ] and < 30 [ severe ] ml / min/1.73 m ) , bmi ( < and median ) , duration of t2 dm ( < and median ) , ethnicity ( hispanic or latino and non - hispanic or non - latino ) , sex , prior antihyperglycemic therapy status ( yes or no ) , and race ( asian , black , white , or other ) . a post hoc analysis evaluated the effect of sitagliptin versus glipizide on a composite end point consisting of glycemic control ( reduction in a1c > 0.5% ) , no body weight gain , and no hypoglycemia . safety measurements included evaluation of adverse events ( aes ) , physical examination and vital signs , and electrocardiograms . blood was collected at baseline ( predose ) and at various time points throughout the treatment period of 54 weeks for efficacy and safety measurements . the primary study end points were change from baseline in a1c , the incidence of hypoglycemia events , and overall safety and tolerability . the ancova model included terms for treatment , renal insufficiency stratum at visit 4/week 2 ( moderate or severe ) , prior diabetes pharmacotherapy ( on or not on aha ) , and a covariate for baseline a1c . sitagliptin was to be declared noninferior to glipizide in lowering a1c at week 54 if the upper bound of the 95% ci around the between - group difference was less than the noninferiority margin of 0.4% . using an sd of 1.1% for a1c change from baseline at week 54 , the study had 90% power to declare noninferiority for a margin of 0.4% at an level of 0.05 , assuming the true mean difference was 0% . a composite end point of a1c reduction from baseline of > 0.5% , no body weight gain , and no hypoglycemia was evaluated post hoc . the odds ratio and 95% ci of achieving the composite end point for sitagliptin versus glipizide were provided using logistic regression , adjusting for treatment , renal insufficiency stratum , prior diabetes pharmacotherapy , and baseline a1c . change from baseline in body weight at week 54 was analyzed using an ancova model similar to that used for the efficacy analyses , based on all patients with measurements both at baseline and week 54 . at week 54 , similar reductions from baseline ( mean of 7.8% in both groups ) in a1c were observed in both treatment groups ( table 1 ) . the difference in least squares ( ls ) mean change for sitagliptin versus glipizide ( 0.11% [ 95% ci 0.29 to 0.06 ] ) met the criterion for noninferiority because the upper bound of the 95% ci was less than the prespecified noninferiority margin ( 0.4% ) . , 47.4% of patients in the sitagliptin group versus 41.5% in the glipizide group met the a1c goal of < 7% ( difference 5.6% [ 95% ci 5.9 to 16.9 ] ) and 17.8% in the sitagliptin group versus 14.8% in the glipizide group met the a1c goal of < 6.5% ( difference 3.3% [ 95% ci 5.7 to 12.1 ] ) . however , greater changes from baseline in a1c were observed in patients with higher a1c at baseline ( baseline a1c 8% ; ls mean change from baseline 1.25% [ 95% ci 1.48 to 1.02 ] in the sitagliptin group and 1.10% [ 1.32 to 0.87 ] in the glipizide group ) relative to those with lower a1c at baseline ( baseline a1c < 8% ; ls mean change from baseline 0.46% [ 95% ci 0.63 to 0.28 ] in the sitagliptin group and 0.38% [ 0.55 to 0.21 ] in the glipizide group ) for both treatment groups . the post hoc composite end point ( reduction in a1c > 0.5% , no body weight gain , and no hypoglycemia ) was achieved in a significantly greater proportion of patients with sitagliptin versus glipizide ( 35.7 vs. 14.2% , respectively ) . the odds ratio for achieving the composite end point with sitagliptin compared with glipizide was 3.4 ( 95% ci 1.96.2 ) . the profiles of mean change from baseline in fpg over time showed similar trends in both groups , beginning with a decrease in the first 12 weeks , followed by generally stable levels for the remainder of the study ( fig . in the glipizide group , increases relative to baseline were observed for total cholesterol , hdl - c , ldl - c , and non hdl - c , and a decrease relative to baseline was observed for triglycerides ( table 1 ) . the proportion of patients reporting aes of symptomatic hypoglycemia was significantly lower ( p = 0.001 ) in the sitagliptin group ( 6.2% ) compared with the glipizide group ( 17.0% ) . overall , 1.4% of patients in the sitagliptin group were reported with a severe episode of hypoglycemia compared with 2.8% in the glipizide group ( between - group difference 1.4 [ 95% ci 4.8 to 1.5 ] ) . for gastrointestinal aes , there were no significant differences between groups in the incidences of abdominal pain , diarrhea , and vomiting and a significantly lower incidence of nausea ( p = 0.025 ) with sitagliptin versus glipizide . in the prespecified analysis adjusting for exposure to treatment , the incidence rate of vascular events was 3.5 incident events per 100 patient - years with sitagliptin compared with 4.8 incident events per 100 patient - years with glipizide . over the study period , body weight decreased in the sitagliptin group ( 0.6 kg ) and increased in the glipizide group ( 1.2 kg ) , resulting in a statistically significant ( p < 0.001 ) between - group difference of 1.8 kg ( fig . of the randomized patients with moderate renal insufficiency at baseline , 28 of 149 ( 18.8% ) in the sitagliptin group and 17 of 154 ( 11.0% ) in the glipizide group transitioned to severe renal insufficiency status during the study . 2b ; the 95% ci around the between - group difference for this analysis included 0 . at week 54 , similar reductions from baseline ( mean of 7.8% in both groups ) in a1c were observed in both treatment groups ( table 1 ) . the difference in least squares ( ls ) mean change for sitagliptin versus glipizide ( 0.11% [ 95% ci 0.29 to 0.06 ] ) met the criterion for noninferiority because the upper bound of the 95% ci was less than the prespecified noninferiority margin ( 0.4% ) . however , greater changes from baseline in a1c were observed in patients with higher a1c at baseline ( baseline a1c 8% ; ls mean change from baseline 1.25% [ 95% ci 1.48 to 1.02 ] in the sitagliptin group and 1.10% [ 1.32 to 0.87 ] in the glipizide group ) relative to those with lower a1c at baseline ( baseline a1c < 8% ; ls mean change from baseline 0.46% [ 95% ci 0.63 to 0.28 ] in the sitagliptin group and 0.38% [ 0.55 to 0.21 ] in the glipizide group ) for both treatment groups . the post hoc composite end point ( reduction in a1c > 0.5% , no body weight gain , and no hypoglycemia ) was achieved in a significantly greater proportion of patients with sitagliptin versus glipizide ( 35.7 vs. 14.2% , respectively ) . the odds ratio for achieving the composite end point with sitagliptin compared with glipizide was 3.4 ( 95% ci 1.96.2 ) . the profiles of mean change from baseline in fpg over time showed similar trends in both groups , beginning with a decrease in the first 12 weeks , followed by generally stable levels for the remainder of the study ( fig . the proportion of patients reporting aes of symptomatic hypoglycemia was significantly lower ( p = 0.001 ) in the sitagliptin group ( 6.2% ) compared with the glipizide group ( 17.0% ) . overall , 1.4% of patients in the sitagliptin group were reported with a severe episode of hypoglycemia compared with 2.8% in the glipizide group ( between - group difference 1.4 [ 95% ci 4.8 to 1.5 ] ) . for gastrointestinal aes , there were no significant differences between groups in the incidences of abdominal pain , diarrhea , and vomiting and a significantly lower incidence of nausea ( p = 0.025 ) with sitagliptin versus glipizide . in the prespecified analysis adjusting for exposure to treatment , the incidence rate of vascular events was 3.5 incident events per 100 patient - years with sitagliptin compared with 4.8 incident events per 100 patient - years with glipizide . over the study period , body weight decreased in the sitagliptin group ( 0.6 kg ) and increased in the glipizide group ( 1.2 kg ) , resulting in a statistically significant ( p < 0.001 ) between - group difference of 1.8 kg ( fig . of the randomized patients with moderate renal insufficiency at baseline , 28 of 149 ( 18.8% ) in the sitagliptin group and 17 of 154 ( 11.0% ) in the glipizide group the change from baseline in urine albumin / creatinine ratio at week 54 is provided in supplementary fig . 2b ; the 95% ci around the between - group difference for this analysis included 0 . no clinically meaningful between - group differences were noted in the proportions of patients with values meeting predefined limits of change criteria for any of the measured chemistry and hematology parameters or in blood pressure or other vital signs . in patients with t2 dm and chronic renal insufficiency , sitagliptin and glipizide provided similar a1c - lowering efficacy after 54 weeks of treatment , confirming noninferiority of sitagliptin relative to glipizide . the results from this study support the favorable safety and tolerability profile of sitagliptin in patients with moderate or severe renal insufficiency . the percent of patients with events of symptomatic hypoglycemia in the glipizide group was almost three times higher and the number of events of hypoglycemia was more than four times higher with glipizide than with sitagliptin , despite the similar a1c reductions in both groups . assessment of vascular events was of particular interest , given the high frequency of such events in patients with t2 dm and chronic renal insufficiency . a thorough assessment of the effects of study therapy on renal function showed no meaningful between - group differences in changes from baseline in egfr , serum creatinine , urine albumin / creatinine ratio , or uric acid or in the number of patients with laboratory values meeting the predefined limits of change for serum creatinine or uric acid . over the 54-week treatment period , treatment with glipizide resulted in weight gain , whereas treatment with sitagliptin resulted in weight loss , resulting in a statistically significant and clinically meaningful between - group difference of 1.8 kg . a significantly greater proportion of patients in the sitagliptin group achieved the composite end point of reduction in a1c > 0.5% , no body weight gain , and no hypoglycemia compared with those in the glipizide group . in summary , sitagliptin provided glycemic control that was noninferior to that observed with glipizide in patients with t2 dm and chronic renal insufficiency who had inadequate glycemic control . the percentage of patients with reported hypoglycemia and the number of events of hypoglycemia were substantially and clinically meaningfully lower with sitagliptin compared with glipizide . treatment with sitagliptin led to a small reduction in body weight , and treatment with glipizide led to weight gain . other than the differences in hypoglycemia and body weight , both agents had similar safety profiles , with a low and similar rate of serious aes and discontinuations due to aes .

insulin , a key hormone in blood glucose homeostasis , has been detected in human colostrum at supraphysiological levels ( 114306 mu / l ) before decreasing to similar concentrations to those found in blood in the fasting state by day 5 postpartum in healthy mothers [ 1 , 2 ] . has demonstrated that insulin is also present in the milk of mothers with type 1 diabetes mellitus , who lack any endogenous insulin secretion from the pancreas . they also demonstrated that levels of insulin are significantly higher ( p < 0.001 ) in milk from type 1 diabetic mothers than that of nondiabetic control mothers , at both days 3 to 7 postpartum and 3 months postpartum . interestingly , insulin levels in the milk of control mothers vary greatly between studies , while those reported by shehadeh et al . showed higher milk insulin concentrations than those reported by tiittanen et al . , suggesting that significant variation exists between mothers regardless of the presence of diabetes mellitus . however , the work by tiittanen et al . and shehadeh et al . does not further the time of collection or fasting state of mothers , which may account for some of the variability observed . in addition , kulski and hartmann have demonstrated that the levels of insulin in breastmilk may vary with the stage of lactation , with high levels present in colostrum and levels decreasing to a plateau in established lactation . despite this , current medical guides state that insulin is a peptide that is too large to be secreted into milk [ 57 ] . given the established role of dietary insulin in the maturation of intestinal epithelium in rats , it can be postulated that insulin present in milk may play a similar role in the development of human intestinal epithelium [ 8 , 9 ] . additionally , work in rats has shown that a trypsin inhibitor present in milk acts to preserve insulin function [ 10 , 11 ] . furthermore , epithelial cell insulin receptors exist in the intestine of both piglets and calves , and insulin has been suggested to play a role in influencing growth and development of the small intestine [ 12 , 13 ] . this would suggest that insulin could retain biological activity when ingested by the infant and that intact insulin may pass over from the gastrointestinal tract of infants into blood . this suggestion is further supported and extensively discussed in a review of the in vivo and in vitro effects of insulin on the gastrointestinal tract by shehadeh et al . . endogenous insulin typically exists in blood in equilibrium between a free monomeric state and a hexamer with zinc ; it is only able to bind to receptors in the free state and has a half - life of ~20 minutes . a recombinant human protein is typically used for therapy . due to the relatively short half - life , a number of short- or long - acting variants have been developed , either by modifying the amino acid sequence to prevent hexamer formation , or through complexing to zinc or protophane to shift the equilibrium towards the hexameric state [ 6 , 16 ] . due to these modifications to the amino acid sequence , specific assays can be used to identify whether the insulin present in a fluid sample is endogenous or a modified exogenous form of insulin [ 17 , 18 ] . . demonstrated that human dietary insulin in milk may have a tolerogenic effect against bovine insulin in formula and downregulate the production of igg antibodies to bovine insulin , protecting against the development of autoimmunity , as shown with decreased levels of igg to insulin in breast - fed children compared to those fed with cow 's milk formula containing bovine insulin . despite this beneficial effect at lower milk insulin levels , they also noted that high endogenous insulin levels in human milk were associated with a significantly increased incidence of -cell autoimmunity in children , in both control mothers ( p = 0.030 ) and mothers with type 1 diabetes ( p = 0.045 ) , which may suggest that exposure to high levels of endogenous insulin can also promote autoimmunity . however , it must be noted that this does not imply that development of type 1 diabetes mellitus is similarly promoted . in contrast to this , other recent work has demonstrated that administration of oral insulin may help protect residual -cell function in children and adults with recent - onset type 1 diabetes [ 4 , 19 , 20 ] . other work has supported the possible role of breast milk in the prevention of type 1 diabetes mellitus , since there is a lower incidence of type 1 diabetes in breast - fed children , but this is still a controversial area and contended by other sources [ 2224 ] . the effects that type 2 and gestational diabetes mellitus may have on insulin concentration in milk have not yet been identified . given that the only known source of insulin in mothers with type 1 diabetes is injected exogenous insulin , this allows the hypothesis that either insulin is being transported into human milk , in contrast to current literature , or that it is being synthesised in the mammary gland and then secreted into the milk . there are currently no studies comparing the levels of exogenous insulin in milk compared to that produced endogenously in mothers with or without diabetes mellitus . the purpose of this study was to identify the origin of the insulin present in the milk of mothers with type 1 diabetes mellitus as either endogenous or exogenous by determining the levels of total insulin , endogenous insulin , and c - peptide in the milk compared to mothers without diabetes mellitus . this study also aimed to compare the levels of insulin in breast milk from mothers with type 2 diabetes and then investigate any relationship between blood glucose , milk insulin , glucose , c - peptide , and sodium content in breast milk in each of the groups . mothers with type 2 diabetes mellitus have been included in order to compare insulin concentrations and as they should have no exogenous insulin present in their milk or blood . samples from five control mothers ( without diabetes mellitus ) all in established lactation between 1 to 6 months were randomly selected from 24-hour production milk stock collected by the lactation research group . exact state of lactation is not further specified , as it was not relevant to the aims of this study in identifying the origin of insulin in milk . mothers with diabetes mellitus were recruited from the diabetes clinic at king edward memorial hospital , western australia . four mothers with type 1 diabetes mellitus were recruited , and all mothers in this group were on insulin replacement therapy . five mothers with type 2 diabetes mellitus were recruited , and all mothers in this group was on dietary , exercise or metformin treatment , or a combination of these . one mother in the type 1 group ( t1 - 04 ) was lost to followup . all mothers in the three groups were breast feeding at the time and within 4 months postpartum . all mothers supplied written informed consent to participate , and the study was approved by the human research ethics committee , king edward memorial hospital . this involved collecting 1 to 2 ml of fore and hind milk from each breast used at each feed for a period of 24 hours . there were a varying number of feeds per day per mother , depending on infant hunger and the breast used ( single versus both ) . samples were collected before and after a feed from each breast used for each feed . other time - points involved a breastfeed from each breast so samples were collected before and after a feed from both the left and right breast . sample analysis in this and previous studies has shown no difference in milk composition between left and right breasts so this data was analysed collectively . all samples were frozen by the mothers , and then transported to the laboratory and stored at 30c prior for analysis . samples were then thawed to 37c , mixed , and centrifuged at 3,000 rpm ( 05pr-22 refrigerated centrifuge , hitachi ) for 10 minutes at 4c in 300 l polypropylene tubes to separate the fat from the skim layer . defatted milk was either immediately used for analysis or stored in appropriately labeled eppendorf tubes ( 0.5 to 1.5 ml ) and frozen at 30c for later analysis . milk samples were analysed for glucose content using the spectrophotometric method described by arthur et al . . defatted milk samples were analysed for total insulin content ( endogenous + exogenous ) using a chemiluminescent microparticle immunoassay ( cmia ) , which was performed at pathwest ( pathwest laboratory medicine wa , qeii medical complex , sir charles gairdner hospital , western australia ) . c - peptide content of milk samples was similarly determined using the mercodia ab c - peptide elisa kit . the isoinsulin assay detects endogenous insulin , along with the exogenous insulin formulations ( insulin aspart , detemir , glargine , glulisine , and lispro ) , compared to the standard insulin assay , which is specific for endogenous insulin only . exogenous insulin content was calculated by subtracting endogenous insulin content from total ( endogenous and all modified forms ) insulin content . two techniques were used to analyse total insulin content ( cmia + elisa ) in order to assess suitability of the cmia method for milk analysis ; if appropriate , it could then be used in a larger - scale study as a more cost + time - effective approach to analysis of milk insulin content . the recovery , detection , and interassay coefficient of variation are presented in table 2 . insulin content was measured for all samples , including fore + hind milk for each feed ( n = 199 ) . total fat content of the milk samples was assessed using the creamatocrit method described by lucas et al . . finally , milk sodium analysis was performed using an atomic absorption spectroscopy approach , using a varian techtron absorption spectrophotometer model a100 . haemoglobin a1c ( hba1c ) was also documented from routine analysis in pregnancy for those mothers with diabetes . although only a small number of mothers were present in each group , the large number of milk samples collected by each mother over the 24 hours periods allows for statistical analysis of differences between groups . differences between types of diabetes and between mothers were calculated using a repeated measures linear mixed - effects model , in order to take into account different numbers of feeds per day and differing numbers of mothers in each group . this was performed using the statistical functions of r ( version 2.5.1 , the r foundation for statistical computing ) . total insulin content of fore and hind milk samples was measured in all samples ( n = 199 ) using the cmia method and showed good correlation between the content of fore and hind milk samples when all mothers were considered , as shown in figure 1 . analysis of individual groups showed no significant difference in insulin content of fore and hind milk between groups ( p = 0.20 , p = 0.98 , and p = 0.14 for control , type 1 , and type 2 , resp . ) . based on this , fore and hind milk insulin levels were averaged for the purpose of investigating any circadian variation ; and this analysis showed no significant variation in insulin levels during different time points during the day . while there was circadian variation evident for all mothers , there was no consistent trend evident when analyzing as a group ( data not shown ) . in order to determine whether the insulin present in the milk of mothers with type 1 diabetes is synthesised in the mammary gland or is transported from blood , total and endogenous insulin levels were measured in all milk samples in this group ( n = 53 ) . mean sem exogenous insulin levels in the milk of mothers with type 1 diabetes were 12.22 1.34 mu / l for mother t1 - 01 , 31.54 4.95 mu / l for mother t1 - 02 , and 17.86 4.34 mu / l for mother t1 - 03 . there was no detectable endogenous insulin content in the milk of mothers with type 1 diabetes ; therefore , all insulin detected in the milk of mothers with type 1 diabetes was of exogenous origin . in order to investigate variations in insulin concentration between control mothers and mothers with diabetes , total insulin content of each milk sample ( fore + hind milk individually ) from each mother there was no significant difference between fore and hind milk insulin content , so further data algorithmically treats each data point as the mean of fore + hind milk for each feed ( time point ) . total insulin content from all samples is presented in figure 2 , along with the mean insulin content of each group . the control mother t0 - 03 demonstrated significantly higher milk insulin content than the other control mothers ( 78.23 3.96 versus 15.46 1.03 there was no significant difference in mean insulin content between control mothers and mothers with type 1 diabetes ( p = 0.74 ) , control mothers and mothers with type 2 diabetes ( p = 0.93 ) , or between mothers with type 1 and type 2 diabetes ( p = 0.62 ) . insulin is produced by cleaving insulin and c - peptide from the precursor molecule proinsulin . hence , c - peptide content of the milk from two mothers with type 1 diabetes and 3 control mothers was measured using the elisa method described ( n = 61 ) . c - peptide was detected in all samples ; however , levels in the milk of control mothers were significantly higher than those in mothers with type 1 diabetes ( 21.09 1.00 versus 14.49 1.47 pmol / l , p < 0.01 ) . there was no significant correlation between c - peptide and total insulin content in the milk of control mothers ( p = 0.79 ) or mothers with type 1 diabetes ( p = 0.85 ) . in order to investigate the transport of insulin into human milk , the sodium content of the milk from all mothers was measured using atomic absorption spectroscopy ( n = 199 ) as a marker of permeability of the paracellular pathway . there was significantly lower sodium content in the milk of mothers with type 1 diabetes compared to control mothers ( p < 0.05 ) , but no significant difference in mean sodium content between control mothers and mothers with type 2 diabetes ( p = 0.09 ) or between types of diabetes ( p = 0.54 ) . there was no significant relationship between sodium and insulin content of milk for control mothers ( p = 0.17 ) or mothers with type 1 diabetes ( p = 0.37 ) , but a mild negative correlation existed for mothers with type 2 diabetes ( r = 0.34 , p < 0.05 ) . additionally , for the outlier control mother t0 - 03 who demonstrated significantly elevated insulin levels in milk , there was no significant relationship between sodium and insulin content in milk ( p = 0.61 ) . in order to investigate any correlation between blood glucose levels and insulin content in the milk of mothers with type 1 and 2 diabetes , mothers were asked to record their blood glucose levels at least 4 times over the course of the same 24-hour period as their milk sample collection . there was a great deal of variation within mothers for each group over a 24-hour period for both milk insulin and blood glucose ; however , no significant consistent trends were identified . however , there was no significant difference ( p = 0.15 ) in mean blood glucose levels between mothers with type 1 and type 2 diabetes . mean blood glucose levels in mothers with type 1 diabetes were 8.52 0.57 mmol / l , and levels for mothers with type 2 diabetes were 7.45 0.26 mmol / l ( mean sem ) . there was no significant relationship between mean blood glucose levels and mean milk insulin levels in mothers with type 1 diabetes ( p = 0.72 ) or mothers with type 2 diabetes ( p = 0.16 ) . by comparing blood glucose levels and milk insulin concentration over time , there appears to be an inverse relationship between the two ; a reduction in blood glucose levels is mirrored by an increase in milk insulin content . mother t2 - 05 ( type 2 ) demonstrated only small variability in milk insulin content , and mother t2 - 03 ( type 2 ) demonstrated large variability in milk insulin content with only small variation in blood glucose levels . limited blood glucose data was provided by mothers due to sample collection difficulties , and further comparison is not possible within the scope of this paper . as the glucose present in human milk derives from blood glucose , there may be fluctuations in milk glucose content that correlate with either blood glucose levels or insulin content of milk . glucose content of each sample ( fore and hind milk ) was measured using the spectrophotometric method described . there was significantly higher glucose content in the milk of mothers with type 1 diabetes compared to control mothers ( p < 0.05 ) and in the milk of mothers with type 2 diabetes compared to control mothers ( p < 0.05 ) , but no significant difference in milk glucose content between types of diabetes ( p = 0.68 ) . there was no significant correlation between milk and blood glucose levels for mothers with type 1 diabetes ( p = 0.60 ) or mothers with type 2 diabetes ( p = 0.33 ) . there was no significant relationship between glucose and insulin content of milk in control mothers ( p = 0.17 ) or mothers with type 2 diabetes ( p = 0.11 ) , but there was a slight negative correlation between glucose and insulin content in milk from mothers with type 1 diabetes ( r = 0.41 , p < 0.01 ) . most current medical references state that insulin is too large to cross over from blood into milk [ 57 ] . however , there have been continuous references in the scientific literature to the presence of insulin in milk and the presence of elevated levels in the milk of mothers with diabetes compared to mothers without diabetes [ 13 , 30 ] . based on this conflicting evidence , it is proposed that insulin could either be transported into milk or be synthesised in the mammary gland and directly secreted into milk . firstly , the type of insulin present in the milk of mothers with type 1 diabetes mellitus was assessed . these mothers should have no endogenous insulin production from the pancreas , and all insulin present in their blood should result from injections of exogenous insulin . analysis of the milk of these mothers revealed that there was no endogenous insulin present , suggesting that it was all derived from injected insulin present in blood . secondly , the presence of c - peptide in the milk of two mothers with type 1 diabetes and three control mothers was measured . as the endogenous elisa used is highly specific for human insulin , it was possible that any insulin synthesised in the mammary gland may be sufficiently structurally different to insulin synthesised in the pancreas as to not be detected by the elisa , as is seen the two human isoforms of apolipoprotein b ( apob48 , synthesized in the small intestine , and apob100 , synthesized in the liver ) . as insulin and c - peptide are cleaved from proinsulin in equimolar quantities , if insulin was synthesised in the mammary gland , then there should be equimolar quantities of both present in milk . c - peptide was identified in milk mothers with type 1 diabetes , but at 15 to 20x lower than reference blood concentrations , despite the common view that patients with type 1 diabetes do not produce insulin . however , many patients with type 1 diabetes still retain some residual pancreatic -cell function , and insulin is secreted at subphysiological levels alongside c - peptide . these factors indicate that the insulin present in milk is derived from insulin in blood . insulin content in milk for all groups was found to be similar to known reference levels for blood . this is the first time insulin levels have been analysed in milk from mothers with type 2 diabetes and is in concordance with previous work investigating insulin concentration in the milk of control mothers and mothers with type 1 diabetes [ 13 ] . in contrast to other serum proteins of similar size , which are normally present in milk at up to 100x lower concentration than found in blood , these results suggest that insulin does not diffuse into milk via the paracellular pathway . this is supported by the lack of any correlation between insulin and sodium content of milk for control mothers and mothers with type 1 diabetes ; as sodium content of milk is an indicator of the permeability of the paracellular pathway , a positive correlation between insulin and sodium content of milk would be expected if insulin was entering via the paracellular pathway . additionally , sodium content was significantly lower in mothers with type 1 diabetes than in controls , suggesting that the paracellular pathway is closed in these mothers . there was no significant difference in sodium content of milk between mothers with type 2 diabetes and control mothers . these findings suggest that insulin must be actively transported into milk in order to achieve these higher concentrations . two insulin transporters have currently been identified ; an active transporter present on the blood brain barrier ( bbb ) and receptor - mediated endocytosis of insulin occurring in skeletal muscle endothelium [ 36 , 37 ] . as the bbb transporter is only capable of establishing a cerebral concentration of less than 10% of the systemic blood concentration , it is unlikely to be involved in transport of insulin in the mammary gland . however , the transport system in the skeletal muscle endothelium is capable of transporting insulin at up to 50% of blood levels . while this is still lower than the ~100% transfer seen in the mammary gland , as milk is synthesised over a period of time , this could potentially account for the high concentrations present in milk . further consideration must be given to the supraphysiological insulin concentrations demonstrated in human colostrum , and how these are achieved . while this study did not assess colostrum insulin content , it can be postulated that the potential milk trypsin inhibitor postulated in rat models may act to preserve and stabilize insulin within milk , leading to these higher concentrations [ 10 , 11 ] . alternately , active transport of insulin into milk may increase in the colostrum phase , particularly if insulin does indeed play a large role in maturation of the infant digestive system [ 13 , 14 ] . further research into insulin transport mechanisms in both colostrum and normal milk is required to better investigate this possibility . there was no significant difference between the levels of insulin in the milk of control mothers , mothers with type 1 , and mothers with type 2 diabetes , despite the insulin present in the type 1 mothers being artificial in nature . this suggests that the exogenous insulin used for treatment of type 1 diabetes is transported into milk with similar affinity to endogenous insulin in mothers without diabetes . as the exact transport mechanism has yet to be scientifically quantified , the significantly elevated milk insulin levels in control mother t0 - 03 can not be readily explained . if milk insulin concentration does reflect or mirror blood insulin concentrations , then the elevated insulin levels seen would typically represent a woman with undiagnosed , untreated type 2 diabetes . however , as the transporter has yet to be identified , the possibility remains that the elevated milk insulin levels in this mother are due to altered insulin transport , rather than elevated blood insulin levels ; as two isoforms of the insulin receptor exist , it is possible that the high specificity isoform may be preferentially expressed in the mammary gland of this mother , thus resulting in increased insulin transport into her milk . varying data exists in the literature as to the glucose content of milk in mothers with diabetes . it has been established by neville et al . that glucose transport into milk is insulin independent and dependent upon blood glucose concentrations . given this , it would be expected that patients with diabetes would have elevated milk glucose levels , reflecting their typically elevated blood glucose levels . this study identified significantly higher milk glucose concentrations in mothers with type 1 and type 2 diabetes compared to control mothers , as was predicted . there was no correlation with maternal blood glucose levels , in contrast to data reported by jackson et al . however , this may be a result of only being able to compare the mean blood and milk glucose levels for each mother ; the study by jackson et al . compared the direct relationship between blood glucose and milk glucose at time of expression . this finding and the inverse relationship have been included as a potential important course of future investigation . this study demonstrated that both exogenous and endogenous insulin , are actively transported into human milk in similar concentrations to those found in blood , in comparison to other serum proteins of similar or smaller size , such as c - peptide . presumably milk insulin must therefore play a functional or developmental role in the infant . studies in a rat model have demonstrated that oral insulin in milk is involved in maturation of intestinal epithelium and induces pancreatic amylase development at weaning [ 8 , 10 , 11 ] . it has also been demonstrated to influence lactase and saccharase activity in the small intestine in a piglet model . a series of papers reported by koldovsk show that human infants demonstrate decreased blood glucose levels in response to milk insulin in early development , suggesting that intact insulin is crossing into the bloodstream of the infant . . also showed that suckling rats exhibit decreased blood glucose levels in response to oral insulin , whereas weaned rats do not . this is of particular importance in neonatal care , as infants of mothers with diabetes are frequently retained in the neonatal intensive care unit and fed expressed breast milk from their mothers . these infants could potentially then remain hypoglycaemic for longer periods , in contrast to the intended aim of the protocol . tiittanen et al . showed that dietary nonhuman insulin can potentially act as an immunogen and predispose the infant to formation of anti - insulin antibodies , while low levels of human insulin in milk can act as a tolerogen to downregulate any immune response to foreign dietary insulin . their study investigated the presence of bovine insulin in formula as the potential immunogen , which is only three amino acids different in sequence to human insulin . given this potential immunogenic role of nonhuman insulin , it is important to consider the potential implications of the presence of artificial insulin in human milk . artificial recombinant human insulins that are currently used as treatment for patients with type 1 diabetes are typically also only one to three amino acids different in sequence to human insulin . it can therefore be hypothesised that the presence of artificial insulin in the milk of mothers with type 1 diabetes may be acting as an immunogen , which could potentially increase the risk of the infant developing auto - immune diseases in later life . as this immunogenic effect was demonstrated in both early and late lactation , it is possible that insulin is being degraded and only peptide fragments are entering the blood from the intestine . however , given the difference noted in immunogenicity , it is likely that those breakdown products that enter blood still contain the sequence regions that differ between recombinant and endogenous insulin . however , the data in the literature of the role of breastfeeding on the development of type 1 diabetes mellitus in control mothers and those with type 1 diabetes mellitus is still controversial and that the rate of diabetes mellitus in children of type 1 diabetic mothers is lower than in the general population , further suggesting a potential tolerogenic effect of milk insulin [ 2224 ] . a number of limitations must be taken into consideration when interpreting the results of the above study . firstly , as a pilot study , there are only a small number of participants and hence a low sample size . a repeated measures model and a large number of samples per participant are used to counter this , but the low sample size may limit the interpretation of trends of other milk components . however , this should not affect interpretation of human milk insulin origin as exogenous or endogenous . further , the specificity of the elisa assays used for identification of human pancreatic insulin compared to modified exogenous insulin forms means that if there were any structural differences between human pancreatic and a hypothetical mammary source , then the mammary insulin may not be detected with this assay . finally , there was a significant lack of blood glucose concentration data to compare to milk glucose and milk insulin , due to collection difficulties with mothers involved in this study . this study has identified that both endogenous and exogenous insulin is transported from blood into human milk . studies in a rat model with labelled artificial insulin could potentially be used to identify the source of transfer into milk , and use of a hyperinsulinaemic clamp model may be used to determine ( a ) the rate of transfer and ( b ) the transport maximum for the mammary gland transporter . given that insulin is actively transported into human milk and is protected from degradation , it presumably plays a functional or developmental role in the infant . given this , the presence of insulin in formula products needs to be reassessed ; as not all mothers will breast - feed their infants , if insulin plays a functional or developmental role then the addition of insulin to formula could potentially act to improve the similarities of formula to human milk and thus be beneficial to the health and development of formula - fed infants .

despite the important role that insulin plays in the human body , very little is known about its presence in human milk . levels rapidly decrease during the first few days of lactation and then , unlike other serum proteins of similar size , achieve comparable levels to those in serum . despite this , current guides for medical treatment suggest that insulin does not pass into milk , raising the question of where the insulin in milk originates . five mothers without diabetes , 4 mothers with type 1 , and 5 mothers with type 2 diabetes collected milk samples over a 24-hour period . samples were analysed for total and endogenous insulin content and for c - peptide content . all of the insulin present in the milk of type 1 mothers was artificial , and c - peptide levels were 100x lower than in serum . this demonstrates that insulin is transported into human milk at comparable concentration to serum , suggesting an active transport mechanism . the role of insulin in milk is yet to be determined ; however , there are a number of potential implications for the infant of the presence of artificial insulins in milk .

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

insulin , a key hormone in blood glucose homeostasis , has been detected in human colostrum at supraphysiological levels ( 114306 mu / l ) before decreasing to similar concentrations to those found in blood in the fasting state by day 5 postpartum in healthy mothers [ 1 , 2 ] . has demonstrated that insulin is also present in the milk of mothers with type 1 diabetes mellitus , who lack any endogenous insulin secretion from the pancreas . they also demonstrated that levels of insulin are significantly higher ( p < 0.001 ) in milk from type 1 diabetic mothers than that of nondiabetic control mothers , at both days 3 to 7 postpartum and 3 months postpartum . interestingly , insulin levels in the milk of control mothers vary greatly between studies , while those reported by shehadeh et al . , suggesting that significant variation exists between mothers regardless of the presence of diabetes mellitus . in addition , kulski and hartmann have demonstrated that the levels of insulin in breastmilk may vary with the stage of lactation , with high levels present in colostrum and levels decreasing to a plateau in established lactation . despite this , current medical guides state that insulin is a peptide that is too large to be secreted into milk [ 57 ] . given the established role of dietary insulin in the maturation of intestinal epithelium in rats , it can be postulated that insulin present in milk may play a similar role in the development of human intestinal epithelium [ 8 , 9 ] . furthermore , epithelial cell insulin receptors exist in the intestine of both piglets and calves , and insulin has been suggested to play a role in influencing growth and development of the small intestine [ 12 , 13 ] . this would suggest that insulin could retain biological activity when ingested by the infant and that intact insulin may pass over from the gastrointestinal tract of infants into blood . due to these modifications to the amino acid sequence , specific assays can be used to identify whether the insulin present in a fluid sample is endogenous or a modified exogenous form of insulin [ 17 , 18 ] . demonstrated that human dietary insulin in milk may have a tolerogenic effect against bovine insulin in formula and downregulate the production of igg antibodies to bovine insulin , protecting against the development of autoimmunity , as shown with decreased levels of igg to insulin in breast - fed children compared to those fed with cow 's milk formula containing bovine insulin . despite this beneficial effect at lower milk insulin levels , they also noted that high endogenous insulin levels in human milk were associated with a significantly increased incidence of -cell autoimmunity in children , in both control mothers ( p = 0.030 ) and mothers with type 1 diabetes ( p = 0.045 ) , which may suggest that exposure to high levels of endogenous insulin can also promote autoimmunity . however , it must be noted that this does not imply that development of type 1 diabetes mellitus is similarly promoted . other work has supported the possible role of breast milk in the prevention of type 1 diabetes mellitus , since there is a lower incidence of type 1 diabetes in breast - fed children , but this is still a controversial area and contended by other sources [ 2224 ] . given that the only known source of insulin in mothers with type 1 diabetes is injected exogenous insulin , this allows the hypothesis that either insulin is being transported into human milk , in contrast to current literature , or that it is being synthesised in the mammary gland and then secreted into the milk . there are currently no studies comparing the levels of exogenous insulin in milk compared to that produced endogenously in mothers with or without diabetes mellitus . the purpose of this study was to identify the origin of the insulin present in the milk of mothers with type 1 diabetes mellitus as either endogenous or exogenous by determining the levels of total insulin , endogenous insulin , and c - peptide in the milk compared to mothers without diabetes mellitus . this study also aimed to compare the levels of insulin in breast milk from mothers with type 2 diabetes and then investigate any relationship between blood glucose , milk insulin , glucose , c - peptide , and sodium content in breast milk in each of the groups . mothers with type 2 diabetes mellitus have been included in order to compare insulin concentrations and as they should have no exogenous insulin present in their milk or blood . exact state of lactation is not further specified , as it was not relevant to the aims of this study in identifying the origin of insulin in milk . four mothers with type 1 diabetes mellitus were recruited , and all mothers in this group were on insulin replacement therapy . five mothers with type 2 diabetes mellitus were recruited , and all mothers in this group was on dietary , exercise or metformin treatment , or a combination of these . all samples were frozen by the mothers , and then transported to the laboratory and stored at 30c prior for analysis . milk samples were analysed for glucose content using the spectrophotometric method described by arthur et al . defatted milk samples were analysed for total insulin content ( endogenous + exogenous ) using a chemiluminescent microparticle immunoassay ( cmia ) , which was performed at pathwest ( pathwest laboratory medicine wa , qeii medical complex , sir charles gairdner hospital , western australia ) . c - peptide content of milk samples was similarly determined using the mercodia ab c - peptide elisa kit . total fat content of the milk samples was assessed using the creamatocrit method described by lucas et al . although only a small number of mothers were present in each group , the large number of milk samples collected by each mother over the 24 hours periods allows for statistical analysis of differences between groups . analysis of individual groups showed no significant difference in insulin content of fore and hind milk between groups ( p = 0.20 , p = 0.98 , and p = 0.14 for control , type 1 , and type 2 , resp . ) based on this , fore and hind milk insulin levels were averaged for the purpose of investigating any circadian variation ; and this analysis showed no significant variation in insulin levels during different time points during the day . in order to determine whether the insulin present in the milk of mothers with type 1 diabetes is synthesised in the mammary gland or is transported from blood , total and endogenous insulin levels were measured in all milk samples in this group ( n = 53 ) . mean sem exogenous insulin levels in the milk of mothers with type 1 diabetes were 12.22 1.34 mu / l for mother t1 - 01 , 31.54 4.95 mu / l for mother t1 - 02 , and 17.86 4.34 mu / l for mother t1 - 03 . there was no detectable endogenous insulin content in the milk of mothers with type 1 diabetes ; therefore , all insulin detected in the milk of mothers with type 1 diabetes was of exogenous origin . in order to investigate variations in insulin concentration between control mothers and mothers with diabetes , total insulin content of each milk sample ( fore + hind milk individually ) from each mother there was no significant difference between fore and hind milk insulin content , so further data algorithmically treats each data point as the mean of fore + hind milk for each feed ( time point ) . the control mother t0 - 03 demonstrated significantly higher milk insulin content than the other control mothers ( 78.23 3.96 versus 15.46 1.03 there was no significant difference in mean insulin content between control mothers and mothers with type 1 diabetes ( p = 0.74 ) , control mothers and mothers with type 2 diabetes ( p = 0.93 ) , or between mothers with type 1 and type 2 diabetes ( p = 0.62 ) . insulin is produced by cleaving insulin and c - peptide from the precursor molecule proinsulin . hence , c - peptide content of the milk from two mothers with type 1 diabetes and 3 control mothers was measured using the elisa method described ( n = 61 ) . c - peptide was detected in all samples ; however , levels in the milk of control mothers were significantly higher than those in mothers with type 1 diabetes ( 21.09 1.00 versus 14.49 1.47 pmol / l , p < 0.01 ) . there was no significant correlation between c - peptide and total insulin content in the milk of control mothers ( p = 0.79 ) or mothers with type 1 diabetes ( p = 0.85 ) . in order to investigate the transport of insulin into human milk , the sodium content of the milk from all mothers was measured using atomic absorption spectroscopy ( n = 199 ) as a marker of permeability of the paracellular pathway . there was significantly lower sodium content in the milk of mothers with type 1 diabetes compared to control mothers ( p < 0.05 ) , but no significant difference in mean sodium content between control mothers and mothers with type 2 diabetes ( p = 0.09 ) or between types of diabetes ( p = 0.54 ) . there was no significant relationship between sodium and insulin content of milk for control mothers ( p = 0.17 ) or mothers with type 1 diabetes ( p = 0.37 ) , but a mild negative correlation existed for mothers with type 2 diabetes ( r = 0.34 , p < 0.05 ) . additionally , for the outlier control mother t0 - 03 who demonstrated significantly elevated insulin levels in milk , there was no significant relationship between sodium and insulin content in milk ( p = 0.61 ) . in order to investigate any correlation between blood glucose levels and insulin content in the milk of mothers with type 1 and 2 diabetes , mothers were asked to record their blood glucose levels at least 4 times over the course of the same 24-hour period as their milk sample collection . there was a great deal of variation within mothers for each group over a 24-hour period for both milk insulin and blood glucose ; however , no significant consistent trends were identified . however , there was no significant difference ( p = 0.15 ) in mean blood glucose levels between mothers with type 1 and type 2 diabetes . mean blood glucose levels in mothers with type 1 diabetes were 8.52 0.57 mmol / l , and levels for mothers with type 2 diabetes were 7.45 0.26 mmol / l ( mean sem ) . there was no significant relationship between mean blood glucose levels and mean milk insulin levels in mothers with type 1 diabetes ( p = 0.72 ) or mothers with type 2 diabetes ( p = 0.16 ) . by comparing blood glucose levels and milk insulin concentration over time , there appears to be an inverse relationship between the two ; a reduction in blood glucose levels is mirrored by an increase in milk insulin content . mother t2 - 05 ( type 2 ) demonstrated only small variability in milk insulin content , and mother t2 - 03 ( type 2 ) demonstrated large variability in milk insulin content with only small variation in blood glucose levels . as the glucose present in human milk derives from blood glucose , there may be fluctuations in milk glucose content that correlate with either blood glucose levels or insulin content of milk . there was significantly higher glucose content in the milk of mothers with type 1 diabetes compared to control mothers ( p < 0.05 ) and in the milk of mothers with type 2 diabetes compared to control mothers ( p < 0.05 ) , but no significant difference in milk glucose content between types of diabetes ( p = 0.68 ) . there was no significant correlation between milk and blood glucose levels for mothers with type 1 diabetes ( p = 0.60 ) or mothers with type 2 diabetes ( p = 0.33 ) . there was no significant relationship between glucose and insulin content of milk in control mothers ( p = 0.17 ) or mothers with type 2 diabetes ( p = 0.11 ) , but there was a slight negative correlation between glucose and insulin content in milk from mothers with type 1 diabetes ( r = 0.41 , p < 0.01 ) . most current medical references state that insulin is too large to cross over from blood into milk [ 57 ] . however , there have been continuous references in the scientific literature to the presence of insulin in milk and the presence of elevated levels in the milk of mothers with diabetes compared to mothers without diabetes [ 13 , 30 ] . based on this conflicting evidence , it is proposed that insulin could either be transported into milk or be synthesised in the mammary gland and directly secreted into milk . firstly , the type of insulin present in the milk of mothers with type 1 diabetes mellitus was assessed . these mothers should have no endogenous insulin production from the pancreas , and all insulin present in their blood should result from injections of exogenous insulin . analysis of the milk of these mothers revealed that there was no endogenous insulin present , suggesting that it was all derived from injected insulin present in blood . secondly , the presence of c - peptide in the milk of two mothers with type 1 diabetes and three control mothers was measured . as insulin and c - peptide are cleaved from proinsulin in equimolar quantities , if insulin was synthesised in the mammary gland , then there should be equimolar quantities of both present in milk . c - peptide was identified in milk mothers with type 1 diabetes , but at 15 to 20x lower than reference blood concentrations , despite the common view that patients with type 1 diabetes do not produce insulin . however , many patients with type 1 diabetes still retain some residual pancreatic -cell function , and insulin is secreted at subphysiological levels alongside c - peptide . these factors indicate that the insulin present in milk is derived from insulin in blood . insulin content in milk for all groups was found to be similar to known reference levels for blood . this is the first time insulin levels have been analysed in milk from mothers with type 2 diabetes and is in concordance with previous work investigating insulin concentration in the milk of control mothers and mothers with type 1 diabetes [ 13 ] . in contrast to other serum proteins of similar size , which are normally present in milk at up to 100x lower concentration than found in blood , these results suggest that insulin does not diffuse into milk via the paracellular pathway . this is supported by the lack of any correlation between insulin and sodium content of milk for control mothers and mothers with type 1 diabetes ; as sodium content of milk is an indicator of the permeability of the paracellular pathway , a positive correlation between insulin and sodium content of milk would be expected if insulin was entering via the paracellular pathway . additionally , sodium content was significantly lower in mothers with type 1 diabetes than in controls , suggesting that the paracellular pathway is closed in these mothers . there was no significant difference in sodium content of milk between mothers with type 2 diabetes and control mothers . these findings suggest that insulin must be actively transported into milk in order to achieve these higher concentrations . as the bbb transporter is only capable of establishing a cerebral concentration of less than 10% of the systemic blood concentration , it is unlikely to be involved in transport of insulin in the mammary gland . while this is still lower than the ~100% transfer seen in the mammary gland , as milk is synthesised over a period of time , this could potentially account for the high concentrations present in milk . while this study did not assess colostrum insulin content , it can be postulated that the potential milk trypsin inhibitor postulated in rat models may act to preserve and stabilize insulin within milk , leading to these higher concentrations [ 10 , 11 ] . alternately , active transport of insulin into milk may increase in the colostrum phase , particularly if insulin does indeed play a large role in maturation of the infant digestive system [ 13 , 14 ] . there was no significant difference between the levels of insulin in the milk of control mothers , mothers with type 1 , and mothers with type 2 diabetes , despite the insulin present in the type 1 mothers being artificial in nature . this suggests that the exogenous insulin used for treatment of type 1 diabetes is transported into milk with similar affinity to endogenous insulin in mothers without diabetes . as the exact transport mechanism has yet to be scientifically quantified , the significantly elevated milk insulin levels in control mother t0 - 03 can not be readily explained . however , as the transporter has yet to be identified , the possibility remains that the elevated milk insulin levels in this mother are due to altered insulin transport , rather than elevated blood insulin levels ; as two isoforms of the insulin receptor exist , it is possible that the high specificity isoform may be preferentially expressed in the mammary gland of this mother , thus resulting in increased insulin transport into her milk . that glucose transport into milk is insulin independent and dependent upon blood glucose concentrations . this study identified significantly higher milk glucose concentrations in mothers with type 1 and type 2 diabetes compared to control mothers , as was predicted . this study demonstrated that both exogenous and endogenous insulin , are actively transported into human milk in similar concentrations to those found in blood , in comparison to other serum proteins of similar or smaller size , such as c - peptide . studies in a rat model have demonstrated that oral insulin in milk is involved in maturation of intestinal epithelium and induces pancreatic amylase development at weaning [ 8 , 10 , 11 ] . a series of papers reported by koldovsk show that human infants demonstrate decreased blood glucose levels in response to milk insulin in early development , suggesting that intact insulin is crossing into the bloodstream of the infant . showed that dietary nonhuman insulin can potentially act as an immunogen and predispose the infant to formation of anti - insulin antibodies , while low levels of human insulin in milk can act as a tolerogen to downregulate any immune response to foreign dietary insulin . their study investigated the presence of bovine insulin in formula as the potential immunogen , which is only three amino acids different in sequence to human insulin . given this potential immunogenic role of nonhuman insulin , it is important to consider the potential implications of the presence of artificial insulin in human milk . it can therefore be hypothesised that the presence of artificial insulin in the milk of mothers with type 1 diabetes may be acting as an immunogen , which could potentially increase the risk of the infant developing auto - immune diseases in later life . however , given the difference noted in immunogenicity , it is likely that those breakdown products that enter blood still contain the sequence regions that differ between recombinant and endogenous insulin . however , the data in the literature of the role of breastfeeding on the development of type 1 diabetes mellitus in control mothers and those with type 1 diabetes mellitus is still controversial and that the rate of diabetes mellitus in children of type 1 diabetic mothers is lower than in the general population , further suggesting a potential tolerogenic effect of milk insulin [ 2224 ] . a number of limitations must be taken into consideration when interpreting the results of the above study . firstly , as a pilot study , there are only a small number of participants and hence a low sample size . this study has identified that both endogenous and exogenous insulin is transported from blood into human milk . studies in a rat model with labelled artificial insulin could potentially be used to identify the source of transfer into milk , and use of a hyperinsulinaemic clamp model may be used to determine ( a ) the rate of transfer and ( b ) the transport maximum for the mammary gland transporter . given that insulin is actively transported into human milk and is protected from degradation , it presumably plays a functional or developmental role in the infant . given this , the presence of insulin in formula products needs to be reassessed ; as not all mothers will breast - feed their infants , if insulin plays a functional or developmental role then the addition of insulin to formula could potentially act to improve the similarities of formula to human milk and thus be beneficial to the health and development of formula - fed infants .

instant noodles , a steamed and deep - oil fried noodle that is also known as ramen in japan and ramyon in korea , originated in japan in the 1950s and are currently produced in over 80 countries . as of 2008 , approximately 93.6 billion servings of instant noodles have been consumed worldwide . chinese consumed 45.2 billion packages of instant noodles in 2008 , representing 51% of the global consumption of instant noodles , whereas indonesians consumed 13.7 billion packages , japanese consumed 5.1 billion packages , americans consumed 4.3 billion packages , and south koreans consumed 3.3 billion packages . south koreans eat the highest per capita quantity of instant noodles at 69 servings per year , which is 4.8 times higher than the consumption of americans , and 1.7 times higher than the per capita consumption in japan . based on the korean national health and nutrition examination survey ( knhanes ) iii report , instant noodles were consumed at a level of 18.1 g per day per capita nationwide , which made this the second largest food type after steamed rice that contributes to the overall energy intake of individuals . a single serving of instant noodles is usually high in carbohydrates but is low in fiber , vitamins , and minerals . instant noodle manufacturers have made efforts to lower the sodium and fat content in response to public health concerns . instant noodles are now promoted as a nutrient vehicle in developing countries by fortifying either the flours used to make the noodles or the seasoning powders consumed with the noodles . the popularity of instant noodles has been expanding very rapidly during recent decades due to their convenience and reasonable price . however , few data are available with respect to the nutritional status of instant noodle consumers in both asian and western countries . as koreans consume the largest quantity of instant noodles , it is necessary to identify the relationship between instant noodle consumption and nutritional status and food consumption patterns of koreans . therefore , we compared food and nutrient intake in korean adults that consume instant noodles with those who do not consume instant noodles using data from knhanes iii to examine the nutritional status of instant noodle consumers ( inc ) . we also evaluated whether the nutrient intake of koreans consuming instant noodles is appropriate in comparison to the korean dietary reference intakes ( kdri ) . we hope that this study will help to construct guidelines for nutritional education regarding instant noodle consumption . the data analyzed in the present study were obtained from knhanes iii , a study conducted by the ministry for health , welfare , and family affairs in korea in 2005 . the surveys were given to stratified multistage samples of the korean population from multiple geographic areas , ages , and genders . in total , 33,848 people responded to the knhanes iii survey ; 6,440 adults aged 20 years and older participated in both the health examination and the nutrition surveys and were selected for the present study . energy and food intake was obtained from the nutritional survey , whereas data on height , weight , and body mass index ( bmi ) were obtained from the health examination survey . as part of the standard knhanes data collection protocol , 24-hour dietary recalls were elicited and were used here to estimate food and nutrient intake . general characteristics , food and nutrient intake , and anthropometric data were compared across the inc and non - instant noodle consumers ( non - inc ) . income groups were categorized according to the average monthly income in 2005 in relation to the minimum cost of living . low income was defined as an average monthly income that was , at most , 1.2 times the minimum cost of living , a middle income was defined as an average monthly income that was 1.2 - 2.5 times the minimum cost of living , and a high income was defined as an average monthly income that was > 2.5 times the minimum cost of living . subjects were categorized based on their educational level , defined as follows : middle school or lower ( 9 years and below ) , high school ( 10 - 12 years ) , and college or higher ( 13 years or more ) . all analyses were conducted using survey weighting to account for the complex survey design , which consisted of multistage , stratified , and clustered sampling . probability sampling weights were used with strata and primary sampling units in the data analysis . subject characteristics were compared between the inc and non - inc groups using a chi - square test . mean values and standard errors of food and nutrient intake were calculated , and t - tests were used to verify the significance of each in the inc and non - inc groups . energy intake was adjusted for age , whereas other nutrient and food intake data were adjusted for age and energy intake . total food and nutrient intake was compared between the two groups to determine their relationship , and nutritional status was then evaluated using the matched kdri references . energy intake was compared with the estimated energy requirement , sodium and potassium were compared with the intake level considered to be adequate , and the remaining nutrients were compared with the recommended intakes . all statistical analyses were performed using the survey procedure of sas software ( version 9.12 , cary , nc , usa ) and the regress procedure of sudaan software ( release 9.0 , research triangle institute , research triangle park , nc , usa ) using a significance level of p > 0.05 . the data analyzed in the present study were obtained from knhanes iii , a study conducted by the ministry for health , welfare , and family affairs in korea in 2005 . the surveys were given to stratified multistage samples of the korean population from multiple geographic areas , ages , and genders . in total , 33,848 people responded to the knhanes iii survey ; 6,440 adults aged 20 years and older participated in both the health examination and the nutrition surveys and were selected for the present study . energy and food intake was obtained from the nutritional survey , whereas data on height , weight , and body mass index ( bmi ) were obtained from the health examination survey . as part of the standard knhanes data collection protocol , 24-hour dietary recalls were elicited and were used here to estimate food and nutrient intake . general characteristics , food and nutrient intake , and anthropometric data were compared across the inc and non - instant noodle consumers ( non - inc ) . income groups were categorized according to the average monthly income in 2005 in relation to the minimum cost of living . low income was defined as an average monthly income that was , at most , 1.2 times the minimum cost of living , a middle income was defined as an average monthly income that was 1.2 - 2.5 times the minimum cost of living , and a high income was defined as an average monthly income that was > 2.5 times the minimum cost of living . subjects were categorized based on their educational level , defined as follows : middle school or lower ( 9 years and below ) , high school ( 10 - 12 years ) , and college or higher ( 13 years or more ) . all analyses were conducted using survey weighting to account for the complex survey design , which consisted of multistage , stratified , and clustered sampling . probability sampling weights were used with strata and primary sampling units in the data analysis . subject characteristics were compared between the inc and non - inc groups using a chi - square test . mean values and standard errors of food and nutrient intake were calculated , and t - tests were used to verify the significance of each in the inc and non - inc groups . energy intake was adjusted for age , whereas other nutrient and food intake data were adjusted for age and energy intake . total food and nutrient intake was compared between the two groups to determine their relationship , and nutritional status was then evaluated using the matched kdri references . energy intake was compared with the estimated energy requirement , sodium and potassium were compared with the intake level considered to be adequate , and the remaining nutrients were compared with the recommended intakes . all statistical analyses were performed using the survey procedure of sas software ( version 9.12 , cary , nc , usa ) and the regress procedure of sudaan software ( release 9.0 , research triangle institute , research triangle park , nc , usa ) using a significance level of p > 0.05 . general characteristics of the participants in the two groups with respect to instant noodle intake are shown in table 1 . the mean age of the participants in the inc group was lower than that in the non - inc group ( 36.2 vs. 44.9 yrs , respectively ) . participants aged 20 - 49 years accounted for 86.8% of the inc group ; this proportion was significantly higher than that of the non - inc group ( p < 0.001 ) . participants aged 50 - 64 years or 65 years and older accounted for only 9.4% and 3.8% of the inc group , respectively . men ( 63.5% ) consumed more instant noodles than women ( 36.5% ) . additionally , participants in the middle and high income group accounted for 41.9% and 40.8% of inc , respectively , whereas the lower income group accounted for 17.3% of the inc group . in total , 84.5% of the participants in the inc group had a high school education or a higher level of education , whereas 31.3% of participants in the non - inc group had a middle school or lower level of education , which was twice the proportion in the inc group ( 15.5% ) . no significant difference in bmi was observed between the inc and the non - inc groups , although the weights and heights of the inc group were relatively higher than those of the non - inc group . as shown in table 2 , the intake of total plant - originating foods , animal - originating foods , and total food in the non - inc group was significantly higher than that of the inc group after adjusting for age and energy intake . when we looked separately at each individual food group , intake of cereals and grain products in the inc group was significantly higher than those in the non - inc group ( p = 0.05 ) . in contrast , the intake of potatoes and starches , sugars and sweets , seeds and nuts , vegetables , mushrooms , fruits , beverages , seasonings , meats , fishes , and oils and fats in the non - inc group was significantly higher than those in the inc group . however , seeds and nuts , mushrooms , and beverages in the inc group did not differ from those of the non - inc group in men , whereas women in the inc group had a significantly lower intake of legumes and their products compared with women in the non - inc group . the daily nutrient intake of the inc and non - inc groups is shown along with the contribution of instant noodles in table 3 . the inc group had a significantly higher total energy intake than that in the non - inc group ( 2,024.6 kcal / day vs. 2,252.3 kcal / day ) after adjusting for age . additionally , the inc group consumed more fat , sodium , thiamine , and riboflavin than those in the non - inc group after adjusting for age and energy intake . in contrast , lower intake of protein , calcium , phosphorus , iron , potassium , vitamin a , niacin , and vitamin c was observed in the inc group . the percentages of energy from carbohydrates and proteins in the non - inc group were higher than those of the inc group , whereas the percentage of energy from fat in the inc group was 4.2% higher than that of the non - inc group ( 17.7% vs. 21.9% , respectively ) . instant noodle consumption contributed 24.8% to total energy intake , 38.4% of fat intake , 31.0% of sodium intake , 41.7% of thiamine intake , and 34.6% of the riboflavin intake in the inc group . both men and women in the inc group had a higher intake of total energy , fat , sodium , thiamine , and riboflavin but a lower intake of protein , calcium , phosphorus , iron , potassium , vitamin a , niacin , and vitamin c when compared with those in the non - inc group . the percentages of energy from carbohydrate and protein in both men and women of the non - inc group were higher than those in the inc group , whereas the percentage of energy from fat in the inc group was approximately 4% higher than that in the non - inc group . table 4 shows a comparison of the nutrient intake of the inc and non - inc groups in korea to that of the kdri . the percentage comparisons to the kdri for total energy , sodium , thiamine , and riboflavin in the inc group were higher than those of the non - inc group . in contrast , the percentage comparisons to the kdri for protein , calcium , phosphorus , iron , potassium , vitamin a , niacin , and vitamin c in the inc group were lower than those of the non - inc group . among them , the intake of calcium , potassium , and vitamin c were lower than the kdri reference values . sodium intake in the inc group was 4.52 times higher than that of the kdri , whereas that of the non - inc group was 3.93 times higher . thiamine and riboflavin from instant noodles provided 63.3% and 36.8% of the kdri , respectively . similar patterns in men and women with those in the total population were observed for various nutrients ; the calcium , potassium , and vitamin c values in the inc group were lower than the kdri reference values in men ( calcium , 75.7% vs. 89.6% ; potassium , 60.9% vs. 71.5% , ; vitamin c , 79.2% vs. 116.3% , respectively ) . in women , the inc group showed lower percentages in comparison with the kdri for calcium , iron , potassium , niacin , and vitamin c than those in the non - inc group , whereas the percentage comparisons to the kdri for energy , sodium , thiamine , and riboflavin were higher than those of the non - inc group . this study revealed that inc consumed significantly lower amounts of potatoes and starches , sugars , seeds and nuts , vegetables , fruits , beverages , seasonings , oils and fats , meats and fishes than those in the non - inc group , with the exception of cereals and grain products , legumes , seaweeds , eggs , and milk and dairy products . the inc group showed significantly higher intake of energy , fat , sodium , thiamine , and riboflavin ; however , the inc group showed a significantly lower intake of calcium , phosphorus , iron , potassium , vitamin a , niacin , and vitamin c when compared to those in the non - inc group . it is rare to find peer - reviewed studies that have evaluated the relationship between instant noodle consumption and nutritional status , although public concerns regarding this food source are high . to our knowledge , this is the first nationwide study to examine the relationship between instant noodle consumption and nutritional status in koreans . however , several interventional studies have reported that noodle consumption by children in korea affects their nutrient intake [ 8 - 11 ] . but , the association between instant noodle consumption and nutritional status has rarely been examined in the adult population . people typically prefer instant noodles to other convenience foods or fast foods when eating time is a constraint . college students consume the largest amount of bowl - type instant noodles among various convenience foods in korea . in addition , the preferred type of fast food for working men is instant noodles . women instant noodle consumers are younger and have a higher socio - economic status than those of other consumers . similarly , participants who consume instant noodles in this study tended to be younger ( 20 - 49 years ) , mostly in the middle or high income group , and with a level of education greater than high school . indeed , participants aged 20 - 49 years consumed 22.2 g instant noodles per day compared to the consumption of persons aged 50 - 64 years or 65 years and older ( 7.6 g or 5.8 g , data not shown ) . men consumed 23.7 g instant noodles per day , whereas women consumed 11.1 g per day ( data not shown ) . gender differences in instant noodle consumption have also been observed in previous studies ; boys prefer instant noodles to other fast foods , and middle - school male students consume more noodles than girls . thirty percent of the total sales of instant noodles in korea are bowl - type instant noodles consumed as a snack . the fat content of instant noodles is in the range of 12 - 15.8% for the bag - type , and 17.8 - 26.0% for the bowl - type , with few exceptions . a study reported that energy intake from fat accounts for 30.8% and 34.1% of the energy available from the bag - type and bowl - type noodles , respectively . increased consumption of the bowl - type of instant noodles is expected due to convenience , and , thus , a higher consumption of energy and fat intake is expected for the same amount of instant noodle consumption . in this study , instant noodle consumption included the consumption of both the bag - type and the bowl - type and was calculated separately with individual nutritional composition tables . individuals who consume the bowl - type of instant noodles need to be cautious regarding the fat and calorie intake of this food . thus , nutritional education would be helpful to reduce the fat and total calorie intake and to select the appropriate meal combination of other dishes required to achieve balanced nutritional status . one study in korea reported that inc ( middle school students , n = 385 ) added eggs and onions and often consumed noodles along with kimchi , steamed rice , radish pickles , or dried laver . this limited consumption of side dishes with instant noodles was related to the lower consumption of potatoes and starches , seeds and nuts , vegetables , fruits , and seasonings . interestingly , individuals who consume instant noodles consume more cereals and grain products , which was coincident with a previous result . in the present study , the sodium intake in the inc group was > 6.4 g per day , which was 3.2 times higher than the recommended kdri value . instant noodle consumption contributed approximately 30% of the total sodium intake ( 2,032.2 mg per day ) . a study was conducted to reduce sodium levels in instant noodles ; that study reported that 20% of the sodium content , approximately 350 mg , from not eating the soup base without changing taste and flavor . recently , the ministry of health and welfare in korea announced " dietary guidelines for korean adults " , which suggest ten dietary goals , six dietary guidelines , and 23 actual guidelines , including consuming a balanced energy intake and < 5 g sodium intake in the diet . the korea food & drug administration has recently established a guide to " the right food selections for korean kids " to ensure healthy dietary habits in later life . the toyama birth cohort study in japan recently reported that junior high school students who frequently consume instant noodles ( at least 3 days / week ) from the age of 3 show a higher risk for a lower quality of life ( odds ratio [ or ] , 1.49 ; p = 0.007 ) . children who have a preference for salty food tend to maintain and reinforce this tendency in their later life , so nutritional education programs promoting proper sodium consumption should be conducted early in life . several studies have been conducted to examine the association between gastric cancer , diabetes , and blood lipid profiles and instant noodle consumption . instant noodle consumption is associated with a higher risk for gastric cancer compared with that of plain noodles ( n = 105 ; or,4.76 ; p < 0.01 ) . however , instant noodle consumption lowers blood glucose in healthy adults compared to consumption of steamed rice ( n = 30 , p < 0.05 ) . middle - aged women ( 40 - 64-years - of - age ; n = 1,308 ) who consumed instant noodles show relatively healthier lipid profiles in knhanes ii than those who do not consume instant noodles . they also reported that these instant noodle consumers were relatively younger and had a higher socioeconomic status ( ses ) . similarly , although the inc group consumed significantly higher amounts of fat and calories , no significant difference in bmi was observed between the two groups . the present study also showed similar characteristics with a previous study for age and ses . however , a change in nutritional status may cause an anthropometric change in later life . thus , fat and calorie consumption should be monitored by inc and management of consumption should be promoted through nutritional education . recently , instant noodles in southeast asia have been fortified with vitamin a , thiamine , riboflavin , niacin , vitamin b6 , folic acid , iron , zinc , and iodine . many studies have reported the retention rate and stability of fortified nutrients including thiamine , riboflavin , and folate [ 25 - 27 ] . since the fortification of instant noodle began in developing countries in 1994 , southeast asian countries including indonesia , thailand , the philippines , and vietnam have voluntarily fortified with micronutrients . the efficacy of fortifying instant noodles has not been fully determined yet , but some results have been reported for these countries . a study in indonesia found that there is a benefit for vitamin a and iron status of pregnant women and children < 5 years of age who consumed instant noodles fortified with 750 g ( 2,500 iu ) vitamin a and 9 mg iron/100 g for 3 months , compared with a control product . in thailand , seasoning powder fortified with zinc ( 5 mg ) , iron ( 5 mg ) , vitamin a ( 270 g ) , and iodine ( 50 g ) per serving enhanced levels of hemoglobin , zinc , and iodine in children ( n = 569 ) aged 5.5 - 13.4 years . in the present study , the iron intake of the inc group was 87.1% of the recommended kdri intake in women , whereas intake in the non - inc group was 110.2% that of women . iron intake should be encouraged through nutritional education in individuals consuming instant noodles ; the fortification of iron is not suitable for korean adults due to the result that iron levels were 87.1% of the recommended value . thus , nutrition education is an appropriate means for improving the intake of iron and vitamin c in korean adults . choosing the appropriate form of a fortificant is important to minimize nutrient - nutrient as well as nutrient - food interactions and any resulting adverse effects . furthermore , regional nutritional issues should be thoroughly examined and considered , and the fortification strategy should then be arranged by stakeholders , manufacturers , and the local government . in korea , thiamine and riboflavin are fortified as part of the manufacturing process , which contributed to 41.7% and 34.6% of the total consumption , respectively , in this study . recently , calcium and dietary fiber have become fortified as part of the manufacturing process , whereas sodium content has been reduced by 20 - 30% . in addition , instant noodles for children fortified with dietary fiber ( 0 - 3 g ) , calcium ( 250 - 275 mg ) , iron ( 6 mg ) , and an oil mixture ( -6 : -3 = 6:1 , 0.6 g ) and contain less sodium ( 550 - 1,380 mg ) , fat ( 0.6 - 5 g ) and calories ( 305 - 365 kcal ) per package ( 88 - 93 g ) by means of a non - frying process have been launched by one of the leading manufacturers . we hope that manufacturers continue to pursue these efforts , and that these efforts are extended to other products for adults . further studies are needed to clarify the effectiveness of fortification or reformulation on the nutritional status of the korean population using updated food composition tables and new , reformulated instant noodle products the main limitations of this study were its cross - sectional design , and that dietary data were collected for 1 day . as single - day dietary recalls are imprecise at the individual level , the typical dietary intake in individual subjects may not have been assessed precisely . this study revealed that consuming instant noodles may lead to an excessive intake of calories , fats , and sodium but may also lead to an increased intake of thiamine and riboflavin . therefore , nutritional education should be implemented to help adults choose a balanced meal while consuming instant noodles .

instant noodles are widely consumed in asian countries . the korean population consumed the largest quantity of instant noodles in the world in 2008 . however , few studies have investigated the relationship between instant noodles and nutritional status in koreans . the objective of this study was to examine the association between instant noodle consumption and food and nutrient intake in korean adults . we used dietary data of 6,440 subjects aged 20 years and older who participated in the korean national health and nutrition examination survey iii . the average age of the instant noodle consumers ( inc ) was 36.2 and that of the non - instant noodle consumers ( non - inc ) was 44.9 ; men consumed more instant noodles than women ( p < 0.001 ) . with the exception of cereals and grain products , legumes , seaweeds , eggs , and milk and dairy products , inc consumed significantly fewer potatoes and starches , sugars , seeds and nuts , vegetables , mushrooms , fruits , seasonings , beverages , meats , fishes , and oils and fats compared with those in the non - inc group . the inc group showed significantly higher nutrient intake of energy , fat , sodium , thiamine , and riboflavin ; however , the inc group showed a significantly lower intake of protein , calcium , phosphorus , iron , potassium , vitamin a , niacin , and vitamin c compared with those in the non - inc group . this study revealed that consuming instant noodles may lead to excessive intake of energy , fats , and sodium but may also cause increased intake of thiamine and riboflavin . therefore , nutritional education helping adults to choose a balanced meal while consuming instant noodles should be implemented . additionally , instant noodle manufacturers should consider nutritional aspects when developing new products .

Introduction Subjects and Methods Study population Statistical analysis Results Discussion

instant noodles , a steamed and deep - oil fried noodle that is also known as ramen in japan and ramyon in korea , originated in japan in the 1950s and are currently produced in over 80 countries . as of 2008 , approximately 93.6 billion servings of instant noodles have been consumed worldwide . chinese consumed 45.2 billion packages of instant noodles in 2008 , representing 51% of the global consumption of instant noodles , whereas indonesians consumed 13.7 billion packages , japanese consumed 5.1 billion packages , americans consumed 4.3 billion packages , and south koreans consumed 3.3 billion packages . south koreans eat the highest per capita quantity of instant noodles at 69 servings per year , which is 4.8 times higher than the consumption of americans , and 1.7 times higher than the per capita consumption in japan . based on the korean national health and nutrition examination survey ( knhanes ) iii report , instant noodles were consumed at a level of 18.1 g per day per capita nationwide , which made this the second largest food type after steamed rice that contributes to the overall energy intake of individuals . a single serving of instant noodles is usually high in carbohydrates but is low in fiber , vitamins , and minerals . instant noodles are now promoted as a nutrient vehicle in developing countries by fortifying either the flours used to make the noodles or the seasoning powders consumed with the noodles . however , few data are available with respect to the nutritional status of instant noodle consumers in both asian and western countries . as koreans consume the largest quantity of instant noodles , it is necessary to identify the relationship between instant noodle consumption and nutritional status and food consumption patterns of koreans . therefore , we compared food and nutrient intake in korean adults that consume instant noodles with those who do not consume instant noodles using data from knhanes iii to examine the nutritional status of instant noodle consumers ( inc ) . we also evaluated whether the nutrient intake of koreans consuming instant noodles is appropriate in comparison to the korean dietary reference intakes ( kdri ) . we hope that this study will help to construct guidelines for nutritional education regarding instant noodle consumption . the surveys were given to stratified multistage samples of the korean population from multiple geographic areas , ages , and genders . in total , 33,848 people responded to the knhanes iii survey ; 6,440 adults aged 20 years and older participated in both the health examination and the nutrition surveys and were selected for the present study . energy and food intake was obtained from the nutritional survey , whereas data on height , weight , and body mass index ( bmi ) were obtained from the health examination survey . as part of the standard knhanes data collection protocol , 24-hour dietary recalls were elicited and were used here to estimate food and nutrient intake . general characteristics , food and nutrient intake , and anthropometric data were compared across the inc and non - instant noodle consumers ( non - inc ) . subjects were categorized based on their educational level , defined as follows : middle school or lower ( 9 years and below ) , high school ( 10 - 12 years ) , and college or higher ( 13 years or more ) . subject characteristics were compared between the inc and non - inc groups using a chi - square test . mean values and standard errors of food and nutrient intake were calculated , and t - tests were used to verify the significance of each in the inc and non - inc groups . total food and nutrient intake was compared between the two groups to determine their relationship , and nutritional status was then evaluated using the matched kdri references . energy intake was compared with the estimated energy requirement , sodium and potassium were compared with the intake level considered to be adequate , and the remaining nutrients were compared with the recommended intakes . the surveys were given to stratified multistage samples of the korean population from multiple geographic areas , ages , and genders . in total , 33,848 people responded to the knhanes iii survey ; 6,440 adults aged 20 years and older participated in both the health examination and the nutrition surveys and were selected for the present study . energy and food intake was obtained from the nutritional survey , whereas data on height , weight , and body mass index ( bmi ) were obtained from the health examination survey . as part of the standard knhanes data collection protocol , 24-hour dietary recalls were elicited and were used here to estimate food and nutrient intake . general characteristics , food and nutrient intake , and anthropometric data were compared across the inc and non - instant noodle consumers ( non - inc ) . subject characteristics were compared between the inc and non - inc groups using a chi - square test . mean values and standard errors of food and nutrient intake were calculated , and t - tests were used to verify the significance of each in the inc and non - inc groups . total food and nutrient intake was compared between the two groups to determine their relationship , and nutritional status was then evaluated using the matched kdri references . energy intake was compared with the estimated energy requirement , sodium and potassium were compared with the intake level considered to be adequate , and the remaining nutrients were compared with the recommended intakes . general characteristics of the participants in the two groups with respect to instant noodle intake are shown in table 1 . the mean age of the participants in the inc group was lower than that in the non - inc group ( 36.2 vs. 44.9 yrs , respectively ) . participants aged 20 - 49 years accounted for 86.8% of the inc group ; this proportion was significantly higher than that of the non - inc group ( p < 0.001 ) . participants aged 50 - 64 years or 65 years and older accounted for only 9.4% and 3.8% of the inc group , respectively . men ( 63.5% ) consumed more instant noodles than women ( 36.5% ) . additionally , participants in the middle and high income group accounted for 41.9% and 40.8% of inc , respectively , whereas the lower income group accounted for 17.3% of the inc group . in total , 84.5% of the participants in the inc group had a high school education or a higher level of education , whereas 31.3% of participants in the non - inc group had a middle school or lower level of education , which was twice the proportion in the inc group ( 15.5% ) . no significant difference in bmi was observed between the inc and the non - inc groups , although the weights and heights of the inc group were relatively higher than those of the non - inc group . as shown in table 2 , the intake of total plant - originating foods , animal - originating foods , and total food in the non - inc group was significantly higher than that of the inc group after adjusting for age and energy intake . when we looked separately at each individual food group , intake of cereals and grain products in the inc group was significantly higher than those in the non - inc group ( p = 0.05 ) . in contrast , the intake of potatoes and starches , sugars and sweets , seeds and nuts , vegetables , mushrooms , fruits , beverages , seasonings , meats , fishes , and oils and fats in the non - inc group was significantly higher than those in the inc group . however , seeds and nuts , mushrooms , and beverages in the inc group did not differ from those of the non - inc group in men , whereas women in the inc group had a significantly lower intake of legumes and their products compared with women in the non - inc group . the daily nutrient intake of the inc and non - inc groups is shown along with the contribution of instant noodles in table 3 . the inc group had a significantly higher total energy intake than that in the non - inc group ( 2,024.6 kcal / day vs. 2,252.3 kcal / day ) after adjusting for age . additionally , the inc group consumed more fat , sodium , thiamine , and riboflavin than those in the non - inc group after adjusting for age and energy intake . in contrast , lower intake of protein , calcium , phosphorus , iron , potassium , vitamin a , niacin , and vitamin c was observed in the inc group . the percentages of energy from carbohydrates and proteins in the non - inc group were higher than those of the inc group , whereas the percentage of energy from fat in the inc group was 4.2% higher than that of the non - inc group ( 17.7% vs. 21.9% , respectively ) . instant noodle consumption contributed 24.8% to total energy intake , 38.4% of fat intake , 31.0% of sodium intake , 41.7% of thiamine intake , and 34.6% of the riboflavin intake in the inc group . both men and women in the inc group had a higher intake of total energy , fat , sodium , thiamine , and riboflavin but a lower intake of protein , calcium , phosphorus , iron , potassium , vitamin a , niacin , and vitamin c when compared with those in the non - inc group . the percentages of energy from carbohydrate and protein in both men and women of the non - inc group were higher than those in the inc group , whereas the percentage of energy from fat in the inc group was approximately 4% higher than that in the non - inc group . table 4 shows a comparison of the nutrient intake of the inc and non - inc groups in korea to that of the kdri . the percentage comparisons to the kdri for total energy , sodium , thiamine , and riboflavin in the inc group were higher than those of the non - inc group . in contrast , the percentage comparisons to the kdri for protein , calcium , phosphorus , iron , potassium , vitamin a , niacin , and vitamin c in the inc group were lower than those of the non - inc group . among them , the intake of calcium , potassium , and vitamin c were lower than the kdri reference values . sodium intake in the inc group was 4.52 times higher than that of the kdri , whereas that of the non - inc group was 3.93 times higher . thiamine and riboflavin from instant noodles provided 63.3% and 36.8% of the kdri , respectively . similar patterns in men and women with those in the total population were observed for various nutrients ; the calcium , potassium , and vitamin c values in the inc group were lower than the kdri reference values in men ( calcium , 75.7% vs. 89.6% ; potassium , 60.9% vs. 71.5% , ; vitamin c , 79.2% vs. 116.3% , respectively ) . in women , the inc group showed lower percentages in comparison with the kdri for calcium , iron , potassium , niacin , and vitamin c than those in the non - inc group , whereas the percentage comparisons to the kdri for energy , sodium , thiamine , and riboflavin were higher than those of the non - inc group . this study revealed that inc consumed significantly lower amounts of potatoes and starches , sugars , seeds and nuts , vegetables , fruits , beverages , seasonings , oils and fats , meats and fishes than those in the non - inc group , with the exception of cereals and grain products , legumes , seaweeds , eggs , and milk and dairy products . the inc group showed significantly higher intake of energy , fat , sodium , thiamine , and riboflavin ; however , the inc group showed a significantly lower intake of calcium , phosphorus , iron , potassium , vitamin a , niacin , and vitamin c when compared to those in the non - inc group . it is rare to find peer - reviewed studies that have evaluated the relationship between instant noodle consumption and nutritional status , although public concerns regarding this food source are high . to our knowledge , this is the first nationwide study to examine the relationship between instant noodle consumption and nutritional status in koreans . however , several interventional studies have reported that noodle consumption by children in korea affects their nutrient intake [ 8 - 11 ] . but , the association between instant noodle consumption and nutritional status has rarely been examined in the adult population . college students consume the largest amount of bowl - type instant noodles among various convenience foods in korea . in addition , the preferred type of fast food for working men is instant noodles . similarly , participants who consume instant noodles in this study tended to be younger ( 20 - 49 years ) , mostly in the middle or high income group , and with a level of education greater than high school . indeed , participants aged 20 - 49 years consumed 22.2 g instant noodles per day compared to the consumption of persons aged 50 - 64 years or 65 years and older ( 7.6 g or 5.8 g , data not shown ) . men consumed 23.7 g instant noodles per day , whereas women consumed 11.1 g per day ( data not shown ) . gender differences in instant noodle consumption have also been observed in previous studies ; boys prefer instant noodles to other fast foods , and middle - school male students consume more noodles than girls . thirty percent of the total sales of instant noodles in korea are bowl - type instant noodles consumed as a snack . the fat content of instant noodles is in the range of 12 - 15.8% for the bag - type , and 17.8 - 26.0% for the bowl - type , with few exceptions . increased consumption of the bowl - type of instant noodles is expected due to convenience , and , thus , a higher consumption of energy and fat intake is expected for the same amount of instant noodle consumption . in this study , instant noodle consumption included the consumption of both the bag - type and the bowl - type and was calculated separately with individual nutritional composition tables . individuals who consume the bowl - type of instant noodles need to be cautious regarding the fat and calorie intake of this food . thus , nutritional education would be helpful to reduce the fat and total calorie intake and to select the appropriate meal combination of other dishes required to achieve balanced nutritional status . this limited consumption of side dishes with instant noodles was related to the lower consumption of potatoes and starches , seeds and nuts , vegetables , fruits , and seasonings . interestingly , individuals who consume instant noodles consume more cereals and grain products , which was coincident with a previous result . in the present study , the sodium intake in the inc group was > 6.4 g per day , which was 3.2 times higher than the recommended kdri value . instant noodle consumption contributed approximately 30% of the total sodium intake ( 2,032.2 mg per day ) . a study was conducted to reduce sodium levels in instant noodles ; that study reported that 20% of the sodium content , approximately 350 mg , from not eating the soup base without changing taste and flavor . recently , the ministry of health and welfare in korea announced " dietary guidelines for korean adults " , which suggest ten dietary goals , six dietary guidelines , and 23 actual guidelines , including consuming a balanced energy intake and < 5 g sodium intake in the diet . the toyama birth cohort study in japan recently reported that junior high school students who frequently consume instant noodles ( at least 3 days / week ) from the age of 3 show a higher risk for a lower quality of life ( odds ratio [ or ] , 1.49 ; p = 0.007 ) . children who have a preference for salty food tend to maintain and reinforce this tendency in their later life , so nutritional education programs promoting proper sodium consumption should be conducted early in life . several studies have been conducted to examine the association between gastric cancer , diabetes , and blood lipid profiles and instant noodle consumption . instant noodle consumption is associated with a higher risk for gastric cancer compared with that of plain noodles ( n = 105 ; or,4.76 ; p < 0.01 ) . however , instant noodle consumption lowers blood glucose in healthy adults compared to consumption of steamed rice ( n = 30 , p < 0.05 ) . they also reported that these instant noodle consumers were relatively younger and had a higher socioeconomic status ( ses ) . similarly , although the inc group consumed significantly higher amounts of fat and calories , no significant difference in bmi was observed between the two groups . however , a change in nutritional status may cause an anthropometric change in later life . thus , fat and calorie consumption should be monitored by inc and management of consumption should be promoted through nutritional education . recently , instant noodles in southeast asia have been fortified with vitamin a , thiamine , riboflavin , niacin , vitamin b6 , folic acid , iron , zinc , and iodine . many studies have reported the retention rate and stability of fortified nutrients including thiamine , riboflavin , and folate [ 25 - 27 ] . since the fortification of instant noodle began in developing countries in 1994 , southeast asian countries including indonesia , thailand , the philippines , and vietnam have voluntarily fortified with micronutrients . a study in indonesia found that there is a benefit for vitamin a and iron status of pregnant women and children < 5 years of age who consumed instant noodles fortified with 750 g ( 2,500 iu ) vitamin a and 9 mg iron/100 g for 3 months , compared with a control product . in thailand , seasoning powder fortified with zinc ( 5 mg ) , iron ( 5 mg ) , vitamin a ( 270 g ) , and iodine ( 50 g ) per serving enhanced levels of hemoglobin , zinc , and iodine in children ( n = 569 ) aged 5.5 - 13.4 years . in the present study , the iron intake of the inc group was 87.1% of the recommended kdri intake in women , whereas intake in the non - inc group was 110.2% that of women . iron intake should be encouraged through nutritional education in individuals consuming instant noodles ; the fortification of iron is not suitable for korean adults due to the result that iron levels were 87.1% of the recommended value . thus , nutrition education is an appropriate means for improving the intake of iron and vitamin c in korean adults . furthermore , regional nutritional issues should be thoroughly examined and considered , and the fortification strategy should then be arranged by stakeholders , manufacturers , and the local government . in korea , thiamine and riboflavin are fortified as part of the manufacturing process , which contributed to 41.7% and 34.6% of the total consumption , respectively , in this study . in addition , instant noodles for children fortified with dietary fiber ( 0 - 3 g ) , calcium ( 250 - 275 mg ) , iron ( 6 mg ) , and an oil mixture ( -6 : -3 = 6:1 , 0.6 g ) and contain less sodium ( 550 - 1,380 mg ) , fat ( 0.6 - 5 g ) and calories ( 305 - 365 kcal ) per package ( 88 - 93 g ) by means of a non - frying process have been launched by one of the leading manufacturers . we hope that manufacturers continue to pursue these efforts , and that these efforts are extended to other products for adults . further studies are needed to clarify the effectiveness of fortification or reformulation on the nutritional status of the korean population using updated food composition tables and new , reformulated instant noodle products the main limitations of this study were its cross - sectional design , and that dietary data were collected for 1 day . this study revealed that consuming instant noodles may lead to an excessive intake of calories , fats , and sodium but may also lead to an increased intake of thiamine and riboflavin . therefore , nutritional education should be implemented to help adults choose a balanced meal while consuming instant noodles .

the acute reaction to aspirin or other nonsteroidal anti - inflammatory drugs ( nsaids ) in aspirin - exacerbated respiratory disease ( aerd ) patients is a life - threatening condition characterized by upper airway symptoms ( nasal obstruction and rhinorrhea ) and lower airway symptoms ( shortness of breath and respiratory distress ) . aerd patients , however , suffer from chronic manifestations of the disease including chronic rhinosinusitis , nasal polyps , and asthma usually resistant to any treatment . the aiane study showed that up to 80% of aerd patients required intermediate to high doses of inhaled steroids , and up to 50% of them had to take oral steroids in order to obtain asthma control . similarly , the tenor study showed that asthma was severe in 66% of aerd subjects , 34% received high systemic steroidal doses , 67% needed antileukotrienes , and 20% of them required orotracheal intubation during acute reactions . it must be noted that inflammation is usually present in both upper and lower airways and some aerd patients can present with a disease limited to the upper respiratory tract and no lower airways symptoms at all [ 1 , 2 ] . chronic rhinosinusitis is a major condition in aerd , which in its initial phases is manifested only as nasal congestion , with asthma starting about two years after the initial nasal symptoms . chronic rhinosinusitis ( crs ) is defined as an inflammatory condition involving the mucosa underlying the nasal cavity and the paranasal sinuses that can also affect the underlying bone . , crs becomes a life - time condition , which is usually difficult to control . the clinical symptoms and signs to evaluate crs are divided into major and minor criteria . the major criteria are nasal obstruction , facial pressure , nasal discharge , and/or postnasal drip . the minor criteria are the presence of purulence , anosmia and/or hyposmia , chronic cough , headache , dental pain , ear pressure , fatigue , and halitosis . the clinical evaluation is based on anterior rhinoscopy and nasal endoscopy that usually reveals mucosal oedema and hyperemia , with or without polyps , and frequently purulent secretions . crs can be divided into two mutually exclusive histological subtypes based on the presence of polyps or glandular hypertrophy . crs with nasal polyps ( crswnp ) affects the full thickness of the nasal mucosa , which is replaced with an oedematous , generally eosinophilic , epithelium - coated bag of interstitial matrix ground substance . in contrast , crs without nasal polyps ( crssnp ) or hyperplastic rhinosinusitis is characterized by glandular hypertrophy as demonstrated by malekzadeh and colleagues [ 4 , 5 ] . indeed , a greater number of these cells have been reported in both the upper and lower airways of patients of aspirin - sensitive patients as compared with aspirin - tolerant patients [ 68 ] . on activation , eosinophils release a vast array of mediators including leukotrienes , basic proteins ( major basic protein and eosinophil cationic protein ) , cytokines , and oxygen - free radicals that cause local tissue damage . found a correlation between the number of infiltrated eosinophils and both epithelial damage and bm thickening . we have found increased levels of eosinophil cationic protein in nasal secretions of aspirin - sensitive patients . a majority of patients with aspirin intolerance will develop nasal polyps during the course of the disease . nasal polyps are inflammatory pseudotumoral masses that most frequently start to grow from the ostiomeatal complex and the cells of the anterior ethmoidal sinus . they can affect the totality of the remaining sinusal cavities including the posterior ethmoidal cells , the maxillary , and the frontal or the sphenoidal sinuses , and they also can extend to the olfactory cleft , the sphenoethmoidal recess , and the nasal cavities ( figure 1 ) . nasal polyposis in aerd patients is present in up to 80 to 90% of patients and tends to be more aggressive and difficult to treat medically , also presenting with higher recurrence rates after surgery . in the aiane study that included 500 asa - intolerant patients from 14 different centres , nasosinusal polyposis was diagnosed on nasal endoscopy in 60% of the patients , but the prevalence rose to 90% when ct - scans of the nose and sinuses were performed . nasal polyps are formed by lax connective tissue stroma with oedema , inflammatory cells with a predominant eosinophil infiltration , mucosal glands , and newly formed vascular structures . the mucosal covering of the nasal polyps is a columnar glandular pseudostratified epithelium that also plays a major role in cytokine and inflammatory mediator release . there is no doubt that eosinophils are the main inflammatory cells found in the tissue specimens of nasal polyps , but neutrophils can also be present in large amounts and can even be predominant , notably in asiatic patients . other inflammatory cells can also be found in nasal polyps , such as lymphocytes , monocytes , plasma cells , and fibroblasts . chronic sinusitis with nasal polyposis is also characterized by an intense oedematous stroma with albumin deposition , formation of pseudocysts , and subepithelial and perivascular inflammatory cell infiltration . remodelling is a dynamic process regulated by diverse mediators among which tgf is the most important . in addition to being a key factor in the generation or the deficit of t - reg cells , tgf is also a critical factor implicated in the remodelling process in the airways through the attraction and proliferation of fibroblasts and upregulation of extracellular matrix synthesis . the full prevalence of aspirin - induced rhinosinusitis can be difficult to assess , as it primarily relies on the clinical history . we believe that this condition is underdiagnosed worldwide and is only confirmed after an asa challenge testing under ideal conditions . false negative results can occur when the patient is receiving systemic steroid or antileukotriene treatment . systematic reviews report an incidence of 21.1% of asa intolerance in asthmatic patients after asa challenge testing . the incidence rises to 24% in patients with severe asthma and up to 40% when the association of asthma and chronic rhinosinusitis with polyposis is present [ 1215 ] . usually asa intolerance appears in 3040-year - old patients with a previous history of chronic rhinosinusitis and/or asthma . some patients refer an acute viral - like nasal episode that never resolved fully afterwards . intrinsic asthma tends to appear after the rhinitis , but before the onset of nasal polyposis ( figure 2 ) . skin testing for allergy can be positive in up to 3060% of aerd patients , but no association between asa intolerance and ige - mediated mechanisms has been identified . nevertheless , a high prevalence of food intolerance or antibiotic allergies has been found [ 1618 ] . the physiopathologic mechanisms of aerd are still not fully understood . however , it is now well established that an alteration of arachidonic acid metabolism takes place , and it is characterized by an imbalance between cyclooxygenase ( cox ) and lipoxygenase pathways that results in an overactive lipoxygenase pathway . thus , cox-1 inhibition after asa or nsaids intake finally results in an overproduction of leukotrienes that leads to airway inflammation . increased levels of leukotriene c4 synthase with elevated levels of ltc4 , ltd4 , and lte4 with an overexpression of leukotriene producing enzymes such as 5-lipoxygenase ( 5-lo ) and ltc4 synthase in nasal polyp tissue increased levels of urinary leukotriene metabolites have also been associated with hyperplasic crssnp and crswnp in both asa - tolerant ( ata ) and asa - intolerant ( aia ) patients . these urinary leukotriene metabolite levels tend to drop significantly after sinus surgery , probably as a consequence of the elimination of a great amount of leukotriene producing cells in the ethmoidal sinus . indeed increased lte4 levels and cyslt1 receptor overexpression have been identified in nasosinusal mucosa of aspirin - sensitive patients . in an in - vitro study , kowalski et al . found that after incubation with 200 g of acetylsalicylic acid , 15-hydroxyeicosatetraenoic ( 15-hete ) was overproduced by nasal polyp epithelial cells and peripheral blood leucocytes of asa - intolerant patients . 15-hete has several proinflammatory effects such as inflammatory mediator release from the mast cells , mucosal glycoprotein release and bronchial smooth muscle contraction and thus could be a major trigger of airway inflammatory reactions . supporting these observations , mastalerz and colleagues show significant upregulation of some arachidonate lipoxygenation products in asthmatic subjects with aspirin hypersensitivity , as manifested by high baseline levels of 5- , 15-hete in exhaled breath condensate . the lxs modulate leukocyte trafficking and vascular tone , and in contrast to cys - lts they have potent anti - inflammatory effects . there has been a significant number of studies indicating that cytokines , in particular th2 cytokines such as il-4 , regulate inflammatory processes in crs . increased il-4 gene expression and il-4 protein have been found in the upper airways in subjects with chronic sinusitis found as compared with controls . il-13 and ifn - gamma also induce ccl24 production but they are less potent stimuli compared with il-4 . have reported increased expression of both eotaxin ( ccl11 ) and eotaxin-2 in nasal polyps of patients suffering aerd , further supporting our findings in nasal polyposis . the role of eotaxins in nasal polyps has been reviewed previously . in a separate study , we have also shown that aerd patients with crs release the eosinophil attractant ccl5 ( rantes ) in nasal secretions , and lysine aspirin nasal challenge further increases the release of this chemokine . consistent with this finding , kupczyk et al . showed that these patients release increased levels of ccl5 and extended our observation by demonstrating the release of the eosinophil activating chemokine mcp-3 . however , they did not show increased levels of ccl5 following lysine - aspirin challenge . we performed nasal challenge using a nitrocellulose filter , while they applied the nasal challenge by aerosol . these methodological differences may explain the differences observed in these two studies using the aspirin challenge . the release of ccl5 by nasal polyps has also been documented [ 25 , 26 ] . il-5 plays a prominent role in eosinophilic - driven processes , and it has been documented in crswnp in both aspirin - sensitive and aspirin - tolerant patients . interestingly , this cytokine is decisive regarding the impact of staphylococcus - aureus-(se- ) derived enterotoxins , which function as superantigens . s. aureus enterotoxin b ( seb ) shifts the cytokine pattern further in nasal polyps toward t - helper-2 cytokines ( increases interleukin-2 , interleukin-4 , and interleukin-5 greater than twofold ) , but it reduces the t - regulatory cytokines interleukin-10 and tgf - beta1 . s. aureus - derived enterotoxins also influence local immunoglobulin synthesis and induce polyclonal immunoglobulin e production , which may contribute to severe inflammation via activation of mast cells . increased specific ige to both s. aureus enterotoxin a ( sea ) and seb has been detected in nasal polyps from both subject groups , but median levels were markedly higher in aia subjects than in ata subjects . using a microarray cdna technique , sekigawa et al . reported allelic associations of single nucleotide polymorphisms ( snps ) of indo and il1r2 , in nasal polyps derived from aspirin - sensitive , but not aspirin - tolerant , patients . identified periostin as a distinctive marker in nasal polyps derived from aspirin - sensitive patients . protein profiling is another approach , which has been used to investigate the pathogenesis of aspirin sensitivity in aerd patients . showed upregulation of -adaptin and heat shock protein 70 ( hsp70 ) in pooled nasal polyps samples from aia using protein microarray . -adaptin has been reported to be one of many proteins that form complexes in clathrin - coated pits and vesicles during receptor - mediated endocytosis , while hsp70 seem to be involved in environmental stress as it occurs during the inflammation process indicating a greater degree of tissue damage . the increased expression of hsp70 may represent a cytoprotective adaptive response and result in altered cell regulation . this , in turn , may contribute to cellular proliferation and ultimately a more aggressive form of nasal polyposis refractory to treatment characteristically seen in aspirin - sensitive patients . however , it is possible that studies supporting this observation may reflect differences between ethnic groups as no association between aerd and autoimmunity markers has been proved . infection of the upper airways of aerd patients by anaerobic bacteria , gram - negative organisms , staphylococcus aureus , and other bacteria is a frequent a complication . these bacteria are sources of antigens , which can form biofilms which in turn may amplify the inflammatory process . for instance , bachert et al . demonstrated the presence of staphylococcus enterotoxins a and b in 50% of patients with polyposis in the homogenized polyp tissue . it is important to note that these same antigens also have the ability to stimulate the production of specific ige antibodies . chronic viral respiratory infections have been suggested to play an important role in aerd via regulation of cytotoxic lymphocytes . to date , however , there is not convincing evidence that this could be the case . because the ethmoidal osteomeatal complex ( omc ) in the nasal middle meatus is a site of deposition of toxic inhalants , viral particles , and airborne allergens , it is tempting to hypothesize that chronic infection of the ethmoidal sinus could play a role in the genesis of the inflammatory process in the upper airways of aerd patients . the diagnosis of chronic sinusitis in aerd can occasionally be confusing in its early stages , since it usually precedes the onset of the aerd triad ( asthma , asa intolerance , and nasal polyposis ) . it can be easily confused with an allergic rhinitis although the skin tests for allergy will often be negative . however , either a nasal smear or blood count with high eosinophil numbers may give an initial diagnosis of nonallergic rhinitis with eosinophilia ( nares ) , an entity described in 1980 by mullarkey et al . and jacobs et al . . nares has been suggested to be the precursor of nasal polyposis in both aspirin - tolerant and intolerant patients . as the disease progresses , bronchial asthma and intolerance to aspirin will appear making the diagnosis of aerd easier ( figure 2 ) . in contrast to the early diagnosis of rhinitis , the diagnosis of rhinosinusitis with polyps is much easier to perform . symptoms such as hyposmia , nasal obstruction , anterior rhinorrhea , postnasal discharge , and occasionally cephalea are suggestive of nasal polyposis . moreover , either anterior rhinoscopy or nasal endoscopy enables the examiner to see the protrusion of gray or pink translucent , multilobulated , nonfriable , and usually clustered tissue , which characterizes the polyps . a ct scan of the paranasal sinuses may be useful to determine the extension of the polyps in the sinusal cavities . it is indicated in patients suffering asthma , rhinosinusitis , and nasal polyposis and in those with a history of near fatal reactions , but with negative history of asa intolerance ( 15% of asthmatic patients with negative history may be intolerant to asa ) . in patients with asthma and negative history of asa intolerance , but with risk factors ( rhinosinusitis , nasal , polyposis , a history of near fatal reactions ) the risk increases and the test is totally required . currently , there are four asa challenges : oral , bronchial , nasal and intravenous . the oral challenge is considered the gold standard , and it is practiced mainly in the usa . we use this last method as a diagnostic tool in our clinic on a routine basis . once aerd has been diagnosed it has been proved that most patients with nasal polyps are sensitized against common allergens . therefore , all patients with aerd should undergo skin or serological tests as part of their initial evaluation and , if necessary , specific immunotherapy should be instituted . undoubtedly , corticosteroids remain the cornerstone in the treatment of chronic hypereosinophilic sinusitis , whether accompanied or not by nasal polyposis [ 4144 ] . nasal topical steroids such as mometasone furoate , triamcinolone acetonide , or budesonide are used almost systematically as they have demonstrated a clear benefit in the control of mucosal oedema and of nasal polyposis . the prescription of topical nasal steroids is also very important for patients after endoscopic sinus surgery for nasal polyposis , as they can decrease the recurrence and growth of polyps . nasal lavages with saline solution or other commercial seawater sprays are also recommended to all patients . these should be performed at least twice a day in order to remove nasal secretions and crusts as much as possible before the application of the nasal steroid , thus facilitating its better absorption . systemic steroids are also commonly used , notably in cases of moderate - to - severe nasal polyposis or poorly controlled asthma , since it has been demonstrated that after their administration there is , in addition to an improvement in fev1 , a decrease in polyp size and a reduction in both obstructive symptoms and rhinorrhea . some patients also show a partial or complete recovery of the sense of smell . it is generally recommended to administer short courses of prednisone calculated up to 1 mg / kg / day , with a maximum of 80 mg a day , which should always be taken in a daily single dose , early in the morning , in order to strengthen the circadian rhythm of endogenous cortisol . we do not recommend steroids in low doses for prolonged periods because of the long - term deleterious effects on the hypothalamic - pituitary - adrenal axis . the prescription of antibiotics should be reserved only for cases of bacterial infection with the presence of purulent discharge and symptoms such as cephalea , rhinorrhea , posterior dripping , and fever . in such cases , the same antibiotics recommended for any acute bacterial nonpolypoid sinusitis are used empirically , specifically directed against haemophilus influenzae , streptococcus pneumonia , and moraxella catarrhalis . the recommended first choice antibiotic is amoxicillin in combination with clavulanic acid ; some other options are second or third generation cephalosporins , macrolides , or fluoroquinolones . some studies have demonstrated a beneficial effect with the administration of low doses of macrolides for prolonged periods in chronic sinusitis with or without nasal polyposis . in fact , it has been observed in some patients , especially of oriental races , that prolonged administration of low doses of these drugs may have an immunomodulatory effect leading to a reduction in the size of nasal polyps . this immunomodulatory effect of macrolides is perhaps less pronounced in caucasian patients , whose nasal polyps have a predominantly eosinophilic tissue infiltration , characteristic of an inflammation mediated by a th2 response . the use of antileukotrienes has been considered an important adjunctive treatment of both bronchial asthma and chronic hypereosinophilic rhinosinusitis with or without polyps in the aerd . however , both our experience and different reports in the literature show that their effect varies greatly from patient to patient . their use can be justified by the overproduction of leukotrienes present in aerd . however , because of their cost and relative effectiveness , we do not consider them as a part of the primary treatment , and we prefer to reserve them for cases in which conventional treatment with nasal lavages , topical nasal steroids , and one to two short courses of systemic steroid per year are insufficient . aspirin desensitization may be beneficial in selected patients , but it must always be performed under supervised conditions . several protocols of desensitization have been proposed allowing the completion of the procedure , usually within three to five days . the standard protocol for desensitization is an extension of the oral aspirin challenge protocol and all the safety precautions recommended for the challenge should be employed . increasing aspirin doses ( 100300 mg ) are generally administered orally , although the intranasal administration is also a good alternative . once desensitized , the patient must take a full dose of the prescribed amount of aspirin daily in order to maintain the desensitization effect . up to 30% of patients will not tolerate the side effects of daily aspirin intake , and other cox-1 inhibitors such as ibuprofen can trigger bronchospasm in these individuals . it has been demonstrated that desensitization can improve asthma control and prevent the progressive growth of nasal polyps . although several biochemical events occur directly after achieving aspirin desensitization , such as downregulation of arachidonic acid metabolism , decreased inflammatory cell activation , downregulation in cysteinyl - leukotriene cyslt1 receptor expression , the real mechanism of aspirin desensitization remains unknown [ 50 , 51 ] . sweet et al . studied 107 aerd patients in a retrospective survey after 6 years of followup . data from this study clearly demonstrated the clinical benefit of desensitization therapy for aspirin - sensitive patients , including a reduction in the number of hospitalization and emergency room visits , upper airway tract infections , sinus surgeries , and also , for many , an improvement in the sense of smell . however , twenty percent of the 65 patients of the therapy group reported upper digestive symptoms . asa desensitization is the only treatment that has shown clear impact on the natural course of aerd . evidence suggests that desensitization reduces the growth and recurrence rate of nasal polyps in aspirin - sensitive patients in the long term . this indeed reduces the need for sinus surgery and allows a decrease in intranasal corticosteroid use . however , the impact of asa desensitization on asthma has not been as consistent or reliable as the effects observed in the upper airway . surgical treatment must also be considered when nasal polyposis fails to respond to medical treatment . nowadays endoscopic sinus surgical techniques are preferred because of their proven safety and reduced morbidity . several studies confirm that endoscopic sinus surgery in aerd patients not only improves nasal symptoms but also enables a better control of asthma . therefore , surgical treatment should be considered in all cases of nasosinusal polyposis when medical treatment fails to improve nasosinusal symptoms , when more than two short courses of systemic steroids per year are needed , or when asthma is not controlled . another special indication for surgery is anosmia or hyposmia , and even more so in patients with professional noses such as chefs , winemakers , sommeliers , and perfume creators . however , the impact of surgery on olfaction is never guaranteed and surgery by itself could increase olfactory losses . currently , there are three types of surgery indicated for nasal polyposis . in simple polypectomy , only the visible polyps in the nasal cavities are removed , without penetrating into the ethmoidal cells . functional ethmoidectomy includes the opening of all the affected ethmoidal cells , with the resection of all polyps , but with minimal removal of the ethmoidal mucosa . finally , the nasalisation technique consists of a complete bilateral ethmoidectomy with eradication of all the ethmoidal mucosa , except the one covering the frontal recess boundaries . wide antrostomy and sphenoidotomy are also performed , and when needed , the middle turbinate can also be included in the resection . the choice of surgical technique will certainly depend on the preference and personal training of each surgeon ; however , increasing reports in the literature favor a more radical removal of the ethmoidal mucosa . in fact , it has been demonstrated that limited ethmoidectomy without mucosal removal offers long - term results comparable to those of a simple polypectomy ; therefore , we do not consider that the risk of a partial ethmoidectomy is justified if a more limited and more secure procedure can achieve similar results . nasalisation appears to offer better long - term results with a lower rate of recurrence of polyposis , less nasal obstruction and an improvement of olfaction that is more stable over time . it is also noteworthy that a radical surgery has a more positive and prolonged impact on the stabilization of bronchial asthma both in asa - tolerant and intolerant patients . jankowski et al . compared nasalisation with conventional ethmoidectomy , proving that a more radical surgery on the ethmoid mucosa does offer better functional results , both on olfaction and general nasal function [ 5658 ] . some authors actually advocate the systematic resection of the middle turbinate , since this provides a more complete removal of the ethmoidal mucosa , resulting in a lower incidence of polyposis recurrence in the long term . however , based on our experience , we believe that the middle turbinate is an important anatomical landmark that is helpful in cases of revision surgery , and also , with its preservation , frontal recess stenosis is less likely to occur . thus , we prefer to resect the middle turbinate only when it is affected severely by polypoid disease or when it has lost its structural support after the ethmoidectomy . in this latter situation , the resultant middle turbinate lateralization could occlude the surgical cavity , thereby impairing sinus lavages or topical steroid penetration and causing polyp relapse and chronic sinusitis . the surgical goals of nasalisation are the eradication of the ethmoidal mucosa and the creation of a cavity as wide as possible , in order to facilitate the subsequent cleaning and the proper dissemination of topical steroid nasal spray . it is also important to promote good mucosal healing through immediate postoperative application of a parenteral depot systemic steroid and the early reinstitution of topical nasal steroid use . clinically , the goals of surgery are first , improvement of nasal obstructive symptoms with , reduced rhinorrhea and postnasal discharge ; second , a stable and sustained improvement of smell ; finally , a better control of bronchial asthma . despite all the medical and surgical treatments described above despite all the major advances in research , understanding fully the mechanism of aerd still remains a challenge to the modern medical world . it is now well established that aspirin causes inhibition of cyclooxygenase-1 ( cox-1 ) associated with excessive metabolism of arachidonic acid ( aa ) to cysteinyl - leukotrienes ( cys - lts ) , while there is a rapid decrease in the synthesis of cox-1 products including prostaglandin e2 , which is known to have bronchodilator and anti - inflammatory properties . moreover , cytokines and staphylococcus superantigens further amplify the inflammatory process in the airways of aerd patients . however , it is important to keep in mind that a multidisciplinary approach is the cornerstone for a successful treatment of these patients .

rhinosinusitis is a feature of aspirin - exacerbated respiratory disease ( aerd ) , which in the initial phase is manifested as nasal congestion , mostly affecting females at the age of around 30 years on average . subsequently , nasal inflammation progresses to chronic eosinophilic rhinosinusitis , asthma , nasal polyposis , and intolerance to aspirin and to other nsaids . while it has been long established that nsaids cause inhibition of cyclooxygenase-1 ( cox-1 ) , leading to excessive metabolism of arachidonic acid ( aa ) to cysteinyl - leukotrienes ( cys - lts ) , there is now evidence that both cytokines and staphylococcus superantigens amplify the inflammatory process exacerbating the disease . this paper gives a brief overview of the development of chronic rhinosinusitis ( crs ) in sensitive patients , and we share our experience in the diagnosis and management of crs in aerd .

1. Introduction 2. Rhinosinusitis in AERD 3. Epidemiology 4. Physiopathology 5. Diagnosis 6. Treatment 7. Conclusion

the acute reaction to aspirin or other nonsteroidal anti - inflammatory drugs ( nsaids ) in aspirin - exacerbated respiratory disease ( aerd ) patients is a life - threatening condition characterized by upper airway symptoms ( nasal obstruction and rhinorrhea ) and lower airway symptoms ( shortness of breath and respiratory distress ) . aerd patients , however , suffer from chronic manifestations of the disease including chronic rhinosinusitis , nasal polyps , and asthma usually resistant to any treatment . the aiane study showed that up to 80% of aerd patients required intermediate to high doses of inhaled steroids , and up to 50% of them had to take oral steroids in order to obtain asthma control . similarly , the tenor study showed that asthma was severe in 66% of aerd subjects , 34% received high systemic steroidal doses , 67% needed antileukotrienes , and 20% of them required orotracheal intubation during acute reactions . chronic rhinosinusitis is a major condition in aerd , which in its initial phases is manifested only as nasal congestion , with asthma starting about two years after the initial nasal symptoms . chronic rhinosinusitis ( crs ) is defined as an inflammatory condition involving the mucosa underlying the nasal cavity and the paranasal sinuses that can also affect the underlying bone . , crs becomes a life - time condition , which is usually difficult to control . the minor criteria are the presence of purulence , anosmia and/or hyposmia , chronic cough , headache , dental pain , ear pressure , fatigue , and halitosis . the clinical evaluation is based on anterior rhinoscopy and nasal endoscopy that usually reveals mucosal oedema and hyperemia , with or without polyps , and frequently purulent secretions . crs with nasal polyps ( crswnp ) affects the full thickness of the nasal mucosa , which is replaced with an oedematous , generally eosinophilic , epithelium - coated bag of interstitial matrix ground substance . indeed , a greater number of these cells have been reported in both the upper and lower airways of patients of aspirin - sensitive patients as compared with aspirin - tolerant patients [ 68 ] . on activation , eosinophils release a vast array of mediators including leukotrienes , basic proteins ( major basic protein and eosinophil cationic protein ) , cytokines , and oxygen - free radicals that cause local tissue damage . we have found increased levels of eosinophil cationic protein in nasal secretions of aspirin - sensitive patients . a majority of patients with aspirin intolerance will develop nasal polyps during the course of the disease . nasal polyps are inflammatory pseudotumoral masses that most frequently start to grow from the ostiomeatal complex and the cells of the anterior ethmoidal sinus . they can affect the totality of the remaining sinusal cavities including the posterior ethmoidal cells , the maxillary , and the frontal or the sphenoidal sinuses , and they also can extend to the olfactory cleft , the sphenoethmoidal recess , and the nasal cavities ( figure 1 ) . nasal polyposis in aerd patients is present in up to 80 to 90% of patients and tends to be more aggressive and difficult to treat medically , also presenting with higher recurrence rates after surgery . in the aiane study that included 500 asa - intolerant patients from 14 different centres , nasosinusal polyposis was diagnosed on nasal endoscopy in 60% of the patients , but the prevalence rose to 90% when ct - scans of the nose and sinuses were performed . nasal polyps are formed by lax connective tissue stroma with oedema , inflammatory cells with a predominant eosinophil infiltration , mucosal glands , and newly formed vascular structures . the mucosal covering of the nasal polyps is a columnar glandular pseudostratified epithelium that also plays a major role in cytokine and inflammatory mediator release . there is no doubt that eosinophils are the main inflammatory cells found in the tissue specimens of nasal polyps , but neutrophils can also be present in large amounts and can even be predominant , notably in asiatic patients . other inflammatory cells can also be found in nasal polyps , such as lymphocytes , monocytes , plasma cells , and fibroblasts . chronic sinusitis with nasal polyposis is also characterized by an intense oedematous stroma with albumin deposition , formation of pseudocysts , and subepithelial and perivascular inflammatory cell infiltration . remodelling is a dynamic process regulated by diverse mediators among which tgf is the most important . in addition to being a key factor in the generation or the deficit of t - reg cells , tgf is also a critical factor implicated in the remodelling process in the airways through the attraction and proliferation of fibroblasts and upregulation of extracellular matrix synthesis . the full prevalence of aspirin - induced rhinosinusitis can be difficult to assess , as it primarily relies on the clinical history . the incidence rises to 24% in patients with severe asthma and up to 40% when the association of asthma and chronic rhinosinusitis with polyposis is present [ 1215 ] . usually asa intolerance appears in 3040-year - old patients with a previous history of chronic rhinosinusitis and/or asthma . intrinsic asthma tends to appear after the rhinitis , but before the onset of nasal polyposis ( figure 2 ) . skin testing for allergy can be positive in up to 3060% of aerd patients , but no association between asa intolerance and ige - mediated mechanisms has been identified . nevertheless , a high prevalence of food intolerance or antibiotic allergies has been found [ 1618 ] . however , it is now well established that an alteration of arachidonic acid metabolism takes place , and it is characterized by an imbalance between cyclooxygenase ( cox ) and lipoxygenase pathways that results in an overactive lipoxygenase pathway . these urinary leukotriene metabolite levels tend to drop significantly after sinus surgery , probably as a consequence of the elimination of a great amount of leukotriene producing cells in the ethmoidal sinus . indeed increased lte4 levels and cyslt1 receptor overexpression have been identified in nasosinusal mucosa of aspirin - sensitive patients . the lxs modulate leukocyte trafficking and vascular tone , and in contrast to cys - lts they have potent anti - inflammatory effects . there has been a significant number of studies indicating that cytokines , in particular th2 cytokines such as il-4 , regulate inflammatory processes in crs . increased il-4 gene expression and il-4 protein have been found in the upper airways in subjects with chronic sinusitis found as compared with controls . have reported increased expression of both eotaxin ( ccl11 ) and eotaxin-2 in nasal polyps of patients suffering aerd , further supporting our findings in nasal polyposis . the role of eotaxins in nasal polyps has been reviewed previously . in a separate study , we have also shown that aerd patients with crs release the eosinophil attractant ccl5 ( rantes ) in nasal secretions , and lysine aspirin nasal challenge further increases the release of this chemokine . il-5 plays a prominent role in eosinophilic - driven processes , and it has been documented in crswnp in both aspirin - sensitive and aspirin - tolerant patients . interestingly , this cytokine is decisive regarding the impact of staphylococcus - aureus-(se- ) derived enterotoxins , which function as superantigens . s. aureus enterotoxin b ( seb ) shifts the cytokine pattern further in nasal polyps toward t - helper-2 cytokines ( increases interleukin-2 , interleukin-4 , and interleukin-5 greater than twofold ) , but it reduces the t - regulatory cytokines interleukin-10 and tgf - beta1 . s. aureus - derived enterotoxins also influence local immunoglobulin synthesis and induce polyclonal immunoglobulin e production , which may contribute to severe inflammation via activation of mast cells . increased specific ige to both s. aureus enterotoxin a ( sea ) and seb has been detected in nasal polyps from both subject groups , but median levels were markedly higher in aia subjects than in ata subjects . reported allelic associations of single nucleotide polymorphisms ( snps ) of indo and il1r2 , in nasal polyps derived from aspirin - sensitive , but not aspirin - tolerant , patients . identified periostin as a distinctive marker in nasal polyps derived from aspirin - sensitive patients . protein profiling is another approach , which has been used to investigate the pathogenesis of aspirin sensitivity in aerd patients . this , in turn , may contribute to cellular proliferation and ultimately a more aggressive form of nasal polyposis refractory to treatment characteristically seen in aspirin - sensitive patients . however , it is possible that studies supporting this observation may reflect differences between ethnic groups as no association between aerd and autoimmunity markers has been proved . infection of the upper airways of aerd patients by anaerobic bacteria , gram - negative organisms , staphylococcus aureus , and other bacteria is a frequent a complication . these bacteria are sources of antigens , which can form biofilms which in turn may amplify the inflammatory process . demonstrated the presence of staphylococcus enterotoxins a and b in 50% of patients with polyposis in the homogenized polyp tissue . chronic viral respiratory infections have been suggested to play an important role in aerd via regulation of cytotoxic lymphocytes . to date , however , there is not convincing evidence that this could be the case . because the ethmoidal osteomeatal complex ( omc ) in the nasal middle meatus is a site of deposition of toxic inhalants , viral particles , and airborne allergens , it is tempting to hypothesize that chronic infection of the ethmoidal sinus could play a role in the genesis of the inflammatory process in the upper airways of aerd patients . the diagnosis of chronic sinusitis in aerd can occasionally be confusing in its early stages , since it usually precedes the onset of the aerd triad ( asthma , asa intolerance , and nasal polyposis ) . however , either a nasal smear or blood count with high eosinophil numbers may give an initial diagnosis of nonallergic rhinitis with eosinophilia ( nares ) , an entity described in 1980 by mullarkey et al . nares has been suggested to be the precursor of nasal polyposis in both aspirin - tolerant and intolerant patients . as the disease progresses , bronchial asthma and intolerance to aspirin will appear making the diagnosis of aerd easier ( figure 2 ) . in contrast to the early diagnosis of rhinitis , the diagnosis of rhinosinusitis with polyps is much easier to perform . symptoms such as hyposmia , nasal obstruction , anterior rhinorrhea , postnasal discharge , and occasionally cephalea are suggestive of nasal polyposis . moreover , either anterior rhinoscopy or nasal endoscopy enables the examiner to see the protrusion of gray or pink translucent , multilobulated , nonfriable , and usually clustered tissue , which characterizes the polyps . a ct scan of the paranasal sinuses may be useful to determine the extension of the polyps in the sinusal cavities . it is indicated in patients suffering asthma , rhinosinusitis , and nasal polyposis and in those with a history of near fatal reactions , but with negative history of asa intolerance ( 15% of asthmatic patients with negative history may be intolerant to asa ) . in patients with asthma and negative history of asa intolerance , but with risk factors ( rhinosinusitis , nasal , polyposis , a history of near fatal reactions ) the risk increases and the test is totally required . currently , there are four asa challenges : oral , bronchial , nasal and intravenous . the oral challenge is considered the gold standard , and it is practiced mainly in the usa . once aerd has been diagnosed it has been proved that most patients with nasal polyps are sensitized against common allergens . undoubtedly , corticosteroids remain the cornerstone in the treatment of chronic hypereosinophilic sinusitis , whether accompanied or not by nasal polyposis [ 4144 ] . nasal topical steroids such as mometasone furoate , triamcinolone acetonide , or budesonide are used almost systematically as they have demonstrated a clear benefit in the control of mucosal oedema and of nasal polyposis . the prescription of topical nasal steroids is also very important for patients after endoscopic sinus surgery for nasal polyposis , as they can decrease the recurrence and growth of polyps . systemic steroids are also commonly used , notably in cases of moderate - to - severe nasal polyposis or poorly controlled asthma , since it has been demonstrated that after their administration there is , in addition to an improvement in fev1 , a decrease in polyp size and a reduction in both obstructive symptoms and rhinorrhea . it is generally recommended to administer short courses of prednisone calculated up to 1 mg / kg / day , with a maximum of 80 mg a day , which should always be taken in a daily single dose , early in the morning , in order to strengthen the circadian rhythm of endogenous cortisol . we do not recommend steroids in low doses for prolonged periods because of the long - term deleterious effects on the hypothalamic - pituitary - adrenal axis . in such cases , the same antibiotics recommended for any acute bacterial nonpolypoid sinusitis are used empirically , specifically directed against haemophilus influenzae , streptococcus pneumonia , and moraxella catarrhalis . in fact , it has been observed in some patients , especially of oriental races , that prolonged administration of low doses of these drugs may have an immunomodulatory effect leading to a reduction in the size of nasal polyps . this immunomodulatory effect of macrolides is perhaps less pronounced in caucasian patients , whose nasal polyps have a predominantly eosinophilic tissue infiltration , characteristic of an inflammation mediated by a th2 response . the use of antileukotrienes has been considered an important adjunctive treatment of both bronchial asthma and chronic hypereosinophilic rhinosinusitis with or without polyps in the aerd . however , both our experience and different reports in the literature show that their effect varies greatly from patient to patient . their use can be justified by the overproduction of leukotrienes present in aerd . however , because of their cost and relative effectiveness , we do not consider them as a part of the primary treatment , and we prefer to reserve them for cases in which conventional treatment with nasal lavages , topical nasal steroids , and one to two short courses of systemic steroid per year are insufficient . aspirin desensitization may be beneficial in selected patients , but it must always be performed under supervised conditions . the standard protocol for desensitization is an extension of the oral aspirin challenge protocol and all the safety precautions recommended for the challenge should be employed . once desensitized , the patient must take a full dose of the prescribed amount of aspirin daily in order to maintain the desensitization effect . it has been demonstrated that desensitization can improve asthma control and prevent the progressive growth of nasal polyps . although several biochemical events occur directly after achieving aspirin desensitization , such as downregulation of arachidonic acid metabolism , decreased inflammatory cell activation , downregulation in cysteinyl - leukotriene cyslt1 receptor expression , the real mechanism of aspirin desensitization remains unknown [ 50 , 51 ] . data from this study clearly demonstrated the clinical benefit of desensitization therapy for aspirin - sensitive patients , including a reduction in the number of hospitalization and emergency room visits , upper airway tract infections , sinus surgeries , and also , for many , an improvement in the sense of smell . however , twenty percent of the 65 patients of the therapy group reported upper digestive symptoms . evidence suggests that desensitization reduces the growth and recurrence rate of nasal polyps in aspirin - sensitive patients in the long term . however , the impact of asa desensitization on asthma has not been as consistent or reliable as the effects observed in the upper airway . surgical treatment must also be considered when nasal polyposis fails to respond to medical treatment . several studies confirm that endoscopic sinus surgery in aerd patients not only improves nasal symptoms but also enables a better control of asthma . currently , there are three types of surgery indicated for nasal polyposis . functional ethmoidectomy includes the opening of all the affected ethmoidal cells , with the resection of all polyps , but with minimal removal of the ethmoidal mucosa . wide antrostomy and sphenoidotomy are also performed , and when needed , the middle turbinate can also be included in the resection . the choice of surgical technique will certainly depend on the preference and personal training of each surgeon ; however , increasing reports in the literature favor a more radical removal of the ethmoidal mucosa . in fact , it has been demonstrated that limited ethmoidectomy without mucosal removal offers long - term results comparable to those of a simple polypectomy ; therefore , we do not consider that the risk of a partial ethmoidectomy is justified if a more limited and more secure procedure can achieve similar results . nasalisation appears to offer better long - term results with a lower rate of recurrence of polyposis , less nasal obstruction and an improvement of olfaction that is more stable over time . some authors actually advocate the systematic resection of the middle turbinate , since this provides a more complete removal of the ethmoidal mucosa , resulting in a lower incidence of polyposis recurrence in the long term . however , based on our experience , we believe that the middle turbinate is an important anatomical landmark that is helpful in cases of revision surgery , and also , with its preservation , frontal recess stenosis is less likely to occur . thus , we prefer to resect the middle turbinate only when it is affected severely by polypoid disease or when it has lost its structural support after the ethmoidectomy . the surgical goals of nasalisation are the eradication of the ethmoidal mucosa and the creation of a cavity as wide as possible , in order to facilitate the subsequent cleaning and the proper dissemination of topical steroid nasal spray . it is now well established that aspirin causes inhibition of cyclooxygenase-1 ( cox-1 ) associated with excessive metabolism of arachidonic acid ( aa ) to cysteinyl - leukotrienes ( cys - lts ) , while there is a rapid decrease in the synthesis of cox-1 products including prostaglandin e2 , which is known to have bronchodilator and anti - inflammatory properties . moreover , cytokines and staphylococcus superantigens further amplify the inflammatory process in the airways of aerd patients .

patients with head and neck cancer ( hnc ) may suffer variable degrees of functional impairment that are related to speaking , swallowing , breathing , taste , and smell , as well as facial disfigurement during treatment and in the illness course . they are at higher risk of having emotional distress than any other form of cancer amid loss of these functions.1,2 emotional disorders might be expected after a diagnosis of cancer and going through a period of enduring adaptation to the illness from weeks to months . understanding the time course for the impairment of these functions to affect the mood is important for the commencement of early intervention . a review of the literature revealed that there was wide variation in reported rate of depression in patients with hnc : from 6% to 48%.3 however , this huge discrepancy was mainly because of differences in 1 ) method of investigation , 2 ) measurement , 3 ) samples , 4 ) degree of severity or staging of the cancer , and 5 ) time points of assessment . in general , higher rates of depression were estimated when using patient self - reported questionnaires , where overrating might be seen in most items of somatic dysfunction , particularly patients suffering from a chronic or acute physical illness.4 krebber et al5 conducted a meta - analysis , and found that the average rate of depression in hnc patients was 20% when measured by self - reported questionnaires , but only approximately half of that ( 11% ) when a diagnostic interview was employed . most of these studies were done with patients at 1 year or later , and there have not been many studies on psychiatric morbidity of hnc patients during the early phase of treatment.6,7 following the illness course and treatment , the quality of life ( ql ) of cancer patients is expected to be affected with the loss of physical and health - related functions . using a standardized structured interview and validated instrument for the assessment of ql , this study followed patients with newly diagnosed hnc , to 1 ) assess psychiatric morbidity and its changes , specifically with anxiety and depressive disorders ( depression ) at pretreatment , 3 months , and 6 months ; 2 ) assess the health - related ql ( hrql ) of the patients with hnc ; and 3 ) identify risk factors for depression with various health - related function losses that are associated with hnc . this was a prospective study of newly diagnosed hnc patients attending the outpatient combined - treatment clinic for hnc at kaohsiung chang gung memorial hospital in southern taiwan between january 2011 and january 2012 . the inclusion criteria were newly diagnosed and untreated hnc , not metastatic cancer , and having the ability to verbalize and write . this study was approved by the human research ethics committee of chang gung memorial hospital ( 98 - 3563b ) . the psychiatric interview was carried out by a research psychiatrist , while the social and demographic information , as well as ql , was also simultaneously collected by a trained research assistant . the patients were prospectively followed and assessed for their psychiatric morbidities and hrql before treatment ( pretreatment ) and at 3 and 6 months . psychiatric morbidity were being assessed and made using the structured clinical interview for the diagnostic and statistical manual of mental disorders ( dsm)-iv ( scid ) , clinician version , a structured diagnostic interview based on dsm - iv criteria.8 the scid is a clinician - administered , semistructured interview for use with psychiatric patients or with community subjects who are undergoing evaluation for possible psychiatric diseases . its main framework consists of nine diagnostic modules . interviewers may choose to eliminate one or more modules to focus selectively on areas of the greatest diagnostic interest . most sections start with a leading question that allows the interviewer to skip the subsequent questions if not met . the scid is regarded as a gold standard for psychiatric diagnosis , and it was conducted by a senior psychiatrist ( yl ) with over 25 years of clinical and research experience . the european organisation for research and treatment of cancer ( eortc ) quality of life questionnaire ( qlq ) was used to assess ql for cancer patients . various modules have been developed for disease - specific treatment measurements , and in this study we used the module that is specific to the hnc ( the qlq head and neck [ h&n]-35).9 this consists of 35 questions on a 4-point scale ( 1= not at all , 4= very much ) , with seven multiple items on pain , swallowing , senses ( taste and smell ) , speech , social eating , social contact , and sexuality , and eleven single items on other health functions related to teeth , mouth opening , dry mouth , sticky saliva , cough , feeling ill , painkillers , nutrition supplements , feeding tube , weight loss , and weight gain . all scales ranged in a transformed score of 0100 . a high score on a symptom scale or single item indicates more severe symptoms or problems . retest reliability and internal consistency.10 the eortc qlq - h&n35 has been widely used for studying hrql in hnc patients in taiwan and european countries.11,12 the demographic characteristics , psychiatric diagnoses , history of substance use ( alcohol , smoking , betel nut ) , clinical staging of cancer , and treatment methods and hrql were first summarized using descriptive statistics . they were then compared between patients with and without depression using or t - tests . with items based on the eortc qlq - h&n35 , hrql was analyzed and compared across time using analysis of variance for repeated measurements . to investigate the effect of depression at each time point on hrql , a generalized mixed - effect model for repeated measurements this has the advantages over traditional methods , such as last observation carried forward , of including all available data across the follow - up period and providing better estimates under a broad assumption of missing data . each item of the eortc qlq - h&n35 at pretreatment and 3- and 6-month assessment was explored as the primary outcome , with depression at each time point used as the dependent variable . post hoc estimation was also applied to compare outcome variables among these three time points . to avoid false positives , this was a prospective study of newly diagnosed hnc patients attending the outpatient combined - treatment clinic for hnc at kaohsiung chang gung memorial hospital in southern taiwan between january 2011 and january 2012 . the inclusion criteria were newly diagnosed and untreated hnc , not metastatic cancer , and having the ability to verbalize and write . this study was approved by the human research ethics committee of chang gung memorial hospital ( 98 - 3563b ) . the psychiatric interview was carried out by a research psychiatrist , while the social and demographic information , as well as ql , was also simultaneously collected by a trained research assistant . the patients were prospectively followed and assessed for their psychiatric morbidities and hrql before treatment ( pretreatment ) and at 3 and 6 months . psychiatric morbidity were being assessed and made using the structured clinical interview for the diagnostic and statistical manual of mental disorders ( dsm)-iv ( scid ) , clinician version , a structured diagnostic interview based on dsm - iv criteria.8 the scid is a clinician - administered , semistructured interview for use with psychiatric patients or with community subjects who are undergoing evaluation for possible psychiatric diseases . its main framework consists of nine diagnostic modules . interviewers may choose to eliminate one or more modules to focus selectively on areas of the greatest diagnostic interest . most sections start with a leading question that allows the interviewer to skip the subsequent questions if not met . the scid is regarded as a gold standard for psychiatric diagnosis , and it was conducted by a senior psychiatrist ( yl ) with over 25 years of clinical and research experience . the european organisation for research and treatment of cancer ( eortc ) quality of life questionnaire ( qlq ) was used to assess ql for cancer patients . various modules have been developed for disease - specific treatment measurements , and in this study we used the module that is specific to the hnc ( the qlq head and neck [ h&n]-35).9 this consists of 35 questions on a 4-point scale ( 1= not at all , 4= very much ) , with seven multiple items on pain , swallowing , senses ( taste and smell ) , speech , social eating , social contact , and sexuality , and eleven single items on other health functions related to teeth , mouth opening , dry mouth , sticky saliva , cough , feeling ill , painkillers , nutrition supplements , feeding tube , weight loss , and weight gain . all scales ranged in a transformed score of 0100 . a high score on a symptom scale or single item indicates more severe symptoms or problems . retest reliability and internal consistency.10 the eortc qlq - h&n35 has been widely used for studying hrql in hnc patients in taiwan and european countries.11,12 psychiatric morbidity were being assessed and made using the structured clinical interview for the diagnostic and statistical manual of mental disorders ( dsm)-iv ( scid ) , clinician version , a structured diagnostic interview based on dsm - iv criteria.8 the scid is a clinician - administered , semistructured interview for use with psychiatric patients or with community subjects who are undergoing evaluation for possible psychiatric diseases . its main framework consists of nine diagnostic modules . interviewers may choose to eliminate one or more modules to focus selectively on areas of the greatest diagnostic interest . most sections start with a leading question that allows the interviewer to skip the subsequent questions if not met . the scid is regarded as a gold standard for psychiatric diagnosis , and it was conducted by a senior psychiatrist ( yl ) with over 25 years of clinical and research experience . the european organisation for research and treatment of cancer ( eortc ) quality of life questionnaire ( qlq ) was used to assess ql for cancer patients . various modules have been developed for disease - specific treatment measurements , and in this study we used the module that is specific to the hnc ( the qlq head and neck [ h&n]-35).9 this consists of 35 questions on a 4-point scale ( 1= not at all , 4= very much ) , with seven multiple items on pain , swallowing , senses ( taste and smell ) , speech , social eating , social contact , and sexuality , and eleven single items on other health functions related to teeth , mouth opening , dry mouth , sticky saliva , cough , feeling ill , painkillers , nutrition supplements , feeding tube , weight loss , and weight gain . all scales ranged in a transformed score of 0100 . a high score on a symptom scale or single item indicates more severe symptoms or problems . retest reliability and internal consistency.10 the eortc qlq - h&n35 has been widely used for studying hrql in hnc patients in taiwan and european countries.11,12 the demographic characteristics , psychiatric diagnoses , history of substance use ( alcohol , smoking , betel nut ) , clinical staging of cancer , and treatment methods and hrql were first summarized using descriptive statistics . they were then compared between patients with and without depression using or t - tests . with items based on the eortc qlq - h&n35 , hrql was analyzed and compared across time using analysis of variance for repeated measurements . to investigate the effect of depression at each time point on hrql , a generalized mixed - effect model for repeated measurements this has the advantages over traditional methods , such as last observation carried forward , of including all available data across the follow - up period and providing better estimates under a broad assumption of missing data . each item of the eortc qlq - h&n35 at pretreatment and 3- and 6-month assessment was explored as the primary outcome , with depression at each time point used as the dependent variable . post hoc estimation was also applied to compare outcome variables among these three time points . to avoid false positives , of the 106 patients who were recruited in the study , 93 successfully completed the study . among those who did not complete the study , three died and ten were unwilling to continue during the follow - up period and excluded . table 1 shows the sociodemographic and clinical characteristics of the patients , with the majority being males ( 86% ) and an average age of 52.79.6 years . three - quarters of them were married and currently employed , and their mean educational level was 9.93.7 years . substance use was commonly seen in these patients , with 63.4% smoking , 41.9% drinking alcohol , and 29% betel - nut chewing . approximately two - thirds of the patients were diagnosed as having advanced clinical stages of cancer ( iii and iv ) , and over half received surgical and/or chemo- and radiotherapy . the time to initial treatment after the diagnosis was 2.21.4 weeks ( table 1 ) . at the pretreatment evaluation , more than half had one or more psychiatric diagnoses . because of the small number of patients , we grouped the diagnoses of adjustment disorder with anxious mood and anxiety disorder not otherwise specified into anxiety disorders ( anxiety ) , and all depressive - spectrum disorders into depressive disorders ( depression ) . the prevalence of psychiatric disorders after the regrouping was as follows : anxiety disorders ( 27.3% ) , alcohol - use disorder ( 18.9% ) , depressive disorders ( 8.5% ) , and primary insomnia ( 2.8% ) . during the 6-month period , there were some changes in the distribution of psychiatric morbidities , with the most significant being the consistently and sharply decreasing rate of anxiety disorders ( from 27.3% to 6.4% and 3.3% at pretreatment and 3 and 6 months , respectively ) . there was not much change in alcohol - use disorders , with rates ranging from 18.9% to 20.4% . depression showed a skew pattern , with a rise in the rate after pretreatment ( 8.5% ) to three times that at the peak at 3 months ( 24.5% ) , but gradually receded nearly to the level of initial assessment at 6 months ( figure 1 ) . psychiatric morbidity with any psychiatric diagnosis declined steadily during the 6-month period ( with rates from 54.7% at pretreatment to 45.8% at 3 months and 38.7% at 6 months ) . the 6-month follow - up of hrql , with results from the assessment on items based on the eortc qlq - h&n35 at pretreatment , 3 months , and 6 months is shown in table 2 . all except the use of feeding tubes were significant over time , with some differences in patterns of distribution . similar to the pattern of the rate of depression , patients felt worse and had more pain and higher frequency of taking painkillers in the first 3 months . levels returned to approximately the level of pretreatment at 6 months , together with dysfunction of mouth opening , swallowing , sticky saliva , sense and speech problems , trouble with social eating and social contact , and weight loss . the problem with teeth varied over time , but coughing and sexual function decreased with time . dry mouth persistently increased during the 6-month period , despite a small but steady increment of weight gain . when comparisons were made between the depressed and nondepressed hnc patients , there were no significant differences in their sociodemographics ( sex , age , marital status , employment , education , and substance use ) or clinical characteristics ( cancer staging , time to initial treatment , and mode of treatment ) ( table 3 ) . despite significant changes in hrql on almost all items of the eortc qlq - h&n35 during the 6-month period , there were some differences between the two groups across time when they were analyzed further using the generalized mixed - effect model . the results showed that over time , loss of functions to sense ( p<0.001 ) , speech ( p<0.001 ) , sexuality ( p<0.001 ) , and dry mouth ( p<0.001 ) were only significantly related to depression , and those with depression had a higher tendency to take painkillers ( p<0.001 ) and nutrition supplements ( p<0.001 ) than nondepressed patients . however , depression was predicted by sticky saliva and problems with social contact at 3 months and social eating at 6 months ( table 4 ) . of the 106 patients who were recruited in the study , 93 successfully completed the study . among those who did not complete the study , three died and ten were unwilling to continue during the follow - up period and excluded . table 1 shows the sociodemographic and clinical characteristics of the patients , with the majority being males ( 86% ) and an average age of 52.79.6 years . three - quarters of them were married and currently employed , and their mean educational level was 9.93.7 years . substance use was commonly seen in these patients , with 63.4% smoking , 41.9% drinking alcohol , and 29% betel - nut chewing . approximately two - thirds of the patients were diagnosed as having advanced clinical stages of cancer ( iii and iv ) , and over half received surgical and/or chemo- and radiotherapy . the time to initial treatment after the diagnosis was 2.21.4 weeks ( table 1 ) . at the pretreatment evaluation , more than half had one or more psychiatric diagnoses . because of the small number of patients , we grouped the diagnoses of adjustment disorder with anxious mood and anxiety disorder not otherwise specified into anxiety disorders ( anxiety ) , and all depressive - spectrum disorders into depressive disorders ( depression ) . the prevalence of psychiatric disorders after the regrouping was as follows : anxiety disorders ( 27.3% ) , alcohol - use disorder ( 18.9% ) , depressive disorders ( 8.5% ) , and primary insomnia ( 2.8% ) . during the 6-month period , there were some changes in the distribution of psychiatric morbidities , with the most significant being the consistently and sharply decreasing rate of anxiety disorders ( from 27.3% to 6.4% and 3.3% at pretreatment and 3 and 6 months , respectively ) . there was not much change in alcohol - use disorders , with rates ranging from 18.9% to 20.4% . depression showed a skew pattern , with a rise in the rate after pretreatment ( 8.5% ) to three times that at the peak at 3 months ( 24.5% ) , but gradually receded nearly to the level of initial assessment at 6 months ( figure 1 ) . psychiatric morbidity with any psychiatric diagnosis declined steadily during the 6-month period ( with rates from 54.7% at pretreatment to 45.8% at 3 months and 38.7% at 6 months ) . the 6-month follow - up of hrql , with results from the assessment on items based on the eortc qlq - h&n35 at pretreatment , 3 months , and 6 months is shown in table 2 . all except the use of feeding tubes were significant over time , with some differences in patterns of distribution . similar to the pattern of the rate of depression , patients felt worse and had more pain and higher frequency of taking painkillers in the first 3 months . levels returned to approximately the level of pretreatment at 6 months , together with dysfunction of mouth opening , swallowing , sticky saliva , sense and speech problems , trouble with social eating and social contact , and weight loss . the problem with teeth varied over time , but coughing and sexual function decreased with time . dry mouth persistently increased during the 6-month period , despite a small but steady increment of weight gain . when comparisons were made between the depressed and nondepressed hnc patients , there were no significant differences in their sociodemographics ( sex , age , marital status , employment , education , and substance use ) or clinical characteristics ( cancer staging , time to initial treatment , and mode of treatment ) ( table 3 ) . despite significant changes in hrql on almost all items of the eortc qlq - h&n35 during the 6-month period , there were some differences between the two groups across time when they were analyzed further using the generalized mixed - effect model . the results showed that over time , loss of functions to sense ( p<0.001 ) , speech ( p<0.001 ) , sexuality ( p<0.001 ) , and dry mouth ( p<0.001 ) were only significantly related to depression , and those with depression had a higher tendency to take painkillers ( p<0.001 ) and nutrition supplements ( p<0.001 ) than nondepressed patients . however , depression was predicted by sticky saliva and problems with social contact at 3 months and social eating at 6 months ( table 4 ) . this prospective study showed that patients with hnc suffered from variable degrees of psychiatric morbidities during the first 6 months of treatment course . more than a quarter had an anxiety disorder ( 27.3% ) at pretreatment , but the rate steadily declined to 3.3% during the 6-month period . depressive disorder , however , did not follow such a pattern , but changed over time and manifested a skew pattern , with a rise in rate during the first 3 months ( from 8.1% to 24.5% ) and then a gradually return to pretreatment level in later months ( 14% ) . when patients were first confronted with the diagnosis of hnc , anticipatory anxiety was commonly seen , along with symptoms of overwhelming information , distractibility , and poor sleep . at this phase , a high rate of anxiety was demonstrated , similar to previous research.13 however , once the patients were more adapted to the disease and with the progress of treatment , the rate of anxiety significantly declined.14 early intervention by the team , which included psychiatrists and psychologists , also helped reduce tension and anxiety of the patients . we used a standardized structured interview in the assessment of psychiatric symptoms and diagnosis ( scid ) instead of a self - reported questionnaire . such a procedure could avoid erroneous rating of symptoms of hnc for depression or because of the adverse effects of the treatment . for example , physical symptoms with appetite and body - weight loss are common in cancer or during the treatment process . it is thus important to differentiate symptoms of losing of the function to eat ( a symptom of hnc ) with losing of the desire to eat ( a symptom of depression ) most self - reported instruments of depressive symptoms nevertheless are not able to make such distinctive differences.4 also , since the scid assessment was conducted consistently by only one rater , there was less concern about the problem of interrater reliability . to further assess various symptoms and function loss that were related to the cancer , we employed a questionnaire on hrql with a module that is specific for hnc : the eortc qlq - h&n35 . it was found that there was significant impairment in all items of hrql during the 6-month period of treatment , except with the use feeding tubes ( table 3 ) . some of these symptoms might have correlated with depression,12,15,16 and this was able to be identified by applying analysis using the generalized mixed - effect model for repeated measurements across time . the results showed that the hnc patients with depression had more significant impairment than nondepressed patients in problems with sense and speech , dry mouth , sexuality , and loss of weight . the use of painkillers , especially those with narcotic analgesics , has been reported to have a higher risk of depression.17,18 the rates of depression in this sample of hnc patients ranging from 8% to 25% are compatible with earlier studies of longer duration.1923 we found that there were no significant differences in sociodemographics ( sex , age , marital status , employment , education , and substance use ) or clinical characteristics ( cancer staging , time to initial treatment , and mode of treatment ) between patients with and without depressive disorders , which might indicate that those factors were not the major cause of depression of hnc patients during the treatment course . in the first 3 months of treatment , depression was significantly predicated in patients with dysfunction in salivation with sticky saliva and trouble with social contact . dry mouth with sticky saliva is regarded as an adverse sign in chinese medical philosophy that reflects an imbalance of the internal organ systems.24 this might have created pessimism in these patients if they had dysfunction in salivation and limitation in the ingestion and chewing of food . as food and eating are intrinsic factors of life and chinese culture , the loss of such functions among these patients further added to negative thoughts about their vitality and survival . this may partly explain the increasing rate of depressive disorders in the first few months of treatment , together with the fear over the uncertainty of their disease course or even death . with treatment progress , trouble with social eating was still the main factor related to depression at the 6-month interval . this illustrated the importance of food , eating , and nutrition for these patients , despite impairment of certain physical functions that were related to hnc . a fifth of the patients abused alcohol , and this rate was consistent throughout the 6-month period . although alcohol has been regarded as one of the risk factors for hnc,22 the rate of alcohol abuse in this study was considered much lower than most reported studies , of 30%90%.25 taiwanese generally have a lower rate of alcoholism than caucasians , due to a lack of related genes for enzymes that are responsible for metabolizing alcohol and acetaldehyde,26 and this could well have accounted for the relatively low prevalence rate of alcoholism in study . in spite of this , ~30% of these patients had the habit of chewing betel nut . although it is not related to any mood disorders , chewing betel nut is however carcinogenic to humans , and has been reported to have association with oral / buccal cancer.27 betel nut contains a substance called arecoline , which has proved to contribute to histologic changes in the oral mucosa . aside from betel - nut habits , it is noteworthy that > 60% of the patients also smoked . this was not related to mood disorders , but might have harmful effects on health and thus to the disease prognosis.27,28 it is believed that one in four terminally ill patients have symptoms of depression , but that up to 80% of this depression may not be recognized and is thus untreated.29 among the symptoms , loss of sexuality , appetite , body weight , and sleep are also core vegetative symptoms of depression . in our society and across this region , sexual function has often been disregarded , and is regrettably not routinely inquired about or assessed . the awareness of depression , the identification of symptoms , and the initiation of treatment are essential if patients are to be offered optimum palliation of psychological as well as physical symptoms.29 in summary , this study of a 6-month experience of patients with hnc showed that psychiatric morbidities were commonly seen in the early phase of treatment . high rates of anxiety were found before treatment , but had a steadily decline following the treatment course . depression , on the other hand , demonstrated a skew pattern , with a peak at 3 months declining to pretreatment level at later months . dysfunction in salivation and problems with social contacts and eating were the three major risk factors for depression at 3 and 6 months , as impairments of sense , speech , dry mouth , and sexuality were more significant in the depressed patients who also consumed more painkillers and nutritional supplements . first , the sample size was relatively small , and further inferences can not be made based on the exact anatomical sites that are related to different side effects from treatment ( eg , eating and swallowing ) . second , there was no random selection ; a consecutive sampling method was used instead , and it included all cases that fitted the criteria . a longer period is needed to prove the long - term outcome of these functions in relation to depression .

objectivewe aimed to assess psychiatric morbidities of patients with head and neck cancer ( hnc ) in a prospective study at pretreatment , and 3 and 6 months after treatment , and to compare their health - related quality of life ( hrql ) between those with and without depressive disorders ( depression).materials and methodspatients with newly diagnosed hnc from a tertiary hospital were recruited into the study . they were assessed for psychiatric morbidities using the structured clinical interview for the diagnostic and statistical manual of mental disorders , fourth edition . their hrql was simultaneously evaluated using the quality of life questionnaire of the european organisation for research and treatment of cancer with a specific module for head and neck cancer ; and depressed and nondepressed hnc patients were compared by using the generalized mixed - effect model for repeated measurements.resultsa total of 106 patients were recruited into this study . high rates of anxiety were found at pretreatment , but steadily declined over time ( from 27.3% to 6.4% , and later 3.3% ) . a skew pattern of depression was observed , with prevalence rates from 8.5% at pretreatment to 24.5% and 14% at 3 and 6 months , respectively , after treatment . we found that loss of sense ( p=0.001 ) , loss of speech ( p<0.001 ) , low libido ( p=0.001 ) , dry mouth ( p<0.001 ) , and weight loss ( p=0.001 ) were related to depression over time . the depressed patients had a higher consumption of painkillers ( p=0.001 ) and nutrition supplements ( p<0.001 ) . the results showed that depression was predicted by sticky saliva ( p<0.001 ) and trouble with social contact ( p<0.001 ) at 3 months , and trouble with social eating ( p<0.001 ) at 6 months.conclusionpatients with hnc experienced different changes in anxiety and depression in the first 6 months of treatment . dysfunction in salivation , problems with eating , and problems with social contacts were major risk factors for depression .

Introduction Materials and methods Subjects Procedures Structured Clinical Interview for DSM-IV, clinician version Assessment of health-related quality of life Statistical analyses Results Characteristics of samples Psychiatric morbidity Six-month HRQL Depression and HRQL Discussion Conclusion

patients with head and neck cancer ( hnc ) may suffer variable degrees of functional impairment that are related to speaking , swallowing , breathing , taste , and smell , as well as facial disfigurement during treatment and in the illness course . a review of the literature revealed that there was wide variation in reported rate of depression in patients with hnc : from 6% to 48%.3 however , this huge discrepancy was mainly because of differences in 1 ) method of investigation , 2 ) measurement , 3 ) samples , 4 ) degree of severity or staging of the cancer , and 5 ) time points of assessment . in general , higher rates of depression were estimated when using patient self - reported questionnaires , where overrating might be seen in most items of somatic dysfunction , particularly patients suffering from a chronic or acute physical illness.4 krebber et al5 conducted a meta - analysis , and found that the average rate of depression in hnc patients was 20% when measured by self - reported questionnaires , but only approximately half of that ( 11% ) when a diagnostic interview was employed . most of these studies were done with patients at 1 year or later , and there have not been many studies on psychiatric morbidity of hnc patients during the early phase of treatment.6,7 following the illness course and treatment , the quality of life ( ql ) of cancer patients is expected to be affected with the loss of physical and health - related functions . using a standardized structured interview and validated instrument for the assessment of ql , this study followed patients with newly diagnosed hnc , to 1 ) assess psychiatric morbidity and its changes , specifically with anxiety and depressive disorders ( depression ) at pretreatment , 3 months , and 6 months ; 2 ) assess the health - related ql ( hrql ) of the patients with hnc ; and 3 ) identify risk factors for depression with various health - related function losses that are associated with hnc . this was a prospective study of newly diagnosed hnc patients attending the outpatient combined - treatment clinic for hnc at kaohsiung chang gung memorial hospital in southern taiwan between january 2011 and january 2012 . the patients were prospectively followed and assessed for their psychiatric morbidities and hrql before treatment ( pretreatment ) and at 3 and 6 months . psychiatric morbidity were being assessed and made using the structured clinical interview for the diagnostic and statistical manual of mental disorders ( dsm)-iv ( scid ) , clinician version , a structured diagnostic interview based on dsm - iv criteria.8 the scid is a clinician - administered , semistructured interview for use with psychiatric patients or with community subjects who are undergoing evaluation for possible psychiatric diseases . the european organisation for research and treatment of cancer ( eortc ) quality of life questionnaire ( qlq ) was used to assess ql for cancer patients . various modules have been developed for disease - specific treatment measurements , and in this study we used the module that is specific to the hnc ( the qlq head and neck [ h&n]-35).9 this consists of 35 questions on a 4-point scale ( 1= not at all , 4= very much ) , with seven multiple items on pain , swallowing , senses ( taste and smell ) , speech , social eating , social contact , and sexuality , and eleven single items on other health functions related to teeth , mouth opening , dry mouth , sticky saliva , cough , feeling ill , painkillers , nutrition supplements , feeding tube , weight loss , and weight gain . retest reliability and internal consistency.10 the eortc qlq - h&n35 has been widely used for studying hrql in hnc patients in taiwan and european countries.11,12 the demographic characteristics , psychiatric diagnoses , history of substance use ( alcohol , smoking , betel nut ) , clinical staging of cancer , and treatment methods and hrql were first summarized using descriptive statistics . they were then compared between patients with and without depression using or t - tests . to investigate the effect of depression at each time point on hrql , a generalized mixed - effect model for repeated measurements this has the advantages over traditional methods , such as last observation carried forward , of including all available data across the follow - up period and providing better estimates under a broad assumption of missing data . to avoid false positives , this was a prospective study of newly diagnosed hnc patients attending the outpatient combined - treatment clinic for hnc at kaohsiung chang gung memorial hospital in southern taiwan between january 2011 and january 2012 . the patients were prospectively followed and assessed for their psychiatric morbidities and hrql before treatment ( pretreatment ) and at 3 and 6 months . psychiatric morbidity were being assessed and made using the structured clinical interview for the diagnostic and statistical manual of mental disorders ( dsm)-iv ( scid ) , clinician version , a structured diagnostic interview based on dsm - iv criteria.8 the scid is a clinician - administered , semistructured interview for use with psychiatric patients or with community subjects who are undergoing evaluation for possible psychiatric diseases . the european organisation for research and treatment of cancer ( eortc ) quality of life questionnaire ( qlq ) was used to assess ql for cancer patients . various modules have been developed for disease - specific treatment measurements , and in this study we used the module that is specific to the hnc ( the qlq head and neck [ h&n]-35).9 this consists of 35 questions on a 4-point scale ( 1= not at all , 4= very much ) , with seven multiple items on pain , swallowing , senses ( taste and smell ) , speech , social eating , social contact , and sexuality , and eleven single items on other health functions related to teeth , mouth opening , dry mouth , sticky saliva , cough , feeling ill , painkillers , nutrition supplements , feeding tube , weight loss , and weight gain . retest reliability and internal consistency.10 the eortc qlq - h&n35 has been widely used for studying hrql in hnc patients in taiwan and european countries.11,12 psychiatric morbidity were being assessed and made using the structured clinical interview for the diagnostic and statistical manual of mental disorders ( dsm)-iv ( scid ) , clinician version , a structured diagnostic interview based on dsm - iv criteria.8 the scid is a clinician - administered , semistructured interview for use with psychiatric patients or with community subjects who are undergoing evaluation for possible psychiatric diseases . the european organisation for research and treatment of cancer ( eortc ) quality of life questionnaire ( qlq ) was used to assess ql for cancer patients . various modules have been developed for disease - specific treatment measurements , and in this study we used the module that is specific to the hnc ( the qlq head and neck [ h&n]-35).9 this consists of 35 questions on a 4-point scale ( 1= not at all , 4= very much ) , with seven multiple items on pain , swallowing , senses ( taste and smell ) , speech , social eating , social contact , and sexuality , and eleven single items on other health functions related to teeth , mouth opening , dry mouth , sticky saliva , cough , feeling ill , painkillers , nutrition supplements , feeding tube , weight loss , and weight gain . retest reliability and internal consistency.10 the eortc qlq - h&n35 has been widely used for studying hrql in hnc patients in taiwan and european countries.11,12 the demographic characteristics , psychiatric diagnoses , history of substance use ( alcohol , smoking , betel nut ) , clinical staging of cancer , and treatment methods and hrql were first summarized using descriptive statistics . they were then compared between patients with and without depression using or t - tests . to investigate the effect of depression at each time point on hrql , a generalized mixed - effect model for repeated measurements this has the advantages over traditional methods , such as last observation carried forward , of including all available data across the follow - up period and providing better estimates under a broad assumption of missing data . to avoid false positives , of the 106 patients who were recruited in the study , 93 successfully completed the study . approximately two - thirds of the patients were diagnosed as having advanced clinical stages of cancer ( iii and iv ) , and over half received surgical and/or chemo- and radiotherapy . because of the small number of patients , we grouped the diagnoses of adjustment disorder with anxious mood and anxiety disorder not otherwise specified into anxiety disorders ( anxiety ) , and all depressive - spectrum disorders into depressive disorders ( depression ) . the prevalence of psychiatric disorders after the regrouping was as follows : anxiety disorders ( 27.3% ) , alcohol - use disorder ( 18.9% ) , depressive disorders ( 8.5% ) , and primary insomnia ( 2.8% ) . during the 6-month period , there were some changes in the distribution of psychiatric morbidities , with the most significant being the consistently and sharply decreasing rate of anxiety disorders ( from 27.3% to 6.4% and 3.3% at pretreatment and 3 and 6 months , respectively ) . depression showed a skew pattern , with a rise in the rate after pretreatment ( 8.5% ) to three times that at the peak at 3 months ( 24.5% ) , but gradually receded nearly to the level of initial assessment at 6 months ( figure 1 ) . psychiatric morbidity with any psychiatric diagnosis declined steadily during the 6-month period ( with rates from 54.7% at pretreatment to 45.8% at 3 months and 38.7% at 6 months ) . the 6-month follow - up of hrql , with results from the assessment on items based on the eortc qlq - h&n35 at pretreatment , 3 months , and 6 months is shown in table 2 . similar to the pattern of the rate of depression , patients felt worse and had more pain and higher frequency of taking painkillers in the first 3 months . levels returned to approximately the level of pretreatment at 6 months , together with dysfunction of mouth opening , swallowing , sticky saliva , sense and speech problems , trouble with social eating and social contact , and weight loss . when comparisons were made between the depressed and nondepressed hnc patients , there were no significant differences in their sociodemographics ( sex , age , marital status , employment , education , and substance use ) or clinical characteristics ( cancer staging , time to initial treatment , and mode of treatment ) ( table 3 ) . despite significant changes in hrql on almost all items of the eortc qlq - h&n35 during the 6-month period , there were some differences between the two groups across time when they were analyzed further using the generalized mixed - effect model . the results showed that over time , loss of functions to sense ( p<0.001 ) , speech ( p<0.001 ) , sexuality ( p<0.001 ) , and dry mouth ( p<0.001 ) were only significantly related to depression , and those with depression had a higher tendency to take painkillers ( p<0.001 ) and nutrition supplements ( p<0.001 ) than nondepressed patients . however , depression was predicted by sticky saliva and problems with social contact at 3 months and social eating at 6 months ( table 4 ) . of the 106 patients who were recruited in the study , 93 successfully completed the study . table 1 shows the sociodemographic and clinical characteristics of the patients , with the majority being males ( 86% ) and an average age of 52.79.6 years . approximately two - thirds of the patients were diagnosed as having advanced clinical stages of cancer ( iii and iv ) , and over half received surgical and/or chemo- and radiotherapy . because of the small number of patients , we grouped the diagnoses of adjustment disorder with anxious mood and anxiety disorder not otherwise specified into anxiety disorders ( anxiety ) , and all depressive - spectrum disorders into depressive disorders ( depression ) . the prevalence of psychiatric disorders after the regrouping was as follows : anxiety disorders ( 27.3% ) , alcohol - use disorder ( 18.9% ) , depressive disorders ( 8.5% ) , and primary insomnia ( 2.8% ) . during the 6-month period , there were some changes in the distribution of psychiatric morbidities , with the most significant being the consistently and sharply decreasing rate of anxiety disorders ( from 27.3% to 6.4% and 3.3% at pretreatment and 3 and 6 months , respectively ) . depression showed a skew pattern , with a rise in the rate after pretreatment ( 8.5% ) to three times that at the peak at 3 months ( 24.5% ) , but gradually receded nearly to the level of initial assessment at 6 months ( figure 1 ) . psychiatric morbidity with any psychiatric diagnosis declined steadily during the 6-month period ( with rates from 54.7% at pretreatment to 45.8% at 3 months and 38.7% at 6 months ) . the 6-month follow - up of hrql , with results from the assessment on items based on the eortc qlq - h&n35 at pretreatment , 3 months , and 6 months is shown in table 2 . similar to the pattern of the rate of depression , patients felt worse and had more pain and higher frequency of taking painkillers in the first 3 months . levels returned to approximately the level of pretreatment at 6 months , together with dysfunction of mouth opening , swallowing , sticky saliva , sense and speech problems , trouble with social eating and social contact , and weight loss . when comparisons were made between the depressed and nondepressed hnc patients , there were no significant differences in their sociodemographics ( sex , age , marital status , employment , education , and substance use ) or clinical characteristics ( cancer staging , time to initial treatment , and mode of treatment ) ( table 3 ) . despite significant changes in hrql on almost all items of the eortc qlq - h&n35 during the 6-month period , there were some differences between the two groups across time when they were analyzed further using the generalized mixed - effect model . the results showed that over time , loss of functions to sense ( p<0.001 ) , speech ( p<0.001 ) , sexuality ( p<0.001 ) , and dry mouth ( p<0.001 ) were only significantly related to depression , and those with depression had a higher tendency to take painkillers ( p<0.001 ) and nutrition supplements ( p<0.001 ) than nondepressed patients . however , depression was predicted by sticky saliva and problems with social contact at 3 months and social eating at 6 months ( table 4 ) . this prospective study showed that patients with hnc suffered from variable degrees of psychiatric morbidities during the first 6 months of treatment course . more than a quarter had an anxiety disorder ( 27.3% ) at pretreatment , but the rate steadily declined to 3.3% during the 6-month period . depressive disorder , however , did not follow such a pattern , but changed over time and manifested a skew pattern , with a rise in rate during the first 3 months ( from 8.1% to 24.5% ) and then a gradually return to pretreatment level in later months ( 14% ) . at this phase , a high rate of anxiety was demonstrated , similar to previous research.13 however , once the patients were more adapted to the disease and with the progress of treatment , the rate of anxiety significantly declined.14 early intervention by the team , which included psychiatrists and psychologists , also helped reduce tension and anxiety of the patients . to further assess various symptoms and function loss that were related to the cancer , we employed a questionnaire on hrql with a module that is specific for hnc : the eortc qlq - h&n35 . some of these symptoms might have correlated with depression,12,15,16 and this was able to be identified by applying analysis using the generalized mixed - effect model for repeated measurements across time . the results showed that the hnc patients with depression had more significant impairment than nondepressed patients in problems with sense and speech , dry mouth , sexuality , and loss of weight . the use of painkillers , especially those with narcotic analgesics , has been reported to have a higher risk of depression.17,18 the rates of depression in this sample of hnc patients ranging from 8% to 25% are compatible with earlier studies of longer duration.1923 we found that there were no significant differences in sociodemographics ( sex , age , marital status , employment , education , and substance use ) or clinical characteristics ( cancer staging , time to initial treatment , and mode of treatment ) between patients with and without depressive disorders , which might indicate that those factors were not the major cause of depression of hnc patients during the treatment course . in the first 3 months of treatment , depression was significantly predicated in patients with dysfunction in salivation with sticky saliva and trouble with social contact . dry mouth with sticky saliva is regarded as an adverse sign in chinese medical philosophy that reflects an imbalance of the internal organ systems.24 this might have created pessimism in these patients if they had dysfunction in salivation and limitation in the ingestion and chewing of food . this may partly explain the increasing rate of depressive disorders in the first few months of treatment , together with the fear over the uncertainty of their disease course or even death . with treatment progress , trouble with social eating was still the main factor related to depression at the 6-month interval . this illustrated the importance of food , eating , and nutrition for these patients , despite impairment of certain physical functions that were related to hnc . although alcohol has been regarded as one of the risk factors for hnc,22 the rate of alcohol abuse in this study was considered much lower than most reported studies , of 30%90%.25 taiwanese generally have a lower rate of alcoholism than caucasians , due to a lack of related genes for enzymes that are responsible for metabolizing alcohol and acetaldehyde,26 and this could well have accounted for the relatively low prevalence rate of alcoholism in study . although it is not related to any mood disorders , chewing betel nut is however carcinogenic to humans , and has been reported to have association with oral / buccal cancer.27 betel nut contains a substance called arecoline , which has proved to contribute to histologic changes in the oral mucosa . this was not related to mood disorders , but might have harmful effects on health and thus to the disease prognosis.27,28 it is believed that one in four terminally ill patients have symptoms of depression , but that up to 80% of this depression may not be recognized and is thus untreated.29 among the symptoms , loss of sexuality , appetite , body weight , and sleep are also core vegetative symptoms of depression . the awareness of depression , the identification of symptoms , and the initiation of treatment are essential if patients are to be offered optimum palliation of psychological as well as physical symptoms.29 in summary , this study of a 6-month experience of patients with hnc showed that psychiatric morbidities were commonly seen in the early phase of treatment . high rates of anxiety were found before treatment , but had a steadily decline following the treatment course . depression , on the other hand , demonstrated a skew pattern , with a peak at 3 months declining to pretreatment level at later months . dysfunction in salivation and problems with social contacts and eating were the three major risk factors for depression at 3 and 6 months , as impairments of sense , speech , dry mouth , and sexuality were more significant in the depressed patients who also consumed more painkillers and nutritional supplements .

chondrocytes are virtually the only cellular component of the cartilage and have a role in maintaining the equilibrium between anabolic production of collagens type ii and aggrecans which make up the extracellular matrix and catabolic enzymes , including aggrecanases and matrix metalloproteinases ( mmps ) , important for cartilage turnover . in osteoarthritis ( oa ) , a relative increase in the production of these enzymes can result in aberrant cartilage destruction . autologous chondrocyte implantation ( aci ) of ex vivo expanded chondrocytes retaining ability to repopulate focal lesions of articular cartilage is an important therapeutic aim in orthopaedics . when cultured in supporting 3d scaffold substitute structures , chondrocytes maintain the production of collagen type ii , aggrecans , and mmp13 , whereas in monolayer cultures they display a progressive loss of these characteristic proteins and eventually gain a fibroblast - like phenotype and switch to collagen type i expression instead of type ii , that reflects a dedifferentiated status . however , if chondrocytes from monolayer cultures are shifted to 3d scaffolds , they can reexpress chondrocyte - specific proteins and regain some degree of differentiation [ 3 , 4 ] . in an effort to gain a better understanding of the molecular mechanisms that govern the phenotypes observed in oa and in the different culture conditions described above , that are still only partially known , we have investigated the expression of a group of zinc finger proteins potentially implicated in the physiological regulation of the homeostasis of articular chondrocytes , namely , znf423 , znf470 , znf521 , and znf780b . znf521/ehzf is a large multifunctional protein with 30 zinc fingers , identified in our laboratory for its selective abundance in immature hematopoietic progenitors compared to mature leukocytes [ 5 , 6 ] . znf521 shows features of a transcriptional corepressor and has been found to modulate the transcriptional induction of erythroid and b - lymphoid differentiation by gata1 and ebf1 [ 7 , 8 ] . in addition to the hematopoietic system , znf521 has recently been demonstrated to drive the generation of neuroectodermal precursors from embryonic stem cells and to contribute to the growth , clonogenicity , and tumorigenicity of medulloblastoma cells . importantly , the murine orthologue of znf521 , termed zfp521 , has been identified as a central cell fate regulator in mesenchymal stem cells where , in a complex interplay with ebf1 and zfp423 , it represses their adipogenic potential and promotes osteoblastic commitment [ 11 , 12 ] . in osteoblasts , zfp521 has been shown to be associated with runx2 and antagonizes its transcriptional activity , thereby delaying their early differentiation steps and promoting later stages of osteoblast maturation . relevantly , zfp521 has also been shown to be an important downstream effector of pthrp in growth plate chondrocytes in which it regulates proliferation and differentiation . zfp521 knock - out mice generated using the cre recombinase driven by the collagen ii promoter showed decreased growth plate chondrocyte proliferation , early hypertrophic transition , and reduced tibial growth plate thickness with less extracellular matrix . znf423/oaz is the paralogue of znf521 , with which it shares a high degree of homology ( > 66% ) , the structural architecture ( including the presence of 30 zinc finger motifs ) , and some overlapping , but not necessarily identical , functions . this factor was originally identified as an inhibitor olf1/ebf1 in the olfactory epithelium [ 1618 ] . it was subsequently found to be an important mediator in the signaling pathway of the bone morphogenetic proteins ( bmp ) 2 and 4 , as well as of retinoic acid and of notch . znf423 is required for cerebellar development [ 2224 ] and implicated in the development of b - lymphoid leukemias [ 25 , 26 ] . importantly , zfp423 has recently risen to prominence as a master factor of the adipogenic commitment of mesenchymal stem cells through the activation of ppar . this effect is abrogated by wisp2 that binds to zfp423 and sequesters it in the cytosol and by zfp52 which inhibits the ebf1-mediated induction of zfp423 expression in mesenchymal stem cells . two additional zinc finger proteins have been included in this study that are less well characterized but have both been linked to cartilage physiology . znf780b is an as yet uncharacterized zinc finger protein sharing 74.2% identity with mouse zfp60 , a zinc finger transcription factor with a krab domain and 19 zinc finger motifs . zfp60 was originally identified in muscle differentiation ; its expression in cultured prehypertrophic chondrocytes coincides with the expression of regulators of chondrocyte maturation such as indian hedgehog ( ihh ) and the pthrp receptor , and its overexpression inhibits cartilage differentiation in the atdc5 chondrogenic cell line . znf470 is a protein with 17 zinc fingers as well as krab - a and krab - b motifs , whose mrna was originally isolated from mesenchymal progenitors induced to differentiate towards chondrocytes in vitro . the znf470 transcript was found transiently expressed during chondrogenesis , with a peak of maximal abundance that coincided with the beginning of the increase in col2a1 expression , and was strongly upregulated during the dedifferentiation of bovine articular chondrocytes in response to retinoic acid treatment . based on the evidence summarized above , these four zinc finger proteins can be considered likely candidate regulators of the homeostasis of differentiated articular chondrocytes that is compromised in oa . therefore , we sought to determine whether the levels of their expression were altered in oa chondrocytes or in culture conditions that promote the emergence of a dedifferentiated phenotype that is reminiscent to some extent to that of osteoarthritic chondrocytes . the cell human chondrocyte cell lines t / c-28a4 and the c-28 derived from t / c-28a4 [ 33 , 34 ] were cultured in adhesion in dmem high glucose supplemented with 10% fetal bovine serum ( fbs ) . the human hematopoietic cell lines , thp1 ( acute monocytic leukemia ) , k562 ( erythroleukemia ) , and im9 ( b cell lymphoma ) , were cultured in suspension in rpm1 , 10% fbs ; the medulloblastoma cell line daoy was cultured in dmem , 10% fbs . all media were supplemented with 100 u / ml penicillin and 50 g / ml streptomycin ; the cells were maintained in 5% co2 at 37c . the study protocol was approved by the local ethics committee and the research was carried out in compliance with the declaration of helsinki . cartilage samples were obtained from a total of 25 osteoarthritic patients with an age range of 4578 years having bmi values from 24.69 to 41.02 . all patients were subjected to total joint replacement and samples of articular cartilage were obtained as discarded surgical material . specifically , cartilage was taken from the femoral head in patients with hip oa and from femoral condyles and the tibial plateau in patients with knee oa . diagnosis of oa was based on established clinical and radiological criteria [ 36 , 37 ] . a radiological classification of each osteoarthritic joint was carried out according to the ahlbck scale . in addition , cartilage was harvested from 4 patients ( 3 underwent hallux valgus and 1 pes cavus deformity correction , resp . ) ; these samples served as a control group ( table 1 ) . after the cartilage was removed from the joint , the tissue was extensively washed with isotonic saline solution to remove residual blood or synovial fluid . specimens were excised from the superficial and deep layers of the articular cartilage avoiding the calcified layer and the subchondral bone . cartilage fragments were cut finely , washed in phosphate buffered saline ( pbs ) , and digested with 0.2% collagenase type 2 ( worthington ) in dmem high glucose for 16 h at 37c . debris was removed by filtration through 40 m cell strainers ( bd biosciences ) . the cell number and viability were assessed by 0.2% trypan blue and cells ( 3,000 cell / cm ) were allowed to adhere to collagen coated ( dil 1 : 45 of 3 mg / ml sigma ) tissue culture dishes in dmem high glucose , 10% fbs . cells were passaged with 0.25% trypsin - edta at weekly intervals until 80% confluency on tissue culture dishes in dmem 10% fbs for up to 5 passages . total rna was prepared with trizol ( life technologies ) treated with dnase i ( rnase free , promega ) 1 u/1 g rna . the purity and amount of rna were determined by spectrophotometric measurement ( od 260/280 ratio ) . cdna was synthesized from 1 g rna using superscript iii reverse transcriptase at 42c and 2.5 m random hexamers ( life technologies ) . q - pcr reactions were carried out with the iq - sybr green supermix ( bio - rad ) in duplicate according to the manufacturer 's instructions and analyzed using the iq5 multicolor detection system ( bio - rad ) . one cycle of 3 min at 95c for activation was followed by 45 cycles of 10 seconds at 95c , 10 seconds at 60c , and 20 seconds at 72c and then a melting curve . the specificity of amplification was confirmed by the dissociation curves of the amplicons and size of the amplified product analyzed on a 2% agarose gels . relative gene expression was determined using the comparative threshold cycles ct method , normalizing for endogenous gapdh , and expression ratio was calculated as 2 . normalized expression values are reported in relative units ru and are used for comparative analysis among the samples . primers for chondrocyte markers , col1 , col2a1 , and aggrecan were as published as well as those for mmp13 . primers for the other zinc finger proteins were designed using the primer design program from ncbi with mpt 6062c spanning exon - intron junctions . primers were for znf423 ( fwd - ggaaaggcacccagacatcg , rev - cggggagtcgaacatctggt ) , for znf780b ( fwd - ctagctgggggagaagcccga , rev - atcaggctgcaggcactccca ) , for znf470 ( fwd - tgtgactgtccggtgcgtgg , rev - tgtgactgtccggtgcgtgg ) , and for ppar ( fwd - ctgtcggtttcagaagtgcct , rev - cccaaacctgatggcattgtgagaca ) . chondrocytes grown in suspension in nonadherent conditions as spheres in petri dishes for 2448 h 1 10/ml cells were encapsulated in 1 : 1 volume alginate solution ( 1.25% alginate acid ( sigma - aldrich ) 20 mm hepes 150 mm nacl ph 7.4 ) and polymerized by dropping , using a 21-gauge needle , into 102 mm cacl2 , 10 mm hepes , ph 7.4 . beads were washed on 40 m strainers with 0.9% saline solution and cultured in dmem 10% fbs in petri dishes with weekly medium changes for 3 weeks . cells were recovered by treating beads with 55 mm edta in 10 mm hepes ph7.4 and then rna was prepared by trizol reagent . lentiviral transduction was performed with chondrocytes encapsulated in alginate beads using lentiviral shrna vectors expressing egfp as a reporter marker , as described [ 8 , 10 , 41 ] . viruses were prepared by cotransfecting 293 t cells with plasmids coding for the viral components pcmv - vsvg and pcmv - deltar8 - 91 . after 48 h , viral supernatants were filtered ( 0.45 m ) and added together with 4 g / ml polybrene to the chondrocyte cultures . the effectiveness of the transduction was monitored by visualization of the chondrocyte spheres for green fluorescent protein expression ( floid microscope life technologies ) . immunofluorescence was performed on cells washed in pbs , fixed in 50% methanol and 50% acetone , dried , and washed in pbs followed by blocking in 10% fbs in pbs . primary antibodies for col2a1 sc2887 and znf521 ( ehzf ) sc84808 were from santa cruz bd biosciences and were used at 1 : 200 for 16 hours and detected with anti - rabbit alexa fluor 498 ( life technologies ) at 1 : 200 binding for 2 hours . student t - test was used to assess the significance of the differences between values . the analysis of variance was used to evaluate the comparisons between groups using two - tailed analyses . chondrocytes from cartilage samples from either hip or knee joints of patients affected by oa ( stages ii iv ) undergoing replacement surgery were compared to a control group of samples from hallux valgus and pes cavus . the strategy used was to culture and amplify the chondrocytes for one passage to obtain sufficient rna for a reliable q - rt - pcr analysis . this strategy yielded results more reliably quantifiable than those obtained using rna prepared directly from cartilage tissue , where the amount extracted was low and problems of degradation were more frequent . the relative amounts of mrna present are compared to the chondrocyte - derived cell lines t / c-28a4 and the c-28 [ 33 , 34 ] which are known to express lower levels of chondrocyte - specific genes . the transcripts encoding matrix proteins col2a1 and aggrecan and the catalytic matrix - degrading protease mmp13 were expressed at considerably higher levels in the primary oa chondrocytes than in either chondrocyte cell line . in osteoarthritic cartilage quiescent chondrocytes become activated and proliferate , forming clusters and producing more of both matrix and matrix - degrading enzymes [ 1 , 42 ] . this was confirmed by the expression of these genes in our group of specimens of oa chondrocytes ( stages ii iv ) that displayed higher expression levels of col2a1 and mmp13 than control cells ( figure 1 ) . sox9 , a transcription factor known to be required for chondrogenesis , was expressed at a similar level in the cell lines and in primary chondrocytes , indicating that it has not been lost when the cells were immortalized . among the zinc finger proteins studied , znf521 was over 100-fold more expressed in primary chondrocytes , with no significant variation between control and oa cells , compared to the t / c-28a4 cell line ( figure 1 ) . such a high level of expression suggests that znf521 may be functionally relevant in primary chondrocytes . znf423 mrna was also abundant in primary cells , but a significant decrease in the amounts of its transcript was observed in oa - derived chondrocytes compared to control cells ( figure 1 ; p = 0.011 ) . in addition , the levels of znf423 mrna showed a significant age - related variation , with a lower expression in chondrocytes derived from patients over 60 than under 60 years old ( figure 2(a ) ; p = 0.0215 ) . this trend was not evident for the other zinc finger proteins transcripts analyzed . znf470 and znf780b , like sox9 , were expressed at comparable levels in both oa and control primary chondrocytes as well as in the chondrocyte cell lines ( figure 1 ) and their expression did not change with age ( figure 2(a ) ) . the nuclear receptor , peroxisome proliferator - activated receptor gamma ( ppar ) , has been shown to exert an anti - inflammatory and chondroprotective action , and the modulation of its expression by interleukin 1 , or the cartilage - specific disruption of its gene in mice , has been linked to the development of oa [ 43 , 44 ] . since ppar is a major target of znf423 in adipocyte precursors , we compared its expression in nine oa samples that showed low levels of znf423 mrna to that of control samples and found a strong decrease in ppar expression in oa chondrocytes , comparable to that of znf423 ( figure 2(b ) ; p = 0.00187 ) . primary chondrocytes , once extracted from the cartilage matrix , can be cultured in adhesion on tissue culture plates . initially two populations are visible : some cells are rounded and partly attached to the tissue culture dish and fewer round cells are present and the majority extend in a flattened fibroblastic fashion onto the dish . over a period of 4 - 5 weeks with weekly passages rna was isolated at each passage during the cultures derived from 10 different oa chondrocyte samples and analyzed by q - pcr for chondrocyte - specific and the zinc finger protein transcripts . this analysis showed that the mrnas for the chondrocyte matrix protein col2a1 and the mmp13 protease decreased significantly during the cell culture passages , whereas aggrecan remained expressed at high levels throughout the culture period . collagen type i , known to be associated with a more undifferentiated phenotype , notably increased as expected . we observed that the zinc finger proteins znf521 and znf423 , which are expressed at very high levels compared to the chondrocytic cell line t / c28a4 , continue to be expressed throughout the dedifferentiation culture period , whereas znf470 and znf780b retained expression levels comparable to those of t / c28a4 cells ( figure 3(a ) ) . immunofluorescence staining for col2a1 showed at early passages a high number of intensely staining cells in the rounded population , which was largely lost at later passages with most cells displaying only a considerably weaker staining . znf521 was expressed in the nucleus of both types of cells throughout the cultures ( figure 3(b ) ) confirming the results from the q - pcr analysis . the expression of znf521 in primary oa chondrocytes ( figure 4(a ) ) was considerably high and comparable to that observed in the hematopoietic cell lines thp1 , k562 and in the medulloblastoma cell line daoy , which have been shown to depend on a functionally active znf521 [ 7 , 10 , 46 ] . therefore , an rnai - based silencing approach was undertaken to assess the role of this protein in cultured primary chondrocytes . to this end , we infected primary chondrocytes with lentiviral vectors constructed to enable the integration in the target cells ' genome of shrnas specific for znf521 together with egfp as a transgene , resulting in egfp - identifiable cells that produce reduced amounts of znf521 mrna ( figure 4(c ) ) and protein . experiments were performed using chondrocytes encapsulated in alginate beads , a matrix in which chondrocytes grow in sphere - like bodies of associated cells , where egfp - expressing cells could be visualised ( figure 4(b ) ) . after 3 weeks of culture rna was extracted and the expression of collagens was compared to that of cells grown in adhesion with weekly passages . the control chondrocytes in alginate beads had retained the differentiated phenotype compared to their counterparts growing in adherence , as shown by the significantly higher col2a1 and lower col1 expression . instead , the cells transduced with either shrna specific for znf521 displayed a more dedifferentiated phenotype , with a decrease in the col2a1 mrna and an increase in col1 transcript , compared to control cells ( figures 4(d ) and 4(e ) ) . in oa the healthy balance of the cartilage homeostasis , normally ensured by articular chondrocytes present within the extra - cellular matrix , is disrupted . chondrocytes embedded in the cartilage matrix normally produce the proteoglycans that compose the matrix , whose damage renders the cartilage tissue vulnerable to protease digestion . the changes in cellular activity such as those observed in the development of oa may be the consequence of discrete changes in the expression of sets of transcription factors that in turn control the expression of genes whose products are responsible for the maintenance of the physiological tissue homeostasis . proteins belonging to the sox and runx families are known to be central players in regulating transcription in chondrogenesis and differentiated chondrocytes present in the articulation cartilage . sox9 is necessary for chondrogenic differentiation before and after mesenchymal condensations , whereas sox5 and sox6 are needed only after mesenchymal condensation . haploinsufficiency of sox9 in humans leads to the skeletal dysmorphology syndrome campomelic dysplasia underlining the crucial role described for sox9 . runx2 is known not only to be important in osteogenesis but also in articular cartilage and oa , where it induces the expression of mmp13 in a process that can be counteracted by tgfbeta [ 4951 ] , and its expression increases with the progression of oa resulting in a more pronounced matrix degradation by mmp13 . other factors including shox / shox2 , dlx5 , and mef2c have been also implicated particularly in the hypertrophy of chondrocytes and may have a role in pathogenesis of oa . it is predicted that additional transcription factors may participate in the control of chondrocyte homeostasis and that an altered balance of their expression / activity is likely to contribute to its disruption that results in the onset of oa . we have focused our attention on a group of zinc finger proteins that , based on their established role in orchestrating the lineage fate choice of mesenchymal stem cells ( znf423 and znf521 ) or on their direct or indirect links with cartilage physiology ( znf470 and of znf780b ) , might play a regulatory role in normal articular chondrocytes and in oa . the expression of these zinc finger proteins was investigated in chondrocytes from an extensive cohort of patients affected by oa at different stages , in comparison with chondrocytes derived from patients with disorders distinct from oa proper , considered as controls . the analyses were conducted both on freshly isolated cells ( after one round of culture , necessary to achieve acceptable rna purity ) and on cells cultured for different periods of time in different conditions . while the expression profiles of znf470 and of znf780b did not display significant variations in any of the conditions analyzed , znf423 mrna was significantly lower in those from oa compared to controls . this was paralleled by a similar strong decrease in the expression of the znf423 target , ppar , a nuclear receptor whose inhibition has been associated with the development of oa [ 43 , 44 ] . whether this decrease is a direct consequence of the low expression of znf423 and whether enforced expression of znf423 in oa chondrocytes may restore normal levels of ppar and attenuate the oa phenotype remain to be determined in future studies . it will also be of interest to conduct an in - depth analysis of the expression and activity of other factors , such as wisp2 and ebf1 , which have been shown to functionally interact with znf423 in the control of ppar expression [ 11 , 27 , 28 ] . in addition to oa , we found that znf423 was also significantly reduced in elderly patients ( over 60 ) compared to younger ones . cartilage chondrocytes are known to be extremely long lived and undergo changes with age , which result in modifications in their anabolic and catabolic processes and include a reduced responsiveness to growth factors , such as tgfbeta , bmps , and wnt . since znf423 has been described to be a prominent effector of bmp2 and bmp4 [ 19 , 55 ] , a lower expression could be part of this mechanism . it must be kept in mind that all four controls , whose chondrocytes show higher levels of znf423 than the oa patients , are in the younger group ; however , even if these subjects were excluded , the znf423 expression of the younger patients remained significantly higher than that of the elderly cohort . taken together , the results illustrated in this paper suggest that znf423 underexpression may represent a useful oa - associated biomarker and possibly be a factor in oa pathogenesis . in this regard , it is interesting to notice that a recent in silico analysis of differentially coexpressed genes identified znf423 among the ten top - ranked transcription factors functionally relevant to oa . the expression of znf521 did not display significant changes among the different patients ' groups classified by age or oa stage ; however , we were intrigued by the high levels of expression in primary chondrocytes compared to the immortalized cell lines . this prompted us to test whether silencing of znf521 expression would modify the phenotype of primary cells . as illustrated in figure 3 , znf521 knockdown with two distinct shrnas in chondrocytes cultured in alginate beads resulted in a phenotype with marked characteristics of dedifferentiation . this suggests that the presence of high levels of znf521 expression may be a requisite for the maintenance of the identity in primary chondrocytes . mechanistically , transcription is controlled by the concerted action of transcription factors and epigenetic regulators , including high - mobility group proteins like hmgb-1 and hmga1 [ 35 , 58 ] that are both increased in later stages of oa reflecting multiple changes at the level of transcription and an involvement in pathogenesis of oa , as well as histone - modifying enzymes such as histone acetylases ( hats ) and deacetylases ( hdacs ) . several features of differentiated chondrocytes are known to be affected by the activity of these effectors ; for instance , collagen type ii expression is controlled by hdac activity in articular chondrocytes as well as by the class iii hdac , sirt1 , a nad - dependent hdac , which regulates cartilage gene expression . both znf521 and znf423 have n - terminal motifs that interact with the hdac - containing nurd complex [ 5 , 6 , 61 ] , and the integrity of this motif has been shown to be essential for the promotion of growth and tumorigenicity of medulloblastoma cells ; in addition , zfp521 is also known to interact with hdac3 and hdac4 that are instrumental in the regulatory functions of zfp521 in growth plate chondrocytes and osteoblasts [ 14 , 15 ] . it will therefore be of interest to investigate whether the association of znf521 with chromatin - remodelling factors may also play a role in articular chondrocytes and in determining the oa phenotype . finally , the functional interactions between znf521 and znf423 in articular chondrocytes warrant further investigation to determine whether the two factors exert antagonistic functions in these cells as documented in mesenchymal stem cells or if their coexpression is required for the maintenance of the differentiated phenotype . a better understanding of the role of these factors in chondrocyte physiology may provide further insight into the molecular mechanisms that control their homeostasis and their potential contribution to the pathophysiology of oa .

articular chondrocytes are responsible for the maintenance of healthy articulations ; indeed , dysregulation of their functions , including the production of matrix proteins and matrix - remodeling proteases , may result in fraying of the tissue and development of osteoarthritis ( oa ) . to explore transcriptional mechanisms that contribute to the regulation of chondrocyte homeostasis and may be implicated in oa development , we compared the gene expression profile of a set of zinc finger proteins potentially linked to the control of chondrocyte differentiation and/or functions ( znf423 , znf470 , znf521 , and znf780b ) in chondrocytes from patients affected by oa and from subjects not affected by oa . this analysis highlighted a significantly lower expression of the transcript encoding znf423 in chondrocytes from oa , particularly in elderly patients . interestingly , this decrease was mirrored by the similarly reduced expression of ppar , a known target of znf423 with anti - inflammatory and chondroprotective properties . the znf521 mrna instead was abundant in all primary chondrocytes studied ; the rnai - mediated silencing of this gene significantly altered the col2a / col1 expression ratio , associated with the maintenance of the differentiated phenotype , in chondrocytes cultivated in alginate beads . these results suggest a role for znf423 and znf521 in the regulation of chondrocyte homeostasis and warrant further investigations to elucidate their mechanism of action .

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

chondrocytes are virtually the only cellular component of the cartilage and have a role in maintaining the equilibrium between anabolic production of collagens type ii and aggrecans which make up the extracellular matrix and catabolic enzymes , including aggrecanases and matrix metalloproteinases ( mmps ) , important for cartilage turnover . in osteoarthritis ( oa ) , a relative increase in the production of these enzymes can result in aberrant cartilage destruction . when cultured in supporting 3d scaffold substitute structures , chondrocytes maintain the production of collagen type ii , aggrecans , and mmp13 , whereas in monolayer cultures they display a progressive loss of these characteristic proteins and eventually gain a fibroblast - like phenotype and switch to collagen type i expression instead of type ii , that reflects a dedifferentiated status . however , if chondrocytes from monolayer cultures are shifted to 3d scaffolds , they can reexpress chondrocyte - specific proteins and regain some degree of differentiation [ 3 , 4 ] . in an effort to gain a better understanding of the molecular mechanisms that govern the phenotypes observed in oa and in the different culture conditions described above , that are still only partially known , we have investigated the expression of a group of zinc finger proteins potentially implicated in the physiological regulation of the homeostasis of articular chondrocytes , namely , znf423 , znf470 , znf521 , and znf780b . in addition to the hematopoietic system , znf521 has recently been demonstrated to drive the generation of neuroectodermal precursors from embryonic stem cells and to contribute to the growth , clonogenicity , and tumorigenicity of medulloblastoma cells . importantly , the murine orthologue of znf521 , termed zfp521 , has been identified as a central cell fate regulator in mesenchymal stem cells where , in a complex interplay with ebf1 and zfp423 , it represses their adipogenic potential and promotes osteoblastic commitment [ 11 , 12 ] . zfp521 knock - out mice generated using the cre recombinase driven by the collagen ii promoter showed decreased growth plate chondrocyte proliferation , early hypertrophic transition , and reduced tibial growth plate thickness with less extracellular matrix . znf423/oaz is the paralogue of znf521 , with which it shares a high degree of homology ( > 66% ) , the structural architecture ( including the presence of 30 zinc finger motifs ) , and some overlapping , but not necessarily identical , functions . it was subsequently found to be an important mediator in the signaling pathway of the bone morphogenetic proteins ( bmp ) 2 and 4 , as well as of retinoic acid and of notch . znf423 is required for cerebellar development [ 2224 ] and implicated in the development of b - lymphoid leukemias [ 25 , 26 ] . importantly , zfp423 has recently risen to prominence as a master factor of the adipogenic commitment of mesenchymal stem cells through the activation of ppar . two additional zinc finger proteins have been included in this study that are less well characterized but have both been linked to cartilage physiology . znf780b is an as yet uncharacterized zinc finger protein sharing 74.2% identity with mouse zfp60 , a zinc finger transcription factor with a krab domain and 19 zinc finger motifs . zfp60 was originally identified in muscle differentiation ; its expression in cultured prehypertrophic chondrocytes coincides with the expression of regulators of chondrocyte maturation such as indian hedgehog ( ihh ) and the pthrp receptor , and its overexpression inhibits cartilage differentiation in the atdc5 chondrogenic cell line . the znf470 transcript was found transiently expressed during chondrogenesis , with a peak of maximal abundance that coincided with the beginning of the increase in col2a1 expression , and was strongly upregulated during the dedifferentiation of bovine articular chondrocytes in response to retinoic acid treatment . based on the evidence summarized above , these four zinc finger proteins can be considered likely candidate regulators of the homeostasis of differentiated articular chondrocytes that is compromised in oa . therefore , we sought to determine whether the levels of their expression were altered in oa chondrocytes or in culture conditions that promote the emergence of a dedifferentiated phenotype that is reminiscent to some extent to that of osteoarthritic chondrocytes . the human hematopoietic cell lines , thp1 ( acute monocytic leukemia ) , k562 ( erythroleukemia ) , and im9 ( b cell lymphoma ) , were cultured in suspension in rpm1 , 10% fbs ; the medulloblastoma cell line daoy was cultured in dmem , 10% fbs . the study protocol was approved by the local ethics committee and the research was carried out in compliance with the declaration of helsinki . specifically , cartilage was taken from the femoral head in patients with hip oa and from femoral condyles and the tibial plateau in patients with knee oa . q - pcr reactions were carried out with the iq - sybr green supermix ( bio - rad ) in duplicate according to the manufacturer 's instructions and analyzed using the iq5 multicolor detection system ( bio - rad ) . the specificity of amplification was confirmed by the dissociation curves of the amplicons and size of the amplified product analyzed on a 2% agarose gels . relative gene expression was determined using the comparative threshold cycles ct method , normalizing for endogenous gapdh , and expression ratio was calculated as 2 . primers for the other zinc finger proteins were designed using the primer design program from ncbi with mpt 6062c spanning exon - intron junctions . primers were for znf423 ( fwd - ggaaaggcacccagacatcg , rev - cggggagtcgaacatctggt ) , for znf780b ( fwd - ctagctgggggagaagcccga , rev - atcaggctgcaggcactccca ) , for znf470 ( fwd - tgtgactgtccggtgcgtgg , rev - tgtgactgtccggtgcgtgg ) , and for ppar ( fwd - ctgtcggtttcagaagtgcct , rev - cccaaacctgatggcattgtgagaca ) . lentiviral transduction was performed with chondrocytes encapsulated in alginate beads using lentiviral shrna vectors expressing egfp as a reporter marker , as described [ 8 , 10 , 41 ] . primary antibodies for col2a1 sc2887 and znf521 ( ehzf ) sc84808 were from santa cruz bd biosciences and were used at 1 : 200 for 16 hours and detected with anti - rabbit alexa fluor 498 ( life technologies ) at 1 : 200 binding for 2 hours . chondrocytes from cartilage samples from either hip or knee joints of patients affected by oa ( stages ii iv ) undergoing replacement surgery were compared to a control group of samples from hallux valgus and pes cavus . the relative amounts of mrna present are compared to the chondrocyte - derived cell lines t / c-28a4 and the c-28 [ 33 , 34 ] which are known to express lower levels of chondrocyte - specific genes . the transcripts encoding matrix proteins col2a1 and aggrecan and the catalytic matrix - degrading protease mmp13 were expressed at considerably higher levels in the primary oa chondrocytes than in either chondrocyte cell line . in osteoarthritic cartilage quiescent chondrocytes become activated and proliferate , forming clusters and producing more of both matrix and matrix - degrading enzymes [ 1 , 42 ] . this was confirmed by the expression of these genes in our group of specimens of oa chondrocytes ( stages ii iv ) that displayed higher expression levels of col2a1 and mmp13 than control cells ( figure 1 ) . sox9 , a transcription factor known to be required for chondrogenesis , was expressed at a similar level in the cell lines and in primary chondrocytes , indicating that it has not been lost when the cells were immortalized . among the zinc finger proteins studied , znf521 was over 100-fold more expressed in primary chondrocytes , with no significant variation between control and oa cells , compared to the t / c-28a4 cell line ( figure 1 ) . such a high level of expression suggests that znf521 may be functionally relevant in primary chondrocytes . znf423 mrna was also abundant in primary cells , but a significant decrease in the amounts of its transcript was observed in oa - derived chondrocytes compared to control cells ( figure 1 ; p = 0.011 ) . in addition , the levels of znf423 mrna showed a significant age - related variation , with a lower expression in chondrocytes derived from patients over 60 than under 60 years old ( figure 2(a ) ; p = 0.0215 ) . this trend was not evident for the other zinc finger proteins transcripts analyzed . znf470 and znf780b , like sox9 , were expressed at comparable levels in both oa and control primary chondrocytes as well as in the chondrocyte cell lines ( figure 1 ) and their expression did not change with age ( figure 2(a ) ) . the nuclear receptor , peroxisome proliferator - activated receptor gamma ( ppar ) , has been shown to exert an anti - inflammatory and chondroprotective action , and the modulation of its expression by interleukin 1 , or the cartilage - specific disruption of its gene in mice , has been linked to the development of oa [ 43 , 44 ] . since ppar is a major target of znf423 in adipocyte precursors , we compared its expression in nine oa samples that showed low levels of znf423 mrna to that of control samples and found a strong decrease in ppar expression in oa chondrocytes , comparable to that of znf423 ( figure 2(b ) ; p = 0.00187 ) . initially two populations are visible : some cells are rounded and partly attached to the tissue culture dish and fewer round cells are present and the majority extend in a flattened fibroblastic fashion onto the dish . collagen type i , known to be associated with a more undifferentiated phenotype , notably increased as expected . we observed that the zinc finger proteins znf521 and znf423 , which are expressed at very high levels compared to the chondrocytic cell line t / c28a4 , continue to be expressed throughout the dedifferentiation culture period , whereas znf470 and znf780b retained expression levels comparable to those of t / c28a4 cells ( figure 3(a ) ) . the expression of znf521 in primary oa chondrocytes ( figure 4(a ) ) was considerably high and comparable to that observed in the hematopoietic cell lines thp1 , k562 and in the medulloblastoma cell line daoy , which have been shown to depend on a functionally active znf521 [ 7 , 10 , 46 ] . therefore , an rnai - based silencing approach was undertaken to assess the role of this protein in cultured primary chondrocytes . to this end , we infected primary chondrocytes with lentiviral vectors constructed to enable the integration in the target cells ' genome of shrnas specific for znf521 together with egfp as a transgene , resulting in egfp - identifiable cells that produce reduced amounts of znf521 mrna ( figure 4(c ) ) and protein . experiments were performed using chondrocytes encapsulated in alginate beads , a matrix in which chondrocytes grow in sphere - like bodies of associated cells , where egfp - expressing cells could be visualised ( figure 4(b ) ) . the control chondrocytes in alginate beads had retained the differentiated phenotype compared to their counterparts growing in adherence , as shown by the significantly higher col2a1 and lower col1 expression . instead , the cells transduced with either shrna specific for znf521 displayed a more dedifferentiated phenotype , with a decrease in the col2a1 mrna and an increase in col1 transcript , compared to control cells ( figures 4(d ) and 4(e ) ) . in oa the healthy balance of the cartilage homeostasis , normally ensured by articular chondrocytes present within the extra - cellular matrix , is disrupted . the changes in cellular activity such as those observed in the development of oa may be the consequence of discrete changes in the expression of sets of transcription factors that in turn control the expression of genes whose products are responsible for the maintenance of the physiological tissue homeostasis . proteins belonging to the sox and runx families are known to be central players in regulating transcription in chondrogenesis and differentiated chondrocytes present in the articulation cartilage . runx2 is known not only to be important in osteogenesis but also in articular cartilage and oa , where it induces the expression of mmp13 in a process that can be counteracted by tgfbeta [ 4951 ] , and its expression increases with the progression of oa resulting in a more pronounced matrix degradation by mmp13 . other factors including shox / shox2 , dlx5 , and mef2c have been also implicated particularly in the hypertrophy of chondrocytes and may have a role in pathogenesis of oa . it is predicted that additional transcription factors may participate in the control of chondrocyte homeostasis and that an altered balance of their expression / activity is likely to contribute to its disruption that results in the onset of oa . we have focused our attention on a group of zinc finger proteins that , based on their established role in orchestrating the lineage fate choice of mesenchymal stem cells ( znf423 and znf521 ) or on their direct or indirect links with cartilage physiology ( znf470 and of znf780b ) , might play a regulatory role in normal articular chondrocytes and in oa . the expression of these zinc finger proteins was investigated in chondrocytes from an extensive cohort of patients affected by oa at different stages , in comparison with chondrocytes derived from patients with disorders distinct from oa proper , considered as controls . while the expression profiles of znf470 and of znf780b did not display significant variations in any of the conditions analyzed , znf423 mrna was significantly lower in those from oa compared to controls . this was paralleled by a similar strong decrease in the expression of the znf423 target , ppar , a nuclear receptor whose inhibition has been associated with the development of oa [ 43 , 44 ] . whether this decrease is a direct consequence of the low expression of znf423 and whether enforced expression of znf423 in oa chondrocytes may restore normal levels of ppar and attenuate the oa phenotype remain to be determined in future studies . it will also be of interest to conduct an in - depth analysis of the expression and activity of other factors , such as wisp2 and ebf1 , which have been shown to functionally interact with znf423 in the control of ppar expression [ 11 , 27 , 28 ] . in addition to oa , we found that znf423 was also significantly reduced in elderly patients ( over 60 ) compared to younger ones . cartilage chondrocytes are known to be extremely long lived and undergo changes with age , which result in modifications in their anabolic and catabolic processes and include a reduced responsiveness to growth factors , such as tgfbeta , bmps , and wnt . since znf423 has been described to be a prominent effector of bmp2 and bmp4 [ 19 , 55 ] , a lower expression could be part of this mechanism . it must be kept in mind that all four controls , whose chondrocytes show higher levels of znf423 than the oa patients , are in the younger group ; however , even if these subjects were excluded , the znf423 expression of the younger patients remained significantly higher than that of the elderly cohort . the expression of znf521 did not display significant changes among the different patients ' groups classified by age or oa stage ; however , we were intrigued by the high levels of expression in primary chondrocytes compared to the immortalized cell lines . as illustrated in figure 3 , znf521 knockdown with two distinct shrnas in chondrocytes cultured in alginate beads resulted in a phenotype with marked characteristics of dedifferentiation . this suggests that the presence of high levels of znf521 expression may be a requisite for the maintenance of the identity in primary chondrocytes . mechanistically , transcription is controlled by the concerted action of transcription factors and epigenetic regulators , including high - mobility group proteins like hmgb-1 and hmga1 [ 35 , 58 ] that are both increased in later stages of oa reflecting multiple changes at the level of transcription and an involvement in pathogenesis of oa , as well as histone - modifying enzymes such as histone acetylases ( hats ) and deacetylases ( hdacs ) . several features of differentiated chondrocytes are known to be affected by the activity of these effectors ; for instance , collagen type ii expression is controlled by hdac activity in articular chondrocytes as well as by the class iii hdac , sirt1 , a nad - dependent hdac , which regulates cartilage gene expression . both znf521 and znf423 have n - terminal motifs that interact with the hdac - containing nurd complex [ 5 , 6 , 61 ] , and the integrity of this motif has been shown to be essential for the promotion of growth and tumorigenicity of medulloblastoma cells ; in addition , zfp521 is also known to interact with hdac3 and hdac4 that are instrumental in the regulatory functions of zfp521 in growth plate chondrocytes and osteoblasts [ 14 , 15 ] . finally , the functional interactions between znf521 and znf423 in articular chondrocytes warrant further investigation to determine whether the two factors exert antagonistic functions in these cells as documented in mesenchymal stem cells or if their coexpression is required for the maintenance of the differentiated phenotype . a better understanding of the role of these factors in chondrocyte physiology may provide further insight into the molecular mechanisms that control their homeostasis and their potential contribution to the pathophysiology of oa .

data were obtained from the ims lifelink program : health plan claims ( u.s . ) database ( formerly known as pharmetrics ) , which is comprised of medical and pharmaceutical claims for over 36 million unique patients from 98 health plans across the u.s for the period june 2005 through march 2009 . the database includes inpatient and outpatient diagnoses ( in icd-9-cm format ) and procedures ( in current procedural terminology , 4th edition [ cpt-4 ] , and healthcare common procedure coding system [ hcpcs ] formats ) and both retail and mail - order prescription records . available data on prescription claims include the national drug code ( ndc ) , days ' supply , and quantity dispensed . additional data include demographic variables ( age , sex , geographic region ) , type of insurance ( e.g. , hmo , preferred provider organization ) , payer type ( e.g. , commercial , self - pay ) , provider specialty , and eligibility dates related to plan enrollment and participation . in compliance with the health insurance portability and accountability act ( hipaa ) , patient data used in the analysis were de - identified ; therefore , this study was exempt from institutional review board review . patients entered the study cohort if they had type 2 diabetes and filled any new glucose - lowering medications on or after 1 june 2005 . a new agent was defined as a prescription filled with no evidence of a previous prescription for that agent in the prior 9 months . patients were assigned to the exenatide or nonexenatide cohort based on the first new prescription filled on or after 1 june 2005 . patients were defined as having type 2 diabetes if they had a claim for an oral glucose - lowering medication or exenatide and met at least one additional criterion during the study period : 1 ) an icd-9-cm diagnosis code of 250.xx on a medical , facility , or surgical claim or 2 ) a claim for insulin or pramlintide . patients were excluded from the study if 1 ) they were not continuously enrolled in a health plan with both medical and pharmacy coverage for a minimum of 9 months precohort entry date and at least 1 day postcohort entry date or 2 ) had acute myocardial infarction ( mi ) , ischemic stroke , or coronary revascularization procedures during the 9 months before the cohort entry date ( fig . 1 ) . patient selection and sample attrition . patients were followed from cohort entry date until the end of their observation period , which was defined as the date of disenrollment , the first occurrence of a cvd event of interest , or end of data stream ( 31 march 2009 ) . to capture complex treatment patterns ( e.g. , exposure to glucose - lowering therapy ) over time , patients were allowed to change cohort membership during the study period . a patient was assigned to the exenatide cohort if the day 's supply for an exenatide prescription plus 31 days covered the end point of the time interval . as a result patients were designated as exenatide patients as long as a prescription for exenatide was found despite the addition or discontinuation of other glucose - lowering agents , including insulin . similarly , nonexenatide patients were so designated despite switching or adding glucose - lowering agents different from the initial agent ( except exenatide ) . cvd events of interest were defined as the first occurrence of mi , ischemic stroke , or coronary revascularization procedure ( angioplasty / atherectomy , percutaneous transluminal coronary angioplasty / stenting , or coronary artery bypass graft ) . incident mi was defined as a hospitalization with an icd-9-cm diagnosis code ( 410.xx excluding 410.7x ) in the primary position . similarly , ischemic stroke was defined as a hospitalization with an icd-9-cm diagnosis code ( 433.x1 , 434.x1 , 435.9 , 436 , 437.1x , 437.9x ) in the primary position . coronary revascularization procedure was defined as a hospitalization or an outpatient visit with one of the following cpt procedure codes ( 3545035459 , 3547035475 , 3548035495 ; 92980 , 92981 , 92982 , 92984 , 92995 , 92996 , g0290 , g0291 , s2211 , s2222 ; 3351033536 , 33572 , s2204s2209 ) . baseline covariates were derived from the pharmacy and medical claims rendered during the 9 months preceding cohort entry , inclusive of claims rendered during the day of cohort entry . the following variables were included : patient demographics ; the calendar quarter of study entry ; indicators of diabetes severity ( number of glucose - lowering medications used , diabetic retinopathy , peripheral neuropathy , renal impairment , and dialysis ) ; indicators of preexisting cvd ( ischemic heart disease , congestive heart failure , acute coronary syndrome , deep vein thrombosis , arrhythmia / conduction - related events ) ; cvd risk factors ( hyperlipidemia , hypertriglyceridemia , and hypertension ) ; other comorbidities ( malignant neoplasms , obesity , depressive disorders , alcohol dependence / abuse , smoking , hyperthyroidism , cardiomyopathy , cerebrovascular disease , chronic obstructive pulmonary disease , asthma , other chronic kidney disease , arthritis , osteoporosis , and open heart surgery ) , filled prescriptions for concomitant therapies ( ace inhibitors , angiotensin - receptor blockers , vasodilators , anti - arrhythmic agents , fibrates , hmg - coa reductase inhibitors [ statins ] , anti - obesity agents , antiplatelet agents , nonsteroidal anti - inflammatory drugs , antithrombolytic agents , and immunomodulating drugs ) ; and patients ' charlson comorbidity index score ( 15 ) . the analyses included ( and adjusted for ) variables that occurred most frequently ( top 50 ) in the database ( primary and secondary diagnosis codes for most recent hospitalization pre - exposure , procedures , icd-9 diagnosis codes , and medications ) , whether they were thought to be causal or not , to optimize balance between the study groups . a propensity score weighted discrete time survival analysis with time - varying exposure to exenatide ( 16 ) was used to compare the hazard of incident cvd events between patients treated with exenatide versus other glucose - lowering therapies , which included all formulations of metformin , thiazolidinediones , sulfonylureas , dipeptidyl - peptidase inhibitors , -glucosidase inhibitors , and insulin . a propensity score model was developed to predict patients ' assignment at cohort entry to ensure cohort balance . the propensity score model included > 300 variables and the interaction effects derived from the baseline covariates ( online appendices 14 , available at http://care.diabetesjournals.org/cgi/content/full/dc10-1393/dc1 ) . the final propensity model was then used to generate a patient - specific propensity score that was used to adjust for potential selection bias in the final analyses . to ensure the stability of results with regard to methodological choices , we completed a primary analysis and sensitivity analyses that used various designs and adjustment techniques . the primary analysis was a discrete time survival analysis using time - varying exposure to exenatide . the estimation procedure was completed using generalized estimating equations to account for the multiplicity of observations within patients . the standardized propensity score weights were calculated for each patient by the inverse of the propensity score , adjusted for the sample size of each cohort . as a sensitivity analysis for the adjustment technique , the same model as in the base case patients were grouped into deciles based on their estimated propensity score , and patients were then compared within each stratum using the pooled logistic regression . the summary hazard ratio ( hr ) was calculated as a weighted average of all point estimates within each stratum , the weight being the inverse of the variance of each estimate . the variance of the weighted average was the inverse of the sum of the weights . as a sensitivity analysis for the design , we estimated and compared the rate of cvd events using intent - to - treat analysis . the intent - to - treat analysis provides a good benchmark to the general practice in a clinical trial and is known to preserve the null hypothesis . all data management and analyses were performed using statistical analysis software ( versions 8.2 and 9.1 ; sas , cary , nc ) . for all analyses , statistical significance was evaluated at a type 1 error of 5% . data were obtained from the ims lifelink program : health plan claims ( u.s . ) database ( formerly known as pharmetrics ) , which is comprised of medical and pharmaceutical claims for over 36 million unique patients from 98 health plans across the u.s for the period june 2005 through march 2009 . the database includes inpatient and outpatient diagnoses ( in icd-9-cm format ) and procedures ( in current procedural terminology , 4th edition [ cpt-4 ] , and healthcare common procedure coding system [ hcpcs ] formats ) and both retail and mail - order prescription records . available data on prescription claims include the national drug code ( ndc ) , days ' supply , and quantity dispensed . additional data include demographic variables ( age , sex , geographic region ) , type of insurance ( e.g. , hmo , preferred provider organization ) , payer type ( e.g. , commercial , self - pay ) , provider specialty , and eligibility dates related to plan enrollment and participation . in compliance with the health insurance portability and accountability act ( hipaa ) , patient data used in the analysis were de - identified ; therefore , this study was exempt from institutional review board review . patients entered the study cohort if they had type 2 diabetes and filled any new glucose - lowering medications on or after 1 june 2005 . a new agent was defined as a prescription filled with no evidence of a previous prescription for that agent in the prior 9 months . patients were assigned to the exenatide or nonexenatide cohort based on the first new prescription filled on or after 1 june 2005 . patients were defined as having type 2 diabetes if they had a claim for an oral glucose - lowering medication or exenatide and met at least one additional criterion during the study period : 1 ) an icd-9-cm diagnosis code of 250.xx on a medical , facility , or surgical claim or 2 ) a claim for insulin or pramlintide . patients were excluded from the study if 1 ) they were not continuously enrolled in a health plan with both medical and pharmacy coverage for a minimum of 9 months precohort entry date and at least 1 day postcohort entry date or 2 ) had acute myocardial infarction ( mi ) , ischemic stroke , or coronary revascularization procedures during the 9 months before the cohort entry date ( fig . patients were followed from cohort entry date until the end of their observation period , which was defined as the date of disenrollment , the first occurrence of a cvd event of interest , or end of data stream ( 31 march 2009 ) . to capture complex treatment patterns ( e.g. , exposure to glucose - lowering therapy ) over time , patients were allowed to change cohort membership during the study period . a patient was assigned to the exenatide cohort if the day 's supply for an exenatide prescription plus 31 days covered the end point of the time interval . as a result patients were designated as exenatide patients as long as a prescription for exenatide was found despite the addition or discontinuation of other glucose - lowering agents , including insulin . similarly , nonexenatide patients were so designated despite switching or adding glucose - lowering agents different from the initial agent ( except exenatide ) . cvd events of interest were defined as the first occurrence of mi , ischemic stroke , or coronary revascularization procedure ( angioplasty / atherectomy , percutaneous transluminal coronary angioplasty / stenting , or coronary artery bypass graft ) . incident mi was defined as a hospitalization with an icd-9-cm diagnosis code ( 410.xx excluding 410.7x ) in the primary position . similarly , ischemic stroke was defined as a hospitalization with an icd-9-cm diagnosis code ( 433.x1 , 434.x1 , 435.9 , 436 , 437.1x , 437.9x ) in the primary position . coronary revascularization procedure was defined as a hospitalization or an outpatient visit with one of the following cpt procedure codes ( 3545035459 , 3547035475 , 3548035495 ; 92980 , 92981 , 92982 , 92984 , 92995 , 92996 , g0290 , g0291 , s2211 , s2222 ; 3351033536 , 33572 , s2204s2209 ) . baseline covariates were derived from the pharmacy and medical claims rendered during the 9 months preceding cohort entry , inclusive of claims rendered during the day of cohort entry . the following variables were included : patient demographics ; the calendar quarter of study entry ; indicators of diabetes severity ( number of glucose - lowering medications used , diabetic retinopathy , peripheral neuropathy , renal impairment , and dialysis ) ; indicators of preexisting cvd ( ischemic heart disease , congestive heart failure , acute coronary syndrome , deep vein thrombosis , arrhythmia / conduction - related events ) ; cvd risk factors ( hyperlipidemia , hypertriglyceridemia , and hypertension ) ; other comorbidities ( malignant neoplasms , obesity , depressive disorders , alcohol dependence / abuse , smoking , hyperthyroidism , cardiomyopathy , cerebrovascular disease , chronic obstructive pulmonary disease , asthma , other chronic kidney disease , arthritis , osteoporosis , and open heart surgery ) , filled prescriptions for concomitant therapies ( ace inhibitors , angiotensin - receptor blockers , vasodilators , anti - arrhythmic agents , fibrates , hmg - coa reductase inhibitors [ statins ] , anti - obesity agents , antiplatelet agents , nonsteroidal anti - inflammatory drugs , antithrombolytic agents , and immunomodulating drugs ) ; and patients ' charlson comorbidity index score ( 15 ) . the analyses included ( and adjusted for ) variables that occurred most frequently ( top 50 ) in the database ( primary and secondary diagnosis codes for most recent hospitalization pre - exposure , procedures , icd-9 diagnosis codes , and medications ) , whether they were thought to be causal or not , to optimize balance between the study groups . a propensity score weighted discrete time survival analysis with time - varying exposure to exenatide ( 16 ) was used to compare the hazard of incident cvd events between patients treated with exenatide versus other glucose - lowering therapies , which included all formulations of metformin , thiazolidinediones , sulfonylureas , dipeptidyl - peptidase inhibitors , -glucosidase inhibitors , and insulin . a propensity score model was developed to predict patients ' assignment at cohort entry to ensure cohort balance . the propensity score model included > 300 variables and the interaction effects derived from the baseline covariates ( online appendices 14 , available at http://care.diabetesjournals.org/cgi/content/full/dc10-1393/dc1 ) . the final propensity model was then used to generate a patient - specific propensity score that was used to adjust for potential selection bias in the final analyses . to ensure the stability of results with regard to methodological choices , we completed a primary analysis and sensitivity analyses that used various designs and adjustment techniques . the primary analysis was a discrete time survival analysis using time - varying exposure to exenatide . the estimation procedure was completed using generalized estimating equations to account for the multiplicity of observations within patients . the standardized propensity score weights were calculated for each patient by the inverse of the propensity score , adjusted for the sample size of each cohort . as a sensitivity analysis for the adjustment technique , the same model as in the base case was estimated using propensity score stratification as the method of adjustment . patients were grouped into deciles based on their estimated propensity score , and patients were then compared within each stratum using the pooled logistic regression . the summary hazard ratio ( hr ) was calculated as a weighted average of all point estimates within each stratum , the weight being the inverse of the variance of each estimate . the variance of the weighted average was the inverse of the sum of the weights . as a sensitivity analysis for the design , we estimated and compared the rate of cvd events using intent - to - treat analysis . the intent - to - treat analysis provides a good benchmark to the general practice in a clinical trial and is known to preserve the null hypothesis . all data management and analyses were performed using statistical analysis software ( versions 8.2 and 9.1 ; sas , cary , nc ) . for all analyses , statistical significance was evaluated at a type 1 error of 5% . more than 1.2 million patients were identified with evidence of at least one glucose - lowering prescription on or after 1 june 2005 . after applying all study criteria , a total of 383,525 patients prescribed glucose - lowering medications were included in the analysis ( fig . 1 ; table 1 ) , including 21,754 exenatide initiators and 361,771 nonexenatide initiators . exenatide initiators appeared to have more severe diabetes based on indicators of diabetes severity ( table 1 ) . specifically , exenatide initiators had evidence of greater use of other glucose - lowering therapies before the initial exenatide prescription and a greater proportion of the exenatide initiators had evidence of diabetic retinopathy , peripheral neuropathy , hyperlipidemia , hypertension , and ischemic heart disease than nonexenatide initiators . medications used to treat cvd risk factors were more commonly listed for exenatide initiators than nonexenatide initiators , including antihypertensive agents , antihyperlipidemic agents , and specifically ace inhibitors , angiotensin - receptor blockers , statins , and fibrates . arthritis was the most common comorbidity in exenatide initiators , and associated nonsteroidal anti - inflammatory drug use was also greater . obesity ( icd-9-cm code 278.0x ) was more prevalent in exenatide initiators than in nonexenatide initiators . baseline clinical characteristics and demographics for patients initiating exenatide twice daily and other glucose - lowering medications data are percent or means sd unless otherwise indicated . . over the course of the study , 39,275 patients and 381,218 patients were exposed to ( treated with ) exenatide and nonexenatide therapies , respectively . the hr for cvd events among the exenatide - treated patients compared with the non exenatide - treated patients is shown in fig . exenatide - treated patients were significantly less likely to have a cvd event ( hr 0.81 ; 95% ci 0.680.95 ; p = 0.01 ) compared with non exenatide - treated patients . results were robust with respect to the statistical method used . in a propensity score stratified analysis , exenatide - treated patients were less likely to have a cvd event ( hr 0.80 ; 0.740.86 ; p < 0.001 ) compared with non exenatide - treated patients . in the intent - to - treat analysis , exenatide initiators were less likely to have a cvd event ( hr 0.86 ; 0.810.92 ; p < 0.001 ) compared with non - exenatide initiators . hrs for cardiovascular events among the exenatide twice daily study cohort versus nonexenatide study cohort resulting from various methodological techniques . error bars represent 95% cis . propensity - score stratified , propensity score , stratified by decile ; itt , intention to treat . consistent with the reduced frequency of cvd events , exenatide initiators had lower rates of hospitalization for cvd - related events ( hr 0.88 ; 0.790.98 ; p = 0.02 ) during the follow - up period compared with the non - exenatide initiators and had a lower rate of all - cause hospitalization ( hr 0.94 ; 0.910.97 ; p < 0.001 ) . over the course of the study , 39,275 patients and 381,218 patients were exposed to ( treated with ) exenatide and nonexenatide therapies , respectively . the hr for cvd events among the exenatide - treated patients compared with the non exenatide - treated patients is shown in fig . exenatide - treated patients were significantly less likely to have a cvd event ( hr 0.81 ; 95% ci 0.680.95 ; p = 0.01 ) compared with non exenatide - treated patients . results were robust with respect to the statistical method used . in a propensity score stratified analysis , exenatide - treated patients were less likely to have a cvd event ( hr 0.80 ; 0.740.86 ; p < 0.001 ) compared with non exenatide - treated patients . in the intent - to - treat analysis , exenatide initiators were less likely to have a cvd event ( hr 0.86 ; 0.810.92 ; p < 0.001 ) compared with non - exenatide initiators . hrs for cardiovascular events among the exenatide twice daily study cohort versus nonexenatide study cohort resulting from various methodological techniques . propensity - score stratified , propensity score , stratified by decile ; itt , intention to treat . consistent with the reduced frequency of cvd events , exenatide initiators had lower rates of hospitalization for cvd - related events ( hr 0.88 ; 0.790.98 ; p = 0.02 ) during the follow - up period compared with the non - exenatide initiators and had a lower rate of all - cause hospitalization ( hr 0.94 ; 0.910.97 ; p < 0.001 ) . in a real world cohort of patients with type 2 diabetes , use of exenatide twice daily was associated with a reduced risk for cvd events and cvd - related hospitalization . the finding that the associated risk reduction is robust using multiple statistical approaches gives credibility to the observation of reduced cvd risk . the observation that lipid levels , blood pressure , obesity , and evidence of prior cvd were higher in patients initially treated with exenatide than in patients initially treated with other agents lends additional support to the concept that exenatide use is associated with favorable effects on cvd outcomes and hospitalization compared with other therapies . these data are consistent with the hypothesis that the glp-1 receptor agonist exenatide may reduce the risk for cvd in patients with type 2 diabetes . several factors may explain the observed reduction in cvd events and hospitalization in exenatide versus non exenatide - treated patients : greater reduction of hyperglycemia with less hypoglycemia and/or improvement in cvd risk factors , including weight , lipids , and blood pressure ( 14 ) . a randomized controlled clinical trial of the cardiovascular outcomes associated with long - term use of exenatide is needed to demonstrate whether exenatide treatment reduces cvd risk and to determine whether changes in a1c , the incidence of hypoglycemia , and/or reductions in cvd risk factors are associated with improved cardiovascular outcomes . although observational studies have generally shown a relationship between hyperglycemia ( even below the threshold for the diagnosis of type 2 diabetes ) and cvd ( 13 ) , clinical trials have not consistently confirmed that reducing hyperglycemia reduces cvd ( 49 ) . the uk prospective diabetes study ( ukpds ) reported a significant reduction in mi in the small cohort treated with metformin ( 6 ) , but effects with sulfonylureas and insulin did not achieve statistical significance until the 10-year follow - up study ( 7,8 ) . three large recently completed intervention trials ( veterans affairs diabetes trial [ vadt ] , action to control cardiovascular risk in diabetes [ accord ] , and action in diabetes and vascular disease : preterax and diamicron mr controlled evaluation [ advance ] ) did not show evidence of cvd risk reduction in the primary outcome ( nonfatal mi , nonfatal stroke , or death from cardiovascular causes ) ( 4,5,9 ) . in fact , accord was terminated early because of increased all - cause mortality in the intensive glycemic treatment arm ( 5 ) . of note , the increased mortality in each of the accord arms was associated with hypoglycemia ( 17 ) . ( 13 ) suggests that when the data from ukpds , accord , advance , and vadt are combined , the reduction in cvd events becomes statistically significant , the effect is small . these observations from accord and vadt suggest that when there is a need to intensify glucose control , hypoglycemia may increase cvd risk and counter the benefits of reducing hyperglycemia . the primary strength of this study was the inclusion of patients with type 2 diabetes who received glucose - lowering therapy in a real - world population , a large and broadly representative source population . this study reflects common clinical practice across the u.s . and provides for more than adequate statistical power and information to adjust for potential confounders . however , the administrative data used in this real - world study present challenges of accurately ascertaining variables of interest . although we sought to mitigate potential sources of bias in the conduct of this study , some limitations remain , including potential misclassification of exposure and/or outcome , and the potential for residual confounding . diagnosis of cvd using icd-9-cm codes may not always represent a clinical diagnosis of the disease ; however , prior publications indicate that the estimated predictive value of administrative data for identifying cardiovascular end points are high ( 95% for acute mi and stroke ) , suggesting that icd-9-cm codes have good positive predictive value ( 18,19 ) . in studies based on administrative data , the misclassification might be assumed to be nondifferential with respect to exposure , with a bias in the hr toward the null value that might obscure an association between exenatide and cvd risk . however , if physicians monitor more closely patients with severe diabetes ( who are more likely to be on exenatide ) for cvd , then the estimated association of exenatide with cvd would be biased away from the null . we controlled for a large set of factors that potentially differed between the groups at baseline using propensity score methodology . nevertheless , the incomplete capture of variables in the claims data , such as weight , smoking , alcohol consumption , and change in other variables associated with cvd risk ( e.g. , lipids , blood pressure , and a1c ) , is a limitation of the present analysis . indeed , the baseline characteristics of the study cohorts suggest that exenatide initiators have a higher prevalence of potential risk factors for cvd , such as obesity , hypertension , and hyperlipidemia . the direction of imbalance in these partially captured variables suggests that remaining ( unmeasured ) confounding would lead to a higher risk of cvd among exenatide users . however , baseline laboratory data and measures of cvd risk markers such as weight , blood pressure , and lipids are needed to adjust for population differences , and we do not have these data . we expect that adjustment for the preindex clinical characteristics including use of antihyperlipidemic and antihypertensive medications serve as proxies for these variables . additionally , the linkage between pharmacy submission of claims and patients ' receipt and consumption of the medication is assumed and not directly measured ; prior work suggests that medication exposure measures can be accurately derived from pharmacy claims ( 20 ) . as an insurance database , the results are most generalizable to similar commercially insured patients , but the results are likely to be relevant to a more general population of patients with type 2 diabetes , unless uninsured patients differ in their response to exenatide . this study does not address potential questions about whether nonexenatide agents may be associated with increased risk ( with possible absence of benefit from exenatide ) or neutral risk of cvd events . in conclusion , in this retrospective epidemiological study , exenatide - treated patients were 19% less likely to have a cvd event than patients treated with other glucose - lowering agents ; exenatide - treated patients were also less likely to experience cvd - related and all - cause hospitalization . the results were robust with respect to the statistical method and support the cvd safety of exenatide twice daily for patients with type 2 diabetes . the improved cv outcomes observed in this retrospective database analysis need to be confirmed in prospective studies of treatment with exenatide .

objectiveto test the hypothesis that exenatide twice daily reduces the relative incidence of cardiovascular disease ( cvd ) events among patients with type 2 diabetes compared with other glucose - lowering agent(s).research design and methodsa retrospective database analysis was performed of the lifelink database of medical and pharmaceutical insurance claims for june 2005 through march 2009 . patients with no history in the preceding 9 months of myocardial infarction , ischemic stroke , or coronary revascularization procedure were assigned to the exenatide - initiated or non exenatide - initiated cohorts based on the first new prescription filled and reassigned if exenatide was prescribed or discontinued . incident cvd events ( myocardial infarction , ischemic stroke , or coronary revascularization procedure ) were identified by icd-9-cm diagnosis codes . patient outcomes were adjusted for differences in clinical and demographic characteristics and compared using propensity score weighted discrete time survival analysis with time - varying exposure to exenatide.resultsa total of 39,275 patients with type 2 diabetes were treated with exenatide twice daily , and 381,218 patients were treated with other glucose - lowering therapies . patients who initiated exenatide were more likely to have prior ischemic heart disease , obesity , hyperlipidemia , hypertension , and/or other comorbidities at baseline . exenatide - treated patients were less likely to have a cvd event than non exenatide - treated patients ( hazard ratio 0.81 ; 95% ci 0.680.95 ; p = 0.01 ) and lower rates of cvd - related hospitalization ( 0.88 ; 0.790.98 ; p = 0.02 ) and all - cause hospitalization ( 0.94 ; 0.910.97 ; p < 0.001).conclusionsexenatide twice - daily treatment was associated with a lower risk of cvd events and hospitalizations than treatment with other glucose - lowering therapies .

RESEARCH DESIGN AND METHODS Source population Cohort formation and exposure definition Definition of study events Baseline covariates Data analysis Primary analyses Sensitivity analyses RESULTS Cardiovascular events and rate of hospitalizations CONCLUSIONS

database ( formerly known as pharmetrics ) , which is comprised of medical and pharmaceutical claims for over 36 million unique patients from 98 health plans across the u.s for the period june 2005 through march 2009 . patients entered the study cohort if they had type 2 diabetes and filled any new glucose - lowering medications on or after 1 june 2005 . a new agent was defined as a prescription filled with no evidence of a previous prescription for that agent in the prior 9 months . patients were assigned to the exenatide or nonexenatide cohort based on the first new prescription filled on or after 1 june 2005 . patients were defined as having type 2 diabetes if they had a claim for an oral glucose - lowering medication or exenatide and met at least one additional criterion during the study period : 1 ) an icd-9-cm diagnosis code of 250.xx on a medical , facility , or surgical claim or 2 ) a claim for insulin or pramlintide . patients were excluded from the study if 1 ) they were not continuously enrolled in a health plan with both medical and pharmacy coverage for a minimum of 9 months precohort entry date and at least 1 day postcohort entry date or 2 ) had acute myocardial infarction ( mi ) , ischemic stroke , or coronary revascularization procedures during the 9 months before the cohort entry date ( fig . patients were followed from cohort entry date until the end of their observation period , which was defined as the date of disenrollment , the first occurrence of a cvd event of interest , or end of data stream ( 31 march 2009 ) . , exposure to glucose - lowering therapy ) over time , patients were allowed to change cohort membership during the study period . a patient was assigned to the exenatide cohort if the day 's supply for an exenatide prescription plus 31 days covered the end point of the time interval . as a result patients were designated as exenatide patients as long as a prescription for exenatide was found despite the addition or discontinuation of other glucose - lowering agents , including insulin . similarly , nonexenatide patients were so designated despite switching or adding glucose - lowering agents different from the initial agent ( except exenatide ) . cvd events of interest were defined as the first occurrence of mi , ischemic stroke , or coronary revascularization procedure ( angioplasty / atherectomy , percutaneous transluminal coronary angioplasty / stenting , or coronary artery bypass graft ) . similarly , ischemic stroke was defined as a hospitalization with an icd-9-cm diagnosis code ( 433.x1 , 434.x1 , 435.9 , 436 , 437.1x , 437.9x ) in the primary position . coronary revascularization procedure was defined as a hospitalization or an outpatient visit with one of the following cpt procedure codes ( 3545035459 , 3547035475 , 3548035495 ; 92980 , 92981 , 92982 , 92984 , 92995 , 92996 , g0290 , g0291 , s2211 , s2222 ; 3351033536 , 33572 , s2204s2209 ) . the following variables were included : patient demographics ; the calendar quarter of study entry ; indicators of diabetes severity ( number of glucose - lowering medications used , diabetic retinopathy , peripheral neuropathy , renal impairment , and dialysis ) ; indicators of preexisting cvd ( ischemic heart disease , congestive heart failure , acute coronary syndrome , deep vein thrombosis , arrhythmia / conduction - related events ) ; cvd risk factors ( hyperlipidemia , hypertriglyceridemia , and hypertension ) ; other comorbidities ( malignant neoplasms , obesity , depressive disorders , alcohol dependence / abuse , smoking , hyperthyroidism , cardiomyopathy , cerebrovascular disease , chronic obstructive pulmonary disease , asthma , other chronic kidney disease , arthritis , osteoporosis , and open heart surgery ) , filled prescriptions for concomitant therapies ( ace inhibitors , angiotensin - receptor blockers , vasodilators , anti - arrhythmic agents , fibrates , hmg - coa reductase inhibitors [ statins ] , anti - obesity agents , antiplatelet agents , nonsteroidal anti - inflammatory drugs , antithrombolytic agents , and immunomodulating drugs ) ; and patients ' charlson comorbidity index score ( 15 ) . the analyses included ( and adjusted for ) variables that occurred most frequently ( top 50 ) in the database ( primary and secondary diagnosis codes for most recent hospitalization pre - exposure , procedures , icd-9 diagnosis codes , and medications ) , whether they were thought to be causal or not , to optimize balance between the study groups . a propensity score weighted discrete time survival analysis with time - varying exposure to exenatide ( 16 ) was used to compare the hazard of incident cvd events between patients treated with exenatide versus other glucose - lowering therapies , which included all formulations of metformin , thiazolidinediones , sulfonylureas , dipeptidyl - peptidase inhibitors , -glucosidase inhibitors , and insulin . the primary analysis was a discrete time survival analysis using time - varying exposure to exenatide . the standardized propensity score weights were calculated for each patient by the inverse of the propensity score , adjusted for the sample size of each cohort . as a sensitivity analysis for the adjustment technique , the same model as in the base case patients were grouped into deciles based on their estimated propensity score , and patients were then compared within each stratum using the pooled logistic regression . as a sensitivity analysis for the design , we estimated and compared the rate of cvd events using intent - to - treat analysis . database ( formerly known as pharmetrics ) , which is comprised of medical and pharmaceutical claims for over 36 million unique patients from 98 health plans across the u.s for the period june 2005 through march 2009 . patients entered the study cohort if they had type 2 diabetes and filled any new glucose - lowering medications on or after 1 june 2005 . a new agent was defined as a prescription filled with no evidence of a previous prescription for that agent in the prior 9 months . patients were assigned to the exenatide or nonexenatide cohort based on the first new prescription filled on or after 1 june 2005 . patients were defined as having type 2 diabetes if they had a claim for an oral glucose - lowering medication or exenatide and met at least one additional criterion during the study period : 1 ) an icd-9-cm diagnosis code of 250.xx on a medical , facility , or surgical claim or 2 ) a claim for insulin or pramlintide . patients were excluded from the study if 1 ) they were not continuously enrolled in a health plan with both medical and pharmacy coverage for a minimum of 9 months precohort entry date and at least 1 day postcohort entry date or 2 ) had acute myocardial infarction ( mi ) , ischemic stroke , or coronary revascularization procedures during the 9 months before the cohort entry date ( fig . patients were followed from cohort entry date until the end of their observation period , which was defined as the date of disenrollment , the first occurrence of a cvd event of interest , or end of data stream ( 31 march 2009 ) . , exposure to glucose - lowering therapy ) over time , patients were allowed to change cohort membership during the study period . a patient was assigned to the exenatide cohort if the day 's supply for an exenatide prescription plus 31 days covered the end point of the time interval . as a result patients were designated as exenatide patients as long as a prescription for exenatide was found despite the addition or discontinuation of other glucose - lowering agents , including insulin . similarly , nonexenatide patients were so designated despite switching or adding glucose - lowering agents different from the initial agent ( except exenatide ) . cvd events of interest were defined as the first occurrence of mi , ischemic stroke , or coronary revascularization procedure ( angioplasty / atherectomy , percutaneous transluminal coronary angioplasty / stenting , or coronary artery bypass graft ) . similarly , ischemic stroke was defined as a hospitalization with an icd-9-cm diagnosis code ( 433.x1 , 434.x1 , 435.9 , 436 , 437.1x , 437.9x ) in the primary position . the following variables were included : patient demographics ; the calendar quarter of study entry ; indicators of diabetes severity ( number of glucose - lowering medications used , diabetic retinopathy , peripheral neuropathy , renal impairment , and dialysis ) ; indicators of preexisting cvd ( ischemic heart disease , congestive heart failure , acute coronary syndrome , deep vein thrombosis , arrhythmia / conduction - related events ) ; cvd risk factors ( hyperlipidemia , hypertriglyceridemia , and hypertension ) ; other comorbidities ( malignant neoplasms , obesity , depressive disorders , alcohol dependence / abuse , smoking , hyperthyroidism , cardiomyopathy , cerebrovascular disease , chronic obstructive pulmonary disease , asthma , other chronic kidney disease , arthritis , osteoporosis , and open heart surgery ) , filled prescriptions for concomitant therapies ( ace inhibitors , angiotensin - receptor blockers , vasodilators , anti - arrhythmic agents , fibrates , hmg - coa reductase inhibitors [ statins ] , anti - obesity agents , antiplatelet agents , nonsteroidal anti - inflammatory drugs , antithrombolytic agents , and immunomodulating drugs ) ; and patients ' charlson comorbidity index score ( 15 ) . the analyses included ( and adjusted for ) variables that occurred most frequently ( top 50 ) in the database ( primary and secondary diagnosis codes for most recent hospitalization pre - exposure , procedures , icd-9 diagnosis codes , and medications ) , whether they were thought to be causal or not , to optimize balance between the study groups . a propensity score weighted discrete time survival analysis with time - varying exposure to exenatide ( 16 ) was used to compare the hazard of incident cvd events between patients treated with exenatide versus other glucose - lowering therapies , which included all formulations of metformin , thiazolidinediones , sulfonylureas , dipeptidyl - peptidase inhibitors , -glucosidase inhibitors , and insulin . the primary analysis was a discrete time survival analysis using time - varying exposure to exenatide . the standardized propensity score weights were calculated for each patient by the inverse of the propensity score , adjusted for the sample size of each cohort . as a sensitivity analysis for the adjustment technique , the same model as in the base case was estimated using propensity score stratification as the method of adjustment . patients were grouped into deciles based on their estimated propensity score , and patients were then compared within each stratum using the pooled logistic regression . as a sensitivity analysis for the design , we estimated and compared the rate of cvd events using intent - to - treat analysis . more than 1.2 million patients were identified with evidence of at least one glucose - lowering prescription on or after 1 june 2005 . after applying all study criteria , a total of 383,525 patients prescribed glucose - lowering medications were included in the analysis ( fig . specifically , exenatide initiators had evidence of greater use of other glucose - lowering therapies before the initial exenatide prescription and a greater proportion of the exenatide initiators had evidence of diabetic retinopathy , peripheral neuropathy , hyperlipidemia , hypertension , and ischemic heart disease than nonexenatide initiators . baseline clinical characteristics and demographics for patients initiating exenatide twice daily and other glucose - lowering medications data are percent or means sd unless otherwise indicated . over the course of the study , 39,275 patients and 381,218 patients were exposed to ( treated with ) exenatide and nonexenatide therapies , respectively . the hr for cvd events among the exenatide - treated patients compared with the non exenatide - treated patients is shown in fig . exenatide - treated patients were significantly less likely to have a cvd event ( hr 0.81 ; 95% ci 0.680.95 ; p = 0.01 ) compared with non exenatide - treated patients . in a propensity score stratified analysis , exenatide - treated patients were less likely to have a cvd event ( hr 0.80 ; 0.740.86 ; p < 0.001 ) compared with non exenatide - treated patients . in the intent - to - treat analysis , exenatide initiators were less likely to have a cvd event ( hr 0.86 ; 0.810.92 ; p < 0.001 ) compared with non - exenatide initiators . hrs for cardiovascular events among the exenatide twice daily study cohort versus nonexenatide study cohort resulting from various methodological techniques . consistent with the reduced frequency of cvd events , exenatide initiators had lower rates of hospitalization for cvd - related events ( hr 0.88 ; 0.790.98 ; p = 0.02 ) during the follow - up period compared with the non - exenatide initiators and had a lower rate of all - cause hospitalization ( hr 0.94 ; 0.910.97 ; p < 0.001 ) . over the course of the study , 39,275 patients and 381,218 patients were exposed to ( treated with ) exenatide and nonexenatide therapies , respectively . the hr for cvd events among the exenatide - treated patients compared with the non exenatide - treated patients is shown in fig . exenatide - treated patients were significantly less likely to have a cvd event ( hr 0.81 ; 95% ci 0.680.95 ; p = 0.01 ) compared with non exenatide - treated patients . in a propensity score stratified analysis , exenatide - treated patients were less likely to have a cvd event ( hr 0.80 ; 0.740.86 ; p < 0.001 ) compared with non exenatide - treated patients . in the intent - to - treat analysis , exenatide initiators were less likely to have a cvd event ( hr 0.86 ; 0.810.92 ; p < 0.001 ) compared with non - exenatide initiators . hrs for cardiovascular events among the exenatide twice daily study cohort versus nonexenatide study cohort resulting from various methodological techniques . consistent with the reduced frequency of cvd events , exenatide initiators had lower rates of hospitalization for cvd - related events ( hr 0.88 ; 0.790.98 ; p = 0.02 ) during the follow - up period compared with the non - exenatide initiators and had a lower rate of all - cause hospitalization ( hr 0.94 ; 0.910.97 ; p < 0.001 ) . in a real world cohort of patients with type 2 diabetes , use of exenatide twice daily was associated with a reduced risk for cvd events and cvd - related hospitalization . the observation that lipid levels , blood pressure , obesity , and evidence of prior cvd were higher in patients initially treated with exenatide than in patients initially treated with other agents lends additional support to the concept that exenatide use is associated with favorable effects on cvd outcomes and hospitalization compared with other therapies . these data are consistent with the hypothesis that the glp-1 receptor agonist exenatide may reduce the risk for cvd in patients with type 2 diabetes . several factors may explain the observed reduction in cvd events and hospitalization in exenatide versus non exenatide - treated patients : greater reduction of hyperglycemia with less hypoglycemia and/or improvement in cvd risk factors , including weight , lipids , and blood pressure ( 14 ) . a randomized controlled clinical trial of the cardiovascular outcomes associated with long - term use of exenatide is needed to demonstrate whether exenatide treatment reduces cvd risk and to determine whether changes in a1c , the incidence of hypoglycemia , and/or reductions in cvd risk factors are associated with improved cardiovascular outcomes . although observational studies have generally shown a relationship between hyperglycemia ( even below the threshold for the diagnosis of type 2 diabetes ) and cvd ( 13 ) , clinical trials have not consistently confirmed that reducing hyperglycemia reduces cvd ( 49 ) . three large recently completed intervention trials ( veterans affairs diabetes trial [ vadt ] , action to control cardiovascular risk in diabetes [ accord ] , and action in diabetes and vascular disease : preterax and diamicron mr controlled evaluation [ advance ] ) did not show evidence of cvd risk reduction in the primary outcome ( nonfatal mi , nonfatal stroke , or death from cardiovascular causes ) ( 4,5,9 ) . in fact , accord was terminated early because of increased all - cause mortality in the intensive glycemic treatment arm ( 5 ) . of note , the increased mortality in each of the accord arms was associated with hypoglycemia ( 17 ) . the primary strength of this study was the inclusion of patients with type 2 diabetes who received glucose - lowering therapy in a real - world population , a large and broadly representative source population . in studies based on administrative data , the misclassification might be assumed to be nondifferential with respect to exposure , with a bias in the hr toward the null value that might obscure an association between exenatide and cvd risk . however , if physicians monitor more closely patients with severe diabetes ( who are more likely to be on exenatide ) for cvd , then the estimated association of exenatide with cvd would be biased away from the null . nevertheless , the incomplete capture of variables in the claims data , such as weight , smoking , alcohol consumption , and change in other variables associated with cvd risk ( e.g. indeed , the baseline characteristics of the study cohorts suggest that exenatide initiators have a higher prevalence of potential risk factors for cvd , such as obesity , hypertension , and hyperlipidemia . as an insurance database , the results are most generalizable to similar commercially insured patients , but the results are likely to be relevant to a more general population of patients with type 2 diabetes , unless uninsured patients differ in their response to exenatide . this study does not address potential questions about whether nonexenatide agents may be associated with increased risk ( with possible absence of benefit from exenatide ) or neutral risk of cvd events . in conclusion , in this retrospective epidemiological study , exenatide - treated patients were 19% less likely to have a cvd event than patients treated with other glucose - lowering agents ; exenatide - treated patients were also less likely to experience cvd - related and all - cause hospitalization . the results were robust with respect to the statistical method and support the cvd safety of exenatide twice daily for patients with type 2 diabetes . the improved cv outcomes observed in this retrospective database analysis need to be confirmed in prospective studies of treatment with exenatide .

in recent years , the reduction of maternal mortality and morbidity has been a major topic in many international symposia and summits . consequently , several countries committed themselves towards the achievement of the millennium development goal 5 , which aimed at reducing maternal mortality by three - quarters by 2015.1 although improvements have been reported in mothers survival , the world health organization still estimated that many women ( 287,000 in 2010 ) continue to die worldwide from various causes related to pregnancy and childbirth.2 a large part of these deaths ( 66,500 ) , which are attributable to unsafe induced abortions,3 occur in sub - saharan countries,4 in which abortion laws are mostly restrictive.5 several studies have addressed the issue and have analyzed the prerequisites for induced abortions in many countries . they have shown that many women turn to abortion to terminate their unwanted pregnancies,6,7 even when laws are prohibitive and methods are unsafe . studies have also reported that hospital admissions resulting from unsafe abortions were frequent.810 moreover , it has been demonstrated that these induced abortions are associated with numerous adverse health problems , such as chronic pelvic inflammatory diseases,11 mental disorders,12 subsequent adverse reproductive outcomes,13 and even secondary infertility.14 in burkina faso , abortion is permitted only in cases of incest , rape , fetal defect , or when the woman s life or physical health is endangered.15,16 because of this , access to safe and legal abortion is difficult to get and women often resort to unsafe procedures,15 at great risk to their health and survival . a previous hospital study revealed that up to 30% of maternal deaths have been the result of unsafe induced abortions.17 additionally , it was estimated that each year , more than 21,800 complications from unsafe induced abortions have been treated in the country s hospital facilities.15 however , these data may largely underestimate the magnitude of the phenomenon , as investigations have highlighted that abortion numbers18 and related morbidity and mortality15 tend to be underreported.18 despite the prevalence of induced abortion , estimated to vary between 25 and 40 per 1,000 women aged 1549 years annually,15,19 it is likely that the extent of the problem has not been thoroughly studied . particularly , information on the conditions and key factors that may precipitate induced abortion is lacking . however , knowledge of these factors is critical in designing appropriate strategies aimed at reducing both unwanted pregnancies and unsafe induced abortions.20 in burkina faso , factors associated with induced abortions have not been studied , in spite of their policy relevance . this study aims at contributing to the literature by investigating the key determinants of induced abortion in a sample of women who resorted to health facilities in ouagadougou for postabortion care . burkina faso , a landlocked country located in the heart of west africa , has a weak health care system.21 the country has a population of 16 million , essentially young and fertile.22 the low purchasing power of the population and of women in particular , limits their access to education , clean water , and health care . maternal mortality is high , with a rate of 300 per 100,000 births.23 contraceptive use is low , with large disparities between poor and rich population groups.24 since 2006 , a national subsidy policy for normal deliveries and emergency obstetric care has been active , in order to reduce financial barriers to care and thereby improve access to qualified care.25 in spite of this policy , utilization of health care services in ouagadougou city is still unsatisfactory , with large inequities between poor and rich women . in order to explore the key determinants of induced abortion , this paper uses data from a cross - sectional study that investigates the costs associated with abortions in ouagadougou . because of the difficulty of recruiting abortion cases in the community,26 participants were prospectively recruited from two hospital facilities . these hospital facilities included one referral - level teaching hospital a top referral hospital in ouagadougou to which complications from abortion are directed for better care and one private health clinic , affiliated with the international planned parenthood federation , with long - standing expertise in treating abortions . a total of 307 women with either a spontaneous or induced abortion were sampled for this study . in each facility , an experienced midwife , generally responsible for the manual vacuum - aspiration ward , was in charge of identifying women with induced or spontaneous abortions based on clinical definitions . additional information on the nature of the abortion was also obtained by interviewing the woman . a case was classified as an induced abortion when the clinical ascertainment was confirmed by the woman herself reporting that she had had an induced abortion . this procedure of classifying the cases may have led to some induced abortions being inaccurately classified as spontaneous.3,27 because of this , women were labeled as certainly induced abortion and reportedly spontaneous abortion . another woman was excluded because she did not complete the interview , leaving a sample size of 304 . after they were identified by the health staff , the women were directed to two female interviewers who were in charge of establishing contact with them for further investigation . all women who met the eligibility criteria were invited to participate in the study . at discharge , subjects who consented to participate in the study were interviewed at the health facility , at the clinic , or at home . the two qualified female interviewers collected data from all the women who had had an induced or a spontaneous abortion , using an interviewer - administered face - to - face questionnaire . prior to fieldwork , interviewers were given comprehensive training on data collection procedures and extraction of clinical data from medical records . during this training session , anticipated difficulties in filling in the questionnaires were thoroughly discussed in order to minimize errors . the main questionnaire that was administered to the women contained a range of questions pertaining to sociodemographic characteristics , pregnancy and birth history , abortion experience , asset ownership and place of residence , and expenditures on abortion and postabortion care , including prereferral costs . the abortion - experience section of the women s questionnaire included questions pertaining to previous abortion experiences and to the type of abortive method used . the women s questionnaire was complemented with a health worker questionnaire , which was intended to extract selected medical information from hospital records . this questionnaire included information related to the gestational age of the pregnancy and the clinical ascertainment of the type of abortion . the dependent variable was the type of abortion , a dummy variable set to 1 when the pregnancy termination was reported as induced or 0 when alleged to be a spontaneous abortion . the empirical literature on factors associated with abortion showed that educated women,2832 young women,2830,3336 unmarried women,28,30,34,37,38 women who had had previous experiences of abortion,31,34,39 women who had living children,29,33,34 and women who did not want the pregnancy28,3941 were more likely to have an induced abortion . therefore , such variables as age , education , marital status , number of children , desire for pregnancy , and previous experience of abortion were considered independent variables . researchers have also demonstrated that women who were experiencing their first pregnancy in life,30 christian women ( compared to muslim women),33,36,39 and women who did not use contraceptives40 were also more likely to have an induced abortion . we therefore also considered the number of pregnancies , the use of contraceptives , and the women s religion as explanatory variables . finally , we included the status of the household - chief ( whether the household is headed by the woman , the husband , or by the woman s parents ) in the analysis of abortion determinants . a descriptive analysis was undertaken in order to understand the distribution of induced and spontaneous abortions relative to each independent variable . chi - squared tests were used to test for significant differences between the groups of women . to identify the key determinants associated with induced abortion for women seeking postabortion care in hospitals in ouagadougou , a two - step analysis consisting of one univariate and one multivariate logistic regression was carried out . the univariate logistic regression was run to determine the association between each of the independent variables and the dependent variable . all the variables that were associated with induced abortion in the univariate logistic regression with a level of significance of 0.05 and 95% confidence were subsequently analyzed in a stepwise multivariate logistic regression . the multivariate regression permitted adjustment among variables and the determination of possible confounding factors . to identify the key factors associated with induced abortion , a downward procedure that minimizes the number of variables while maximizing the accuracy of the model was followed.42 all analyses were conducted on stata version 11.2 ( statacorp , college station , tx , usa ) . burkina faso , a landlocked country located in the heart of west africa , has a weak health care system.21 the country has a population of 16 million , essentially young and fertile.22 the low purchasing power of the population and of women in particular , limits their access to education , clean water , and health care . maternal mortality is high , with a rate of 300 per 100,000 births.23 contraceptive use is low , with large disparities between poor and rich population groups.24 since 2006 , a national subsidy policy for normal deliveries and emergency obstetric care has been active , in order to reduce financial barriers to care and thereby improve access to qualified care.25 in spite of this policy , utilization of health care services in ouagadougou city is still unsatisfactory , with large inequities between poor and rich women . in order to explore the key determinants of induced abortion , this paper uses data from a cross - sectional study that investigates the costs associated with abortions in ouagadougou . because of the difficulty of recruiting abortion cases in the community,26 participants were prospectively recruited from two hospital facilities . these hospital facilities included one referral - level teaching hospital a top referral hospital in ouagadougou to which complications from abortion are directed for better care and one private health clinic , affiliated with the international planned parenthood federation , with long - standing expertise in treating abortions . a total of 307 women with either a spontaneous or induced abortion were sampled for this study . in each facility , an experienced midwife , generally responsible for the manual vacuum - aspiration ward , was in charge of identifying women with induced or spontaneous abortions based on clinical definitions . additional information on the nature of the abortion was also obtained by interviewing the woman . a case was classified as an induced abortion when the clinical ascertainment was confirmed by the woman herself reporting that she had had an induced abortion . this procedure of classifying the cases may have led to some induced abortions being inaccurately classified as spontaneous.3,27 because of this , women were labeled as certainly induced abortion and reportedly spontaneous abortion . another woman was excluded because she did not complete the interview , leaving a sample size of 304 . after they were identified by the health staff , the women were directed to two female interviewers who were in charge of establishing contact with them for further investigation . all women who met the eligibility criteria were invited to participate in the study . at discharge , subjects who consented to participate in the study were interviewed at the health facility , at the clinic , or at home . the two qualified female interviewers collected data from all the women who had had an induced or a spontaneous abortion , using an interviewer - administered face - to - face questionnaire . prior to fieldwork , interviewers were given comprehensive training on data collection procedures and extraction of clinical data from medical records . during this training session , anticipated difficulties in filling in the questionnaires were thoroughly discussed in order to minimize errors . the main questionnaire that was administered to the women contained a range of questions pertaining to sociodemographic characteristics , pregnancy and birth history , abortion experience , asset ownership and place of residence , and expenditures on abortion and postabortion care , including prereferral costs . the abortion - experience section of the women s questionnaire included questions pertaining to previous abortion experiences and to the type of abortive method used . the women s questionnaire was complemented with a health worker questionnaire , which was intended to extract selected medical information from hospital records . this questionnaire included information related to the gestational age of the pregnancy and the clinical ascertainment of the type of abortion . the dependent variable was the type of abortion , a dummy variable set to 1 when the pregnancy termination was reported as induced or 0 when alleged to be a spontaneous abortion . the empirical literature on factors associated with abortion showed that educated women,2832 young women,2830,3336 unmarried women,28,30,34,37,38 women who had had previous experiences of abortion,31,34,39 women who had living children,29,33,34 and women who did not want the pregnancy28,3941 were more likely to have an induced abortion . therefore , such variables as age , education , marital status , number of children , desire for pregnancy , and previous experience of abortion were considered independent variables . researchers have also demonstrated that women who were experiencing their first pregnancy in life,30 christian women ( compared to muslim women),33,36,39 and women who did not use contraceptives40 were also more likely to have an induced abortion . we therefore also considered the number of pregnancies , the use of contraceptives , and the women s religion as explanatory variables . finally , we included the status of the household - chief ( whether the household is headed by the woman , the husband , or by the woman s parents ) in the analysis of abortion determinants . a descriptive analysis was undertaken in order to understand the distribution of induced and spontaneous abortions relative to each independent variable . chi - squared tests were used to test for significant differences between the groups of women . to identify the key determinants associated with induced abortion for women seeking postabortion care in hospitals in ouagadougou , a two - step analysis consisting of one univariate and one multivariate logistic regression was carried out . the univariate logistic regression was run to determine the association between each of the independent variables and the dependent variable . all the variables that were associated with induced abortion in the univariate logistic regression with a level of significance of 0.05 and 95% confidence were subsequently analyzed in a stepwise multivariate logistic regression . the multivariate regression permitted adjustment among variables and the determination of possible confounding factors . to identify the key factors associated with induced abortion , a downward procedure that minimizes the number of variables while maximizing the accuracy of the model was followed.42 all analyses were conducted on stata version 11.2 ( statacorp , college station , tx , usa ) . women who had had an induced abortion constituted 12% of the sample ; 61% of the women were married , and 63% were under 30 years old . education ( primary to university level ) was not common in our sample , with only 33% of the women being formally educated . among the women , 28% declared that their pregnancies were unwanted , and 17% had had a previous experience of abortion , while 29% were on their first pregnancy , 77% were living in households headed by their parents , and up to 80% were not using any form of contraception . table 2 shows associations of induced abortion with the analyzed variables . induced abortions tended to be more prevalent in the educated ( 16% ) , under 30 years old ( 17% ) , single or never married ( 24% ) , and widowed or divorced ( 57% ) women , compared to the uneducated , the married , and women over the age of 30 years . we also found that induced abortion was more prevalent in women who had had a previous experience of abortion ( 68% ) , in women who did not want the pregnancy ( 37% ) , in women who were experiencing their first pregnancy ( 26% ) , in those with living children ( 24% ) , in women under parents guardianship ( 41% ) , and in christians ( 20% ) . the univariate analysis further showed that all the considered variables , except the use of contraceptives , were associated with induced abortion . therefore , being educated , aged less than 30 years , widowed or divorced , and in a household headed by parents all tended to increase the odds of having an induced abortion . we also found that having a previous experience of abortion or having living children , being on the first pregnancy , or the pregnancy being unwanted increased the odds of having an induced abortion . on the contrary , being married or a muslim tended to decrease the odds of having an induced abortion by 97% and 68% , relative to being single or a christian , respectively . moreover , in the multivariate analysis , we found that three key factors , comprising the desire for pregnancy , living in a household headed by the woman s parents , and the woman s marital status , were all associated with induced abortion ( table 3 ) . the effect of the desire for pregnancy was the most important , with the odds of having an induced abortion being ten times higher for women who did not want the pregnancy compared to those who did want it ( odds ratio [ or ] 10.45 , 95% confidence interval [ ci ] 3.5930.41 ) . further , the odds of having an induced abortion were seven times higher for women living in households headed by their parents ( or 6.83 , 95% ci 2.4219.24 ) . the odds of having an induced abortion also increased by three times for divorced or widowed women ( or 3.47 , 95% ci 1.0811.10 ) compared to single or never - married women . on the contrary , married women were 83% less likely to have an induced abortion , even when the pregnancy was not desired ( or 0.17 , 95% ci 0.030.89 ) , compared to single or never - married women . finally , we found that age , education , previous experience of abortion , number of living children , number of pregnancies , and religion had no significant effect on having an induced abortion . women who had had an induced abortion constituted 12% of the sample ; 61% of the women were married , and 63% were under 30 years old . education ( primary to university level ) was not common in our sample , with only 33% of the women being formally educated . among the women , 28% declared that their pregnancies were unwanted , and 17% had had a previous experience of abortion , while 29% were on their first pregnancy , 77% were living in households headed by their parents , and up to 80% were not using any form of contraception . table 2 shows associations of induced abortion with the analyzed variables . induced abortions tended to be more prevalent in the educated ( 16% ) , under 30 years old ( 17% ) , single or never married ( 24% ) , and widowed or divorced ( 57% ) women , compared to the uneducated , the married , and women over the age of 30 years . we also found that induced abortion was more prevalent in women who had had a previous experience of abortion ( 68% ) , in women who did not want the pregnancy ( 37% ) , in women who were experiencing their first pregnancy ( 26% ) , in those with living children ( 24% ) , in women under parents guardianship ( 41% ) , and in christians ( 20% ) . the univariate analysis further showed that all the considered variables , except the use of contraceptives , were associated with induced abortion . therefore , being educated , aged less than 30 years , widowed or divorced , and in a household headed by parents all tended to increase the odds of having an induced abortion . we also found that having a previous experience of abortion or having living children , being on the first pregnancy , or the pregnancy being unwanted increased the odds of having an induced abortion . on the contrary , being married or a muslim tended to decrease the odds of having an induced abortion by 97% and 68% , relative to being single or a christian , respectively . moreover , in the multivariate analysis , we found that three key factors , comprising the desire for pregnancy , living in a household headed by the woman s parents , and the woman s marital status , were all associated with induced abortion ( table 3 ) . the effect of the desire for pregnancy was the most important , with the odds of having an induced abortion being ten times higher for women who did not want the pregnancy compared to those who did want it ( odds ratio [ or ] 10.45 , 95% confidence interval [ ci ] 3.5930.41 ) . further , the odds of having an induced abortion were seven times higher for women living in households headed by their parents ( or 6.83 , 95% ci 2.4219.24 ) . the odds of having an induced abortion also increased by three times for divorced or widowed women ( or 3.47 , 95% ci 1.0811.10 ) compared to single or never - married women . on the contrary , married women were 83% less likely to have an induced abortion , even when the pregnancy was not desired ( or 0.17 , 95% ci 0.030.89 ) , compared to single or never - married women . finally , we found that age , education , previous experience of abortion , number of living children , number of pregnancies , and religion had no significant effect on having an induced abortion . this study reports that 12% of the women who received postabortion care in hospitals had had induced abortions . this proportion was comparable to those found by majlessi et al ( 2008)41 in iran and lema et al ( 1996)28 in kenya , who found that 15.7% and 12% , respectively , of the women they interviewed had had confirmed induced abortions . furthermore , the distribution of the cases indicated that induced abortion is mostly prevalent among young , educated , unmarried women . several other studies also reported the same findings.2831,33,34,3638 it was also prevalent among women with previous abortion experience , among those on their first pregnancy , those who did not want the pregnancy , those with living children , and christians , as well as among those living with their parents . again , these findings were consistent with those from previous studies.30,33,36,40 among the women who sought postabortion care , three key factors the desire for pregnancy ( whether wanted or not ) , the status of the head - of - household ( whether the household was headed by the woman s parents or by either the woman herself or her partner / husband ) , and the woman s marital status this finding was consistent with previous study findings.28,3941 unavailable , unused , or failed contraceptives , as well as males involvement may explain the decision to abort . this high level of unmet need for contraception was consistent with previous research findings in burkina faso24 and in other sub - saharan regions.43 since safe and legal abortions are difficult to get in burkina faso , the only choice left to these women is to go for an illegal abortion whenever they feel that they can not bear their pregnancies . induced abortion was also associated with the status of the head - of - household . this result corroborates that of a previous study that highlighted the parents role in abortion - seeking behavior.36 despite the country s pronatalist mentality , sexuality and pregnancy outside marriage are seen as dishonorable by families , who ultimately may disown daughters who fail to stay virgins until marriage . because of fear of disownment , many women , particularly unmarried women living with their parents , may be more likely to have an induced abortion to end an unwanted pregnancy . such practices are prevalent in many african societies , in which cultural beliefs make it a point of honor to remain chaste until marriage.44 moreover , induced abortion was associated with women s marital status . compared to single or never - married women , divorced or widowed women were more likely to resort to induced abortion . on the contrary , this finding is consistent with several other studies that highlighted the role of marital status in abortion decision making.28,30,34,37,38 the desire not to have any more children may explain the increased likelihood of induced abortion in divorced or widowed women . additionally , because widowed women are still considered as belonging to the deceased husband s family , a new pregnancy may be subject to censure and even the isolation of the woman , particularly if the husband s death is still fresh . we did not find any significant associations between induced abortion and age , education , or the use of contraceptives in the multivariate logistic regression , while various studies have highlighted the critical role of these variables.2831,33,36,37,40,45,46 this may be attributable to the relatively small sample size . in addition , the multivariate modeling may have also adjusted effects between independent variables , eventually removing some correlated variables . this may also explain the failure to find associations between induced abortion and previous abortions , the number of living children , number of pregnancies , and religion . the survey was primarily conducted with the aim of estimating the costs associated with abortions to households and not specifically to explore the determinants of induced abortion . because of this , some possible important variables , such as data on the availability of contraceptives or the women s sexual practices , are missing . its findings may not be generalizable to the whole population of women who have had an induced abortion , as many of them may not present to hospitals for postabortion care . moreover , the study failed to capture information on the male responsible for the pregnancy . they may be a key factor in abortion decision making , considering the roles that men may play as supporters,47,48 instigators , facilitators , collaborators , or advisors.48 unfortunately , the information we collected that was available on men was exclusively that of the official partner or husband of the women . analyzing these data may have led to additional biases : firstly , because information on single women s partners was missing , and secondly because the official partner or husband may not be the father of the pregnancy , as some women may have more than one sexual partner . therefore , other possibly important variables , such as the number of sexual partners of the woman , may be missing . furthermore , the face - to - face interview we conducted with the women may have led to classification biases , as some women who had had an induced abortion may have intentionally reported having had spontaneous abortions.27 this may have consequences for the findings of the study in the sense that misclassification of the cases will decrease the difference in size between the groups of women and therefore affect the odds ratio . some women , particularly the most educated and wealthy , may have sought care in high - standard private clinics rather than the two health facilities in which women were recruited . the comparison group we used was appropriate for the analysis . because a spontaneous abortion may happen to any woman , irrespective of her level of education , wealth , age , etc , it is likely that women falling into this group may be more representative of pregnant women . moreover , this study is the first study to examine the key determinants of induced abortion in burkina faso . its findings may therefore be helpful in the fight against induced abortion and its consequences . finally , it can help in designing a larger , population - based study of the determinants of induced abortions in the whole of burkina faso . the survey was primarily conducted with the aim of estimating the costs associated with abortions to households and not specifically to explore the determinants of induced abortion . because of this , some possible important variables , such as data on the availability of contraceptives or the women s sexual practices , are missing . its findings may not be generalizable to the whole population of women who have had an induced abortion , as many of them may not present to hospitals for postabortion care . moreover , the study failed to capture information on the male responsible for the pregnancy . they may be a key factor in abortion decision making , considering the roles that men may play as supporters,47,48 instigators , facilitators , collaborators , or advisors.48 unfortunately , the information we collected that was available on men was exclusively that of the official partner or husband of the women . analyzing these data may have led to additional biases : firstly , because information on single women s partners was missing , and secondly because the official partner or husband may not be the father of the pregnancy , as some women may have more than one sexual partner . therefore , other possibly important variables , such as the number of sexual partners of the woman , may be missing . furthermore , the face - to - face interview we conducted with the women may have led to classification biases , as some women who had had an induced abortion may have intentionally reported having had spontaneous abortions.27 this may have consequences for the findings of the study in the sense that misclassification of the cases will decrease the difference in size between the groups of women and therefore affect the odds ratio . some women , particularly the most educated and wealthy , may have sought care in high - standard private clinics rather than the two health facilities in which women were recruited . this may also have contributed to distorting the findings . nevertheless , this study has strengths . because a spontaneous abortion may happen to any woman , irrespective of her level of education , wealth , age , etc , it is likely that women falling into this group may be more representative of pregnant women . moreover , this study is the first study to examine the key determinants of induced abortion in burkina faso . its findings may therefore be helpful in the fight against induced abortion and its consequences . finally , it can help in designing a larger , population - based study of the determinants of induced abortions in the whole of burkina faso . this study showed a positive association between induced abortion and unintended pregnancy . in burkina faso , the restrictive abortion law does not prevent women from practicing abortion in cases of unplanned and unwanted pregnancy . this is the case for single women , for women who are divorced or widowed , and women living with their parents , for whom the risk associated with induced abortion is high , especially when the pregnancy is unwanted . this study importantly points to the fact that many women who are sexually active and who do not want to be pregnant do not use any contraception . we believe that improved provision of family planning counseling and methods of contraception , and better availability of contraceptives , may reduce the prevalence of unsafe abortions .

introductiondespite the universal recognition of unsafe abortion as a major public health problem , very little research has been conducted to document its precipitating factors in burkina faso . our aim was to investigate the key determinants of induced abortion in a sample of women who sought postabortion care.materials and methodsa cross - sectional household survey was carried out from february to september 2012 in ouagadougou , burkina faso . data of 37 women who had had an induced abortion and 267 women who had had a spontaneous abortion were prospectively collected on sociodemographic characteristics , pregnancy and birth history , abortion experience , including previous abortion experience , and selected clinical information , including the type of abortion . a two - step regression analysis consisting of a univariate and a multivariate logistic regression was run on stata version 11.2 in order to identify the key determinants of induced abortion.resultsthe findings indicated that 12% of all abortions were certainly induced . three key factors were significantly and positively associated with the probability of having an induced abortion : whether the woman reported that her pregnancy was unwanted ( odds ratio [ or ] 10.45 , 95% confidence interval [ ci ] 3.5930.41 ) ; whether the woman reported was living in a household headed by her parents ( or 6.83 , 95% ci 2.4219.24 ) ; and if the woman reported was divorced or widowed ( or 3.47 , 95% ci 1.0811.10 ) . on the contrary , being married was protective against induced abortion , with women who reported being married having an 83% ( or 0.17 , ci 0.030.89 ) lower chance of having an induced abortion , even when the pregnancy was unwanted.conclusionthis study has identified three major determinants of induced abortion in ouagadougou , burkina faso . improved targeted programs on family planning counseling , methods of contraception , and availability of contraceptives should be widely promoted .

Introduction Materials and methods Study area Study participants and data collection Dependent variable studied Independent variables analyzed Statistical analysis Results Population characteristics Estimation of associations Discussion Limitations and strengths Conclusion

studies have also reported that hospital admissions resulting from unsafe abortions were frequent.810 moreover , it has been demonstrated that these induced abortions are associated with numerous adverse health problems , such as chronic pelvic inflammatory diseases,11 mental disorders,12 subsequent adverse reproductive outcomes,13 and even secondary infertility.14 in burkina faso , abortion is permitted only in cases of incest , rape , fetal defect , or when the woman s life or physical health is endangered.15,16 because of this , access to safe and legal abortion is difficult to get and women often resort to unsafe procedures,15 at great risk to their health and survival . a previous hospital study revealed that up to 30% of maternal deaths have been the result of unsafe induced abortions.17 additionally , it was estimated that each year , more than 21,800 complications from unsafe induced abortions have been treated in the country s hospital facilities.15 however , these data may largely underestimate the magnitude of the phenomenon , as investigations have highlighted that abortion numbers18 and related morbidity and mortality15 tend to be underreported.18 despite the prevalence of induced abortion , estimated to vary between 25 and 40 per 1,000 women aged 1549 years annually,15,19 it is likely that the extent of the problem has not been thoroughly studied . particularly , information on the conditions and key factors that may precipitate induced abortion is lacking . however , knowledge of these factors is critical in designing appropriate strategies aimed at reducing both unwanted pregnancies and unsafe induced abortions.20 in burkina faso , factors associated with induced abortions have not been studied , in spite of their policy relevance . this study aims at contributing to the literature by investigating the key determinants of induced abortion in a sample of women who resorted to health facilities in ouagadougou for postabortion care . burkina faso , a landlocked country located in the heart of west africa , has a weak health care system.21 the country has a population of 16 million , essentially young and fertile.22 the low purchasing power of the population and of women in particular , limits their access to education , clean water , and health care . maternal mortality is high , with a rate of 300 per 100,000 births.23 contraceptive use is low , with large disparities between poor and rich population groups.24 since 2006 , a national subsidy policy for normal deliveries and emergency obstetric care has been active , in order to reduce financial barriers to care and thereby improve access to qualified care.25 in spite of this policy , utilization of health care services in ouagadougou city is still unsatisfactory , with large inequities between poor and rich women . in order to explore the key determinants of induced abortion , this paper uses data from a cross - sectional study that investigates the costs associated with abortions in ouagadougou . these hospital facilities included one referral - level teaching hospital a top referral hospital in ouagadougou to which complications from abortion are directed for better care and one private health clinic , affiliated with the international planned parenthood federation , with long - standing expertise in treating abortions . a total of 307 women with either a spontaneous or induced abortion were sampled for this study . a case was classified as an induced abortion when the clinical ascertainment was confirmed by the woman herself reporting that she had had an induced abortion . this procedure of classifying the cases may have led to some induced abortions being inaccurately classified as spontaneous.3,27 because of this , women were labeled as certainly induced abortion and reportedly spontaneous abortion . the two qualified female interviewers collected data from all the women who had had an induced or a spontaneous abortion , using an interviewer - administered face - to - face questionnaire . the main questionnaire that was administered to the women contained a range of questions pertaining to sociodemographic characteristics , pregnancy and birth history , abortion experience , asset ownership and place of residence , and expenditures on abortion and postabortion care , including prereferral costs . the abortion - experience section of the women s questionnaire included questions pertaining to previous abortion experiences and to the type of abortive method used . this questionnaire included information related to the gestational age of the pregnancy and the clinical ascertainment of the type of abortion . the dependent variable was the type of abortion , a dummy variable set to 1 when the pregnancy termination was reported as induced or 0 when alleged to be a spontaneous abortion . the empirical literature on factors associated with abortion showed that educated women,2832 young women,2830,3336 unmarried women,28,30,34,37,38 women who had had previous experiences of abortion,31,34,39 women who had living children,29,33,34 and women who did not want the pregnancy28,3941 were more likely to have an induced abortion . researchers have also demonstrated that women who were experiencing their first pregnancy in life,30 christian women ( compared to muslim women),33,36,39 and women who did not use contraceptives40 were also more likely to have an induced abortion . finally , we included the status of the household - chief ( whether the household is headed by the woman , the husband , or by the woman s parents ) in the analysis of abortion determinants . a descriptive analysis was undertaken in order to understand the distribution of induced and spontaneous abortions relative to each independent variable . to identify the key determinants associated with induced abortion for women seeking postabortion care in hospitals in ouagadougou , a two - step analysis consisting of one univariate and one multivariate logistic regression was carried out . the univariate logistic regression was run to determine the association between each of the independent variables and the dependent variable . all the variables that were associated with induced abortion in the univariate logistic regression with a level of significance of 0.05 and 95% confidence were subsequently analyzed in a stepwise multivariate logistic regression . to identify the key factors associated with induced abortion , a downward procedure that minimizes the number of variables while maximizing the accuracy of the model was followed.42 all analyses were conducted on stata version 11.2 ( statacorp , college station , tx , usa ) . burkina faso , a landlocked country located in the heart of west africa , has a weak health care system.21 the country has a population of 16 million , essentially young and fertile.22 the low purchasing power of the population and of women in particular , limits their access to education , clean water , and health care . maternal mortality is high , with a rate of 300 per 100,000 births.23 contraceptive use is low , with large disparities between poor and rich population groups.24 since 2006 , a national subsidy policy for normal deliveries and emergency obstetric care has been active , in order to reduce financial barriers to care and thereby improve access to qualified care.25 in spite of this policy , utilization of health care services in ouagadougou city is still unsatisfactory , with large inequities between poor and rich women . in order to explore the key determinants of induced abortion , this paper uses data from a cross - sectional study that investigates the costs associated with abortions in ouagadougou . a total of 307 women with either a spontaneous or induced abortion were sampled for this study . a case was classified as an induced abortion when the clinical ascertainment was confirmed by the woman herself reporting that she had had an induced abortion . this procedure of classifying the cases may have led to some induced abortions being inaccurately classified as spontaneous.3,27 because of this , women were labeled as certainly induced abortion and reportedly spontaneous abortion . the two qualified female interviewers collected data from all the women who had had an induced or a spontaneous abortion , using an interviewer - administered face - to - face questionnaire . the main questionnaire that was administered to the women contained a range of questions pertaining to sociodemographic characteristics , pregnancy and birth history , abortion experience , asset ownership and place of residence , and expenditures on abortion and postabortion care , including prereferral costs . the abortion - experience section of the women s questionnaire included questions pertaining to previous abortion experiences and to the type of abortive method used . this questionnaire included information related to the gestational age of the pregnancy and the clinical ascertainment of the type of abortion . the dependent variable was the type of abortion , a dummy variable set to 1 when the pregnancy termination was reported as induced or 0 when alleged to be a spontaneous abortion . the empirical literature on factors associated with abortion showed that educated women,2832 young women,2830,3336 unmarried women,28,30,34,37,38 women who had had previous experiences of abortion,31,34,39 women who had living children,29,33,34 and women who did not want the pregnancy28,3941 were more likely to have an induced abortion . therefore , such variables as age , education , marital status , number of children , desire for pregnancy , and previous experience of abortion were considered independent variables . researchers have also demonstrated that women who were experiencing their first pregnancy in life,30 christian women ( compared to muslim women),33,36,39 and women who did not use contraceptives40 were also more likely to have an induced abortion . finally , we included the status of the household - chief ( whether the household is headed by the woman , the husband , or by the woman s parents ) in the analysis of abortion determinants . a descriptive analysis was undertaken in order to understand the distribution of induced and spontaneous abortions relative to each independent variable . to identify the key determinants associated with induced abortion for women seeking postabortion care in hospitals in ouagadougou , a two - step analysis consisting of one univariate and one multivariate logistic regression was carried out . the univariate logistic regression was run to determine the association between each of the independent variables and the dependent variable . all the variables that were associated with induced abortion in the univariate logistic regression with a level of significance of 0.05 and 95% confidence were subsequently analyzed in a stepwise multivariate logistic regression . to identify the key factors associated with induced abortion , a downward procedure that minimizes the number of variables while maximizing the accuracy of the model was followed.42 all analyses were conducted on stata version 11.2 ( statacorp , college station , tx , usa ) . women who had had an induced abortion constituted 12% of the sample ; 61% of the women were married , and 63% were under 30 years old . among the women , 28% declared that their pregnancies were unwanted , and 17% had had a previous experience of abortion , while 29% were on their first pregnancy , 77% were living in households headed by their parents , and up to 80% were not using any form of contraception . we also found that induced abortion was more prevalent in women who had had a previous experience of abortion ( 68% ) , in women who did not want the pregnancy ( 37% ) , in women who were experiencing their first pregnancy ( 26% ) , in those with living children ( 24% ) , in women under parents guardianship ( 41% ) , and in christians ( 20% ) . therefore , being educated , aged less than 30 years , widowed or divorced , and in a household headed by parents all tended to increase the odds of having an induced abortion . we also found that having a previous experience of abortion or having living children , being on the first pregnancy , or the pregnancy being unwanted increased the odds of having an induced abortion . on the contrary , being married or a muslim tended to decrease the odds of having an induced abortion by 97% and 68% , relative to being single or a christian , respectively . moreover , in the multivariate analysis , we found that three key factors , comprising the desire for pregnancy , living in a household headed by the woman s parents , and the woman s marital status , were all associated with induced abortion ( table 3 ) . the effect of the desire for pregnancy was the most important , with the odds of having an induced abortion being ten times higher for women who did not want the pregnancy compared to those who did want it ( odds ratio [ or ] 10.45 , 95% confidence interval [ ci ] 3.5930.41 ) . further , the odds of having an induced abortion were seven times higher for women living in households headed by their parents ( or 6.83 , 95% ci 2.4219.24 ) . the odds of having an induced abortion also increased by three times for divorced or widowed women ( or 3.47 , 95% ci 1.0811.10 ) compared to single or never - married women . on the contrary , married women were 83% less likely to have an induced abortion , even when the pregnancy was not desired ( or 0.17 , 95% ci 0.030.89 ) , compared to single or never - married women . finally , we found that age , education , previous experience of abortion , number of living children , number of pregnancies , and religion had no significant effect on having an induced abortion . women who had had an induced abortion constituted 12% of the sample ; 61% of the women were married , and 63% were under 30 years old . among the women , 28% declared that their pregnancies were unwanted , and 17% had had a previous experience of abortion , while 29% were on their first pregnancy , 77% were living in households headed by their parents , and up to 80% were not using any form of contraception . we also found that induced abortion was more prevalent in women who had had a previous experience of abortion ( 68% ) , in women who did not want the pregnancy ( 37% ) , in women who were experiencing their first pregnancy ( 26% ) , in those with living children ( 24% ) , in women under parents guardianship ( 41% ) , and in christians ( 20% ) . therefore , being educated , aged less than 30 years , widowed or divorced , and in a household headed by parents all tended to increase the odds of having an induced abortion . we also found that having a previous experience of abortion or having living children , being on the first pregnancy , or the pregnancy being unwanted increased the odds of having an induced abortion . on the contrary , being married or a muslim tended to decrease the odds of having an induced abortion by 97% and 68% , relative to being single or a christian , respectively . moreover , in the multivariate analysis , we found that three key factors , comprising the desire for pregnancy , living in a household headed by the woman s parents , and the woman s marital status , were all associated with induced abortion ( table 3 ) . the effect of the desire for pregnancy was the most important , with the odds of having an induced abortion being ten times higher for women who did not want the pregnancy compared to those who did want it ( odds ratio [ or ] 10.45 , 95% confidence interval [ ci ] 3.5930.41 ) . further , the odds of having an induced abortion were seven times higher for women living in households headed by their parents ( or 6.83 , 95% ci 2.4219.24 ) . the odds of having an induced abortion also increased by three times for divorced or widowed women ( or 3.47 , 95% ci 1.0811.10 ) compared to single or never - married women . on the contrary , married women were 83% less likely to have an induced abortion , even when the pregnancy was not desired ( or 0.17 , 95% ci 0.030.89 ) , compared to single or never - married women . finally , we found that age , education , previous experience of abortion , number of living children , number of pregnancies , and religion had no significant effect on having an induced abortion . this study reports that 12% of the women who received postabortion care in hospitals had had induced abortions . several other studies also reported the same findings.2831,33,34,3638 it was also prevalent among women with previous abortion experience , among those on their first pregnancy , those who did not want the pregnancy , those with living children , and christians , as well as among those living with their parents . again , these findings were consistent with those from previous studies.30,33,36,40 among the women who sought postabortion care , three key factors the desire for pregnancy ( whether wanted or not ) , the status of the head - of - household ( whether the household was headed by the woman s parents or by either the woman herself or her partner / husband ) , and the woman s marital status this finding was consistent with previous study findings.28,3941 unavailable , unused , or failed contraceptives , as well as males involvement may explain the decision to abort . induced abortion was also associated with the status of the head - of - household . compared to single or never - married women , divorced or widowed women were more likely to resort to induced abortion . on the contrary , this finding is consistent with several other studies that highlighted the role of marital status in abortion decision making.28,30,34,37,38 the desire not to have any more children may explain the increased likelihood of induced abortion in divorced or widowed women . we did not find any significant associations between induced abortion and age , education , or the use of contraceptives in the multivariate logistic regression , while various studies have highlighted the critical role of these variables.2831,33,36,37,40,45,46 this may be attributable to the relatively small sample size . the survey was primarily conducted with the aim of estimating the costs associated with abortions to households and not specifically to explore the determinants of induced abortion . because of this , some possible important variables , such as data on the availability of contraceptives or the women s sexual practices , are missing . its findings may not be generalizable to the whole population of women who have had an induced abortion , as many of them may not present to hospitals for postabortion care . furthermore , the face - to - face interview we conducted with the women may have led to classification biases , as some women who had had an induced abortion may have intentionally reported having had spontaneous abortions.27 this may have consequences for the findings of the study in the sense that misclassification of the cases will decrease the difference in size between the groups of women and therefore affect the odds ratio . moreover , this study is the first study to examine the key determinants of induced abortion in burkina faso . the survey was primarily conducted with the aim of estimating the costs associated with abortions to households and not specifically to explore the determinants of induced abortion . because of this , some possible important variables , such as data on the availability of contraceptives or the women s sexual practices , are missing . its findings may not be generalizable to the whole population of women who have had an induced abortion , as many of them may not present to hospitals for postabortion care . furthermore , the face - to - face interview we conducted with the women may have led to classification biases , as some women who had had an induced abortion may have intentionally reported having had spontaneous abortions.27 this may have consequences for the findings of the study in the sense that misclassification of the cases will decrease the difference in size between the groups of women and therefore affect the odds ratio . moreover , this study is the first study to examine the key determinants of induced abortion in burkina faso . in burkina faso , the restrictive abortion law does not prevent women from practicing abortion in cases of unplanned and unwanted pregnancy . this is the case for single women , for women who are divorced or widowed , and women living with their parents , for whom the risk associated with induced abortion is high , especially when the pregnancy is unwanted . we believe that improved provision of family planning counseling and methods of contraception , and better availability of contraceptives , may reduce the prevalence of unsafe abortions .

microglia are the resident innate immune cells of the central nervous system ( cns ) and play an important role in immune surveillance and in the pathogenesis and progression of a number of cns disorders . microglia are constantly mobile , spending time scanning the extracellular space of the cns . in response to brain injury or immunological stimuli , these cells become activated and undergo dramatic morphological and functional changes , which are highly dependent on the context of their activation . activated microglia phagocytose debris and peptides , present antigens , and produce a number of soluble factors . these factors may be inflammatory , regulatory , or cytotoxic in nature and include reactive oxygen species ( ros ) , nitric oxide ( no ) , proinflammatory and anti - inflammatory cytokines , prostaglandins , and growth factors [ 4 , 5 ] . microglia can be both neuroprotective or neurotoxic when activated , depending on the factors they produce and the quantity and context in which they are released , with prolonged or excessive activation of these cells being associated with neuroinflammation and the progression of a number of cns disorders . the mechanisms behind enhanced microglial activation in these disorders and the features determining the balance between neuroprotection and neurotoxicity are not fully understood . the p2x7 receptor is a trimeric ligand - gated cation channel belonging to the p2x family of purinergic receptors . p2x7 is predominately expressed on mononuclear leukocytes including macrophages and microglia and plays a role in inflammation and immunity . in particular activation of p2x7 by extracellular adenosine-5-triphosphate ( atp ) , or the most potent p2x7 agonist , 2(3)-o-(4-benzoylbenzoyl ) atp ( bzatp ) , causes the passage of small cations including ca , na , and k across the plasma membrane , as well as organic cations , such as the fluorescent dyes ethidium and yo - pro-1 . compared to other p2x receptors , p2x7 requires relatively high atp concentrations for activation , with a half maximal effective concentration ( ec50 ) of 100300 m . activation of p2x7 leads to a number of cell - specific downstream signalling events , including the formation of ros and reactive nitrogen species , and either cell proliferation or death [ 11 , 12 ] . using molecular , immunochemical , and pharmacological techniques , we demonstrate in the current study that the murine microglial eoc13 cell line expresses functional p2x7 . activation of p2x7 by atp in this cell line induces the uptake of organic cations , ros formation , and cell death . rpmi-1640 and dmem / f12 media , glutamax , normal horse serum ( nhs ) , 0.05% trypsin , yo - pro-1 , 2,7-dichlorodihydrofluorescein diacetate ( h2dcfda ) , and 2,7-difluorofluorescein diacetate ( daf - fm da ) were from invitrogen ( grand island , ny ) . fetal bovine serum ( fbs ) ( heat - inactivated before use ) was from bovogen biologicals ( east keilor , australia ) . atp , bzatp , ethidium bromide , dimethyl sulfoxide ( dmso ) , glycerol gelatin , and the p2x7 antagonist brilliant blue g ( bbg ) were from sigma - aldrich ( st . louis , mo ) . the p2x7 antagonists a438079 , az10606120 , and az11645373 were from tocris bioscience ( ellisville , mo ) . protease inhibitor cocktail tablets ( complete , mini , edta - free ) and annexin - v - fluorescein were from roche diagnostics ( penzberg , germany ) . the viability dye 7-aminoactinomycin d ( 7aad ) and the ros scavenger n - acetyl - l - cysteine ( nac ) were from enzo life sciences ( plymouth meeting , pa ) . the broad - spectrum ros inhibitor diphenyleneiodonium ( dpi ) was from cayman chemical ( ann arbor , mi ) . phenyl - methyl - sulfonyl - fluoride ( pmsf ) , n - dodecyl -d - maltoside , and ethylene glycol tetraacetic acid ( egta ) were from amresco ( solon , oh ) . cells preincubated with h2o soluble compounds ( bbg , a438079 , and az10606120 ) were compared to cells preincubated in the absence of each compound . cells preincubated with dmso soluble compounds ( az11645373 and dpi ) were compared to cells preincubated with dmso alone . solutions containing 40 mm nac were prepared in nacl medium ( 140 mm nacl , 5 mm naoh , 5 mm kcl , 10 mm hepes , and 5 mm glucose , ph 7.4 ) and adjusted to ph 7.4 ; cells were then preincubated in nacl medium with or without nac . rabbit anti - mouse p2x7 ( extracellular epitope ) polyclonal antibody ( ab ) and rabbit anti - rat p2x7 ( c - termini epitope ) ab ( and corresponding blocking peptide ) were from alomone labs ( jerusalem , israel ) . peroxidase - conjugated goat anti - rabbit igg ab was from rockland immunochemicals ( gilbertsville , pa ) . cy3-conjugated donkey anti - rabbit igg ab was from jackson immunoresearch ( west grove , pa ) . rat anti - mouse p2x7 monoclonal antibody ( mab ) ( clone hano43 ) was from enzo life sciences . rat igg2b isotype control mab and allophycocyanin- ( apc- ) conjugated donkey anti - rat igg ab were from ebioscience ( san diego , ca ) . the murine macrophage j774 cell line , the murine microglial eoc13 cell line , and the murine lymphoblast ladmac cell line , all originally obtained from the american type culture collection ( manassas , va ) , were kindly provided by jasmyn dunn ( university of queensland , brisbane , australia ) ( j774 ) and iain campbell ( university of sydney , sydney , australia ) ( eoc13 and ladmac ) . j774 cells were maintained in rpmi-1640 medium containing 10% fbs and 2 mm glutamax ( complete rpmi medium ) . eoc13 cells were maintained in dmem / f12 supplemented with 10% fbs , 2 mm glutamax , and 20% ladmac conditioned medium ( complete dmem medium ) . cell lines were maintained at 37c and 95% air/5% co2 and passaged every 3 - 4 days . quarterly mycoplasma testing was carried out using the mycoalert mycoplasma detection kit ( lonza , rockland , me ) , as per the manufacturer 's instructions . for experiments , cells were washed in nacl medium ( 300 g for 5 min ) , resuspended in nacl medium , and equilibrated at 37c for 5 min ( 1 10 cells/1 ml / tube ) . cells were then incubated with 25 m ethidium ( or 1 m yo - pro-1 where indicated ) in the absence or presence of the p2x7 agonists atp or bzatp ( as indicated ) for 5 min . in some experiments , atp - induced cation uptake was assessed with cells suspended in kcl medium ( 150 mm kcl , 5 mm glucose , and 10 mm hepes , ph 7.4 ) or in nacl medium containing 1 mm cacl2 or 100 m egta . in other experiments , cells were preincubated in the absence or presence of p2x7 antagonists or the ros scavenger nac ( as indicated ) for 15 and 30 min , respectively , prior to cation and atp addition . incubations with nucleotides were stopped by the addition of an equal volume of ice - cold nacl medium containing 20 mm mgcl2 ( mgcl2 medium ) followed by centrifugation ( 300 g for 5 min ) . cells were washed once with nacl medium and events collected using a lsr ii flow cytometer ( bd biosciences , san diego , ca ) ( excitation 488 nm , emission collected with 575/26 and 515/20 band - pass filters for ethidium and yo - pro-1 , resp . ) . the mean fluorescence intensity ( mfi ) of relative cation uptake total rna isolation from cells was performed using the rneasy mini kit ( qiagen , hilden , germany ) as per the manufacturer 's instructions . pcr amplification was performed as described previously using superscript iii one - step rt - pcr system platinum taq dna polymerase ( invitrogen ) with 500 ng of rna , and p2x7 forward ( 5-atatccacttccccggccac-3 ) and reverse ( 5-tcggcagtgatgggaccag-3 ) primers for 42 cycles ( 94c , 1 min ; 68c , 1 min ; 72c , 1 min ) . pcr products were separated on a 2% agarose gel in tris - acetate edta buffer and visualised with ethidium bromide staining . images of gels were collected using a gel logic 212 pro imaging system ( carestream health , rochester , ny ) . cells were washed three times with phosphate - buffered saline ( pbs ) ( 300 g for 5 min ) and lysed ( 1 10 cells / ml ) over 60 min in ice - cold lysis buffer ( 50 mm bistris , 750 mm 6-aminohexanoic acid , 1% n - dodecyl -d - maltoside , 1 mm pmsf , and protease inhibitor cocktail , ph 7.0 ) . cells were sheared by passing ten times through a 21 g needle and stored at 20c until needed . cells were then thawed and cleared ( 16,000 g at 4c for 10 min ) . supernatants ( 25 g protein / lane ) were separated under reducing conditions ( 5% -mercaptoethanol ) using a discontinuous sds - page system with a 4% stacking gel and 10% separating gel . proteins were then transferred to nitrocellulose membranes ( bio - rad , hercules , ca ) and blocked at 4c overnight with tris - buffered saline ( 250 mm nacl and 50 mm tris , ph 7.5 ) containing 0.2% tween-20 and 5% milk powder . the following day , nitrocellulose membranes were incubated at room temperature for 2 h with anti - mouse p2x7 ab ( 1 : 500 ) in tris - buffered saline containing 0.2% tween-20 and 5% milk powder . membranes were washed three times over 30 min with tris - buffered saline containing 0.2% tween-20 and then incubated at room temperature for 1 h with peroxidise - conjugated anti - igg ab ( 1 : 1000 ) in tris - buffered saline containing 0.2% tween-20 and 5% milk powder . membranes were washed as above and visualised using chemiluminescent substrate and amersham hyperfilm ecl ( ge healthcare , little chalfont , buckinghamshire , uk ) . images of films were collected using a gs-800 calibrated densitometer ( bio - rad ) . cells in nacl medium containing 10% nhs and 0.02% nan3 ( 1 10 cells/200 l / tube ) were incubated with anti - p2x7 or rat igg2b isotype control mab ( 5 g / ml ) at room temperature for 30 min . cells were then washed twice with nacl medium ( 300 g for 5 min ) and incubated with apc - conjugated anti - rat igg ab ( 1.3 g / ml ) and 7aad ( to exclude dead cells ) for 30 min protected from light . events were then collected using a lsr ii flow cytometer ( excitation 633 nm , emission collected with 660/20 band - pass filter for apc ; excitation 488 nm , emission collected with 695/40 band - pass filter for 7aad ) . relative cell - surface p2x7 was determined using flowjo software and is expressed as the difference in the mfi of specific mab labelling and isotype control labelling . eoc13 or j774 cells in their respective complete culture medium were plated into 24-well plates with 13 mm glass coverslips ( 5 10 cells/0.5 ml / well ) and incubated at 37c , 95% air/5% co2 overnight to allow time to adhere . the following day , cells were fixed with 4% paraformaldehyde in pbs at room temperature for 15 min and then washed three times with pbs over 10 min . cells were incubated with permeabilisation solution ( pbs containing 0.1% dmso , 2% nhs , and 0.1% triton x-100 ) at room temperature for 10 min and washed three times with pbs . cells were then blocked with 20% nhs in pbs at room temperature for 20 min . cells were incubated at 4c overnight with anti - rat p2x7 ab ( 5 g / ml ; preincubated for 1 h in the absence or presence of blocking peptide as per the manufacturer 's instruction ) in pbs containing 1% bsa , 0.2% nhs , and 0.05% nan3 . cells were then washed as above and incubated at room temperature for 1 h with cy3-conjugated anti - rabbit igg ab ( 15 g / ml ) in pbs containing 0.2% nhs . cells were washed as above and then the coverslips mounted onto slides with 50% ( v / v ) glycerol gelatin in pbs . cells were visualised using a dm ibre inverted microscope and tcs sp confocal imaging system ( leica , mannheim , germany ) ( excitation 488 nm , emission collected at 560600 nm ) . eoc13 cells in complete dmem medium were plated into 24-well plates ( 5 10 cells/0.5 ml / well ) and incubated at 37c , cells were then incubated with nacl medium containing 10 m h2dcfda ( 0.5 ml / well ) at 37c , 95% air/5% co2 , protected from light for 30 min . the medium was removed , and cells were further incubated in nacl medium ( containing 1 mm cacl2 ) in the absence or presence of 2 mm atp at 37c , 95% air/5% co2 for 15 min . incubations were stopped by the addition of an equal volume of ice - cold mgcl2 medium . cells were harvested using 0.05% trypsin ( 5 min , 37c ) and were washed once with nacl medium . events were collected using a lsr ii flow cytometer ( excitation 488 nm , emission collected at 515/20 nm ) and the mfi of relative dichlorofluorescein ( dcf ) determined using flowjo software . in some experiments , atp - induced ros formation was assessed in kcl medium , in nacl medium in the absence of 1 mm cacl2 or presence of 100 m egta , or in complete dmem medium in the absence or presence of 10 m az10606120 ( 15 min preincubation , prior to atp addition ) . as free ca lowers the concentration of atp , cells incubated in the absence of 1 mm ca were incubated with 1.4 mm atp to provide equimolar atp concentrations ( 575 m ) , as calculated using the bound and determined program . in other experiments , cells were preincubated in the absence or presence of az10606120 , nac , or dpi ( as indicated ) for 15 , 30 , and 30 min , respectively , prior to atp addition . cells prior to harvesting were also visualised by differential interference contrast ( dic ) imaging using an eclipse te2000 inverted microscope ( nikon , tokyo , japan ) to examine cell morphology , and dic images were captured using image - pro ams ( version 6.1 ) ( media cybernetics , rockville , md ) . eoc13 cells in complete dmem medium were plated into 24-well plates ( 5 10 cells/0.5 ml / well ) and incubated at 37c , 95% air/5% co2 overnight . cells were then incubated with nacl medium containing 10 m daf - fm da ( 0.5 ml / well ) at 37c , 95% air/5% co2 , protected from light for 30 min . cells were then preincubated with nacl medium in the absence or presence of 10 m az10606120 at 37c , 95% air/5% co2 for 15 min . following this , cells were further incubated in the absence or presence of 1.4 mm atp for 15 min . incubations were stopped by the addition of an equal volume of ice - cold mgcl2 medium . cells were harvested using 0.05% trypsin ( 5 min , 37c ) and were washed once with nacl medium . events were collected using a lsr ii flow cytometer ( excitation 488 nm , emission collected at 515/20 nm ) and the mfi of relative benzotriazole derivative determined using flowjo software . eoc13 cells in complete dmem medium were plated into 24-well plates ( 5 10 cells/0.5 ml / well ) and incubated at 37c , 95% air/5% co2 overnight . cells were then incubated with filter sterile atp ( as indicated ) at 37c , 5% co2 for 24 h. in some experiments , cells were preincubated in the absence or presence of 10 m az10606120 or 40 mm nac for 15 or 30 min , respectively , prior to atp addition . in other experiments , cells were preincubated in the absence or presence of 40 mm nac for 90 min , with 2 mm atp added in the final 1560 min , and then the medium replaced and cells incubated at 37c , 95% air/5% co2 for a further 24 h. following the 24 h incubations , cells were harvested from wells using 0.05% trypsin ( 5 min , 37c ) and washed once with annexin - v binding medium ( nacl medium containing 5 mm cacl2 ) . cells were then incubated with annexin - v binding medium containing annexin - v - fluorescein and 7aad at room temperature protected from light for 15 min . events were collected using a lsr ii flow cytometer ( excitation 488 nm , emission collected with 515/20 and 695/40 band - pass filters for annexin - v - fluorescein and 7aad , resp . ) and the mfi of annexin - v - fluorescein and 7aad determined using flowjo software . quadrant markers were used to determine the percentage of annexin - v/7aad , annexin - v/7aad , and annexin - v/7aad cells . in some experiments , cells prior to harvesting were visualised by dic imaging to examine cell morphology , and dic images captured as outlined in section 2.8 . differences between multiple treatments were compared by anova paired with tukey 's hsd posttest using prism 5 for windows ( version 5.04 ) ( graphpad software , san diego , ca ) , with differences considered significant for p < 0.05 . concentration response and inhibition curves were fitted using prism 5 and assuming a variable slope , with normalised and nonnormalised response curves , respectively , selected to obtain the best fit . estimates of ec50 values and half maximal inhibitory concentrations ( ic50 ) were obtained from individual fits of these plots . the murine macrophage j774 cell line is well known to express functional p2x7 . moreover , our group has demonstrated the presence of functional p2x7 in various cell types using a fixed - time fluorescent cation uptake assay ( e.g. , [ 14 , 18 ] ) . therefore , this technique was used to confirm the presence of p2x7 in j774 cells and to validate the use of this cell line as a positive control . incubation of j774 cells with the p2x7 agonist atp and the most potent p2x7 agonist bzatp induced significant ethidium uptake into these cells compared to cells incubated in the absence of nucleotide ( figure 1(a ) ) . in addition , incubation of j774 cells with atp induced significant yo - pro-1 uptake compared to cells incubated in the absence of atp ( figure 1(b ) ) . however , atp - induced yo - pro-1 uptake was significantly lower than atp - induced ethidium uptake ( figure 1(b ) ) . a number of highly specific p2x7 antagonists , including a438079 , az10606120 , and az11645373 , have recently become available . in addition , bbg is commonly used as a p2x7 antagonist in vitro and in vivo ( e.g. , [ 22 , 23 ] ) . therefore , to determine the optimum concentrations of these antagonists required to inhibit murine p2x7 , j774 cells were preincubated in the absence or presence of varying concentrations of bbg , a438079 , az10606120 , and az11645373 and the atp - induced ethidium uptake assessed . each antagonist impaired 1 mm atp - induced ethidium uptake in a concentration - dependent manner , with ic50 values of 1.8 0.2 , 7.9 0.4 , 1.0 0.1 , and 1.5 0.1 m , respectively ( figure 1(c ) ) . az10606120 and a438079 completely inhibited ethidium uptake at respective concentrations of 10 and 100 m . in contrast , bbg and az11645373 were partial antagonists at the atp concentration used ( 1 mm ) . to determine whether eoc13 microglial cells express p2x7 , a series of experiments using j774 cells as a positive control were performed . firstly , rna was isolated from eoc13 and j774 cells and amplified by rt - pcr using primers for p2x7 . similar to j774 cells , eoc13 cells were found to express p2x7 mrna , as evident from the 230 base pair band corresponding to the size of the predicted product ( figure 2(a ) ) . the presence of total p2x7 protein was determined by probing separated whole lysates of both cell lines with an anti - p2x7 ab . immunoblotting revealed one major protein band of 75 kda for both eoc13 and j774 cells ( figure 2(b ) ) , corresponding to the predicted size of glycosylated p2x7 . moreover , both cell lines were incubated with an anti - p2x7 mab and the presence of cell - surface p2x7 determined by flow cytometry . immunolabelling demonstrated cell - surface p2x7 on both eoc13 and j774 cells , with mfis of 13 2 and 14 4 , respectively ( n = 3 ) ( figure 2(c ) ) . finally , both cell lines were stained with an anti - p2x7 ab and analysed by confocal microscopy . this similarly demonstrated the presence of cell - surface p2x7 , as well as intracellular p2x7 , with bright staining observed on all cells ( figure 2(d ) ) . preincubation of the anti - p2x7 ab with blocking peptide completely abrogated the detection of p2x7 in both cell lines ( data not shown ) . together , these results indicate that p2x7 is expressed in eoc13 cells . to determine whether p2x7 was functional in eoc13 cells , the fixed - time ethidium uptake assay was performed . both atp and bzatp were found to induce significant ethidium uptake into eoc13 cells compared to cells incubated in the absence of nucleotide ( figure 3(a ) ) . atp induced ethidium uptake in a concentration - dependent manner , with maximal uptake obtained at an atp concentration of 1 mm and with an ec50 of 130 30 m ( figure 3(b ) ) . subsequent characterisations of p2x7 in eoc13 microglia were performed using this maximal concentration of atp ( 1 mm ) . to determine if the observed atp - induced ethidium uptake into eoc13 cells was mediated by p2x7 , cells were preincubated in the absence or presence of p2x7 antagonists at inhibitory concentrations optimal for 1 mm atp - induced ethidium uptake in j774 cells , as demonstrated above ( figure 1(c ) ) . preincubation of eoc13 cells with 30 m bbg , 100 m a438079 , 10 m az10606120 , and 30 m az11645373 resulted in significant impairment of atp - induced ethidium uptake by 75 2 , 90 1 , 100 0 , and 99 1% , respectively ( figure 3(c ) ) . none of the p2x7 antagonists except az11645373 significantly altered the basal ethidium uptake into eoc13 cells . again with the exception of az11645373 , which reduced the amount of gated viable cells by ~30% , cell viability ( as assessed by forward and side scatter ) was similar between treatments ( data not shown ) . to determine if p2x7 activation could induce the uptake of a second organic cation into eoc13 cells , cells were preincubated in the absence or presence of az10606120 , and atp - induced yo - pro-1 uptake examined . similar to ethidium uptake , 1 mm atp induced significant yo - pro-1 uptake into eoc13 cells compared to cells incubated in the absence of atp ( figure 3(d ) ) . moreover , preincubation of cells with 10 m az10606120 resulted in complete inhibition of atp - induced yo - pro-1 uptake ( figure 3(d ) ) . incubation with az10606120 did not significantly alter the basal yo - pro-1 uptake ( figure 3(d ) ) . furthermore , cell viability ( as assessed by forward and side scatter ) was similar between treatments ( data not shown ) . p2x7 activation has been reported to induce ros formation in a number of cell types , including primary microglia [ 24 , 25 ] . thus , atp - induced ros formation in the eoc13 cell line was investigated using the ros sensitive probe dcf . cells loaded with h2dcfda ( which is converted to dcf inside cells ) were incubated in the absence or presence of atp , and the subsequent ros formation analysed by flow cytometry . as extracellular ca has been reported to be important for p2x7-induced ros formation in a number of cell types [ 24 , 26 , 27 ] , assays were initially conducted in the presence of 1 mm ca . however , due to the inhibitory action of ca on p2x7 , assays were initially conducted with 2 mm atp . incubation with 2 mm atp induced significant ros formation in eoc13 cells compared to cells incubated in the absence of atp ( mfi of ros formation 16.3 0.6 and 5.18 0.06 , resp . furthermore , preincubation of cells with 10 m az10606120 resulted in complete inhibition of atp - induced ros formation ( figure 4(a ) ) , indicating that this process is mediated by p2x7 activation . as for cation uptake ( figure 3 ) , az10606120 did not significantly alter the basal ros formation ( figure 4(a ) ) or cell viability ( as assessed by forward and side scatter ) ( data not shown ) . p2x7 is a ligand - gated cation channel ; therefore the possible roles for cation fluxes in p2x7-induced ros formation were next investigated . p2x7-induced ros formation has been reported to be partially dependent on ca influx in human promyelocytes and rat submandibular gland cells . thus , to determine if ca influx is required for p2x7-mediated ros formation in eoc13 cells , atp - induced ros formation in the absence and presence of ca was investigated . for this comparison , equivalent amounts of atp ( 575 m ) were used by adding 1.4 or 2 mm atp to nacl medium nominally free of ca or containing 1 mm ca , respectively . atp induced significant ros formation in both the absence and presence of 1 mm ca compared to similarly treated cells in the absence of atp ( figure 4(b ) ) . cells incubated in the absence of ca had significantly higher atp - induced ros formation compared to those incubated in the presence of ca . in contrast , atp - induced ethidium uptake ( p2x7 function ) was similar in cells incubated in the absence or presence of ca ( figure 4(b ) ) , indicating that the differences in atp - induced ros formation were not due to altered p2x7 function . thus , to further exclude a role for ca in p2x7-mediated ros formation in eoc13 cells , atp - induced ros formation was investigated in the absence and presence of the ca chelator egta . incubation with 1.4 mm atp induced significant but similar amounts of ros formation in both the absence and presence of 100 m egta compared to similarly treated cells in the absence of atp ( figure 4(c ) ) . again , atp - induced ethidium uptake was similar in cells incubated in the absence or presence of egta ( figure 4(c ) ) . finally , the role of k in p2x7-induced ros formation in eoc13 cells was investigated . both ros and k efflux have been reported to be involved in interleukin-1 ( il-1 ) release from monocytes , although whether these downstream processes are linked is yet to be established . thus , to determine if k efflux is involved in p2x7-mediated ros formation in eoc13 cells , atp - induced ros formation was compared with cells in nacl medium and kcl medium , which prevents the loss of intracellular k. incubation with 1.4 mm atp induced significant ros formation in both nacl and kcl media , with similar levels of ros formation in both media ( figure 4(d ) ) . likewise , atp - induced ethidium uptake was similar in nacl and kcl media ( figure 4(d ) ) , indicating that the inability of high extracellular k to impair atp - induced ros formation was not due to altered p2x7 function . to confirm that p2x7 activation induced ros formation in eoc13 microglia , dcf - loaded cells in nacl medium were preincubated in the absence or presence of the ros scavenger nac , before incubation in the absence or presence of atp . as above ( figure 4 ) , 1.4 mm atp induced significant ros formation ( figure 5(a ) ) . preincubation with 40 mm nac inhibited atp - induced ros formation by 73.7 0.3% ( figure 5(a ) ) . basal ros formation ( figure 5(a ) ) and cell viability ( as assessed by forward and side scatter ) ( data not shown ) were similar between treatments . preincubation of cells with 40 mm nac inhibited atp - induced ethidium uptake by 30 2% ( figure 5(b ) ) . thus , the inhibitory effect of nac on p2x7-induced ros formation may be partially attributed to inhibition of p2x7 itself . however , incubation with nac and atp , but not either compound alone , reduced the amount of gated viable cells by ~40% in the ethidium uptake assay ( as assessed by forward and side scatter ) ( data not shown ) . this suggests that the inhibitory action of nac on atp - induced ethidium uptake may be a result of cytotoxicity in this assay . to confirm that nac did not induce morphological changes or cause cytotoxicity under the conditions used for the ros assay , dic images of adherent cells were acquired following incubation in the absence or presence of atp . cells incubated in the absence or presence of nac ( without atp ) displayed discrete cell bodies with long , spindled shaped processes ( figure 5(c ) ) , as previously observed . cells incubated in the absence or presence of nac ( with atp ) also displayed discrete cell bodies , but with retracted and branched processes ( figure 5(c ) ) , typical of atp causing membrane changes . therefore , in the ros assay , eoc13 cell morphology was not altered by nac when compared to the corresponding treatment . dcf - loaded cells were also preincubated in the absence or presence of the broad - spectrum ros inhibitor dpi and the atp - induced ros formation investigated . however , a 30 min preincubation with dpi at various concentrations ( 540 m ) led to high amounts of cell death ( data not shown ) , and thus this compound was not examined further . to further verify that p2x7 activation induces the formation of reactive species in eoc13 cells , atp - induced no formation was investigated using the no sensitive probe daf - fm da . cells loaded with daf - fm da ( which reacts with no to form a fluorescent benzotriazole ) were preincubated in the absence or presence of az10606120 , followed by incubation in the absence or presence of atp , and the subsequent no formation analysed by flow cytometry . incubation with 1.4 mm atp induced significant no formation in eoc13 cells compared to cells incubated in the absence of atp ( figure 6 ) . furthermore , preincubation of cells with 10 m az10606120 inhibited atp - induced no formation by 82 11% ( figure 6 ) , indicating that this process is mediated by p2x7 activation . again , az10606120 did not significantly alter the basal no formation ( figure 6 ) or cell viability ( as assessed by forward and side scatter ) ( data not shown ) . p2x7 activation results in the death of various cell types [ 11 , 12 ] . to determine whether atp induces the death of eoc13 microglia , cells in complete dmem medium were incubated in the absence or presence of atp for 24 h , and then the percentage of annexin - v/7aad , annexin - v/7aad , and annexin - v/7aad cells examined by flow cytometry ( figure 7(a ) ) . cell death is expressed as the total of dying ( annexin - v/7aad ) and dead ( annexin - v/7aad and annexin - v/7aad ) cells . incubation with either 2 or 3 mm atp but not 1 mm atp resulted in significantly higher percentages of total cell death compared to cells incubated in the absence of atp ( figure 7(a ) ) . next , to determine if the atp - induced eoc13 death was mediated by p2x7 activation , cells were preincubated in the absence or presence of az10606120 and then incubated in the absence or presence of atp for 24 h. as above ( figure 7(a ) ) , 2 mm atp induced significant cell death , with higher percentages of total cell death compared to cells incubated in the absence of atp ( figure 7(b ) ) . preincubation with 10 m az10606120 completely inhibited atp - induced cell death ( figure 7(b ) ) , indicating that this process is mediated by p2x7 activation . p2x7-induced death of murine raw264.7 macrophages is mediated by ros formation . therefore , a role for ros in p2x7-induced death of eoc13 microglia was investigated . to confirm that p2x7 induced ros formation under conditions used to induce cell death , dcf - loaded eoc13 cells in complete dmem medium were incubated in the absence or presence of atp , and then subsequent ros formation determined by flow cytometry . similar to atp - induced cell death ( figure 7(a ) ) , incubation with 2 or 3 but not 1 mm atp induced significant ros formation in eoc13 cells compared to cells incubated in the absence of atp ( figure 7(c ) ) . the requirement for higher atp concentrations to induce cell death ( figure 7(a ) ) or ros formation ( figure 7(c ) ) compared to pore formation ( figure 3(b ) ) is in line with the inhibitory action of divalent cations , which are present in the culture medium but not in the nacl medium used . to confirm that this ros formation was mediated by p2x7 , cells were preincubated with az10606120 and the amounts of atp - induced ros formation determined . again , atp induced significant ros formation compared to cells incubated in the absence of atp ( figure 7(d ) ) . preincubation with 10 m az10606120 completely inhibited this atp - induced ros formation ( figure 7(d ) ) . eoc13 cells were preincubated in the absence or presence of nac followed by atp for 24 h. however , 24 h incubation with 40 mm nac in the absence of atp led to significant amounts of eoc13 cell death ( data not shown ) . therefore , to reduce the total exposure to 40 mm nac , cells were incubated in the absence or presence of nac for 90 min , with atp added in the final 1560 min . the medium was then replaced with fresh complete dmem medium and the cells incubated for 24 h. incubation with 2 mm atp for 30 or 60 min but not 15 min resulted in significant cell death compared to cells incubated in the absence of atp ( figure 7(e ) ) . in contrast to the 24 h incubation with nac , 90 min incubation with 40 mm nac ( without atp ) did not induce significant cell death compared to cells incubated for the same length of time in the absence of both nac and atp ( figure 7(e ) ) . a 60 min preincubation with nac inhibited cell death induced by 30 min incubation with atp by 99 6% ( figure 7(e ) ) . in contrast , a 75 and 30 min preincubation with nac , followed by 15 and 60 min atp treatment , respectively , had no effect on the percentage of cell death compared to cells incubated for the same time length with atp in the absence of nac ( figure 7(e ) ) . to further confirm that nac did not induce morphological changes or cause cytotoxicity under the conditions used for the cell death assay , dic images of adherent cells were acquired following the 24 h incubation . as above ( figure 5(c ) ) , cells incubated in the absence or presence of nac ( without atp ) displayed discrete cell bodies with long , spindled shaped processes ( figure 7(f ) ) . in addition , cells incubated in the presence of nac and atp displayed a similar morphology to that of cells incubated in the absence of atp ( figure 7(f ) ) . in contrast , cells preincubated with atp alone displayed rounded and granular cell bodies with no or few processes ( figure 7(f ) ) , characteristic of cell death . furthermore , wells preincubated with atp alone had a high amount of nonadherent cells compared to the other treatments ( data not shown ) . thus , preincubation with nac prevented the morphological changes associated with atp incubation , but nac alone had no effect on cell morphology . the current study demonstrates for the first time that the murine microglial eoc13 cell line expresses functional p2x7 . firstly , the presence of p2x7 mrna and protein was established using rt - pcr and immunoblotting techniques . in addition , the presence of cell - surface p2x7 was demonstrated using immunofluorescence staining . moreover , p2x7 on eoc13 microglia was functional , as the p2x7 agonists atp and bzatp induced significant ethidium or yo - pro-1 uptake into these cells . in these experiments , atp induced ethidium uptake with an ec50 value which falls within the typical range for atp - induced cation fluxes mediated by recombinant murine p2x7 . furthermore , pretreatment of cells with p2x7 antagonists inhibited atp - induced organic cation uptake . lastly , atp could induce ros formation in and the death of eoc13 cells , and both of these events , which are often associated with p2x7 activation [ 1012 ] , could be inhibited by the p2x7 antagonist az10606120 . the presence of functional p2x7 on eoc13 microglia is consistent with the presence of this receptor on primary microglia and microglial cell lines ( including n9 , n13 , bv-2 , and ntw8 cells ) [ 3235 ] . ros formation has been reported in primary microglia [ 24 , 25 ] , and a role for this process in microglia has been highlighted by several studies . p2x7 activation induces the production of the ros , superoxide , in primary rat microglia , while this receptor is upregulated in a transgenic mouse model of alzheimer 's disease . although a direct link between p2x7 , superoxide , and alzheimer 's disease was not established , the authors proposed a link between these molecules and this disease . this link is supported by subsequent observations by others , where fibrillar -amyloid peptide , which is associated with alzheimer 's disease , caused atp release and autocrine activation of p2x7 leading to ros formation in primary rat microglia . in addition , another group demonstrated that p2x7-induced superoxide release from primary rat microglia induced injury of rat cortical neurons . collectively , this data indicates that p2x7-induced ros formation from microglia may be involved in various neuroinflammatory and neurodegenerative disorders . this may be of particular importance in diseases where microglial p2x7 is reported to be upregulated such as in alzheimer 's disease , multiple sclerosis , and amyotrophic lateral sclerosis [ 25 , 37 ] . it should be noted , however , that dcf , as employed in the current study and as widely used by others to detect ros , can also propagate ros formation . nevertheless , our observation that p2x7 activation also induces the formation of no in eoc13 cells supports a role for this receptor in the formation of reactive species . the current study excluded an essential role for ca influx in p2x7-induced ros formation in eoc13 microglia . this finding is similar to other observations with other murine cell types , including submandibular glands and erythroid cells . in contrast , p2x7-induced ros formation in primary rat microglia and rat submandibular glands is dependent on an influx of ca . the reason for this difference between these two species remains unknown but may reflect differences in experimental protocols or differences in signalling molecules between mice and rats . the current study also excluded an essential role for k efflux in p2x7-induced ros formation in eoc13 microglia . both ros formation and k efflux are involved in il-1 release from monocytes , although whether these downstream processes are linked has not been established . thus , our results indicate that p2x7-induced ros formation does not require k efflux and that ros formation and k efflux may be independent events in il-1 release from myeloid cells . use of an annexin - v/7aad assay suggested that this process was mediated by apoptosis . however , in the absence of other markers of apoptosis and necrosis , this remains to be established , especially since p2x7 activation induces both apoptosis and necrosis in the microglial n13 cell line . nevertheless , our observations support previous studies in which p2x7 activation induced death in primary microglia and other microglial cell lines [ 41 , 42 ] . the physiological role of p2x7-induced microglia death is unclear . further obscuring this is the known role of p2x7 activation in inducing the proliferation of microglia . this paradoxical role of p2x7 is thought to be related to the relative atp concentration , with high concentrations promoting cell death and low concentrations promoting cell proliferation . in support of this , our study observed that atp only induced eoc13 cell death at 2 or 3 but not 1 mm atp . moreover , our data also showed that a transient incubation with atp of 3060 but not 15 min induced cell death in eoc13 microglia . this suggests that transient atp release and subsequent p2x7 activation may be sufficient to kill microglia in vivo . the current study examined a potential link between p2x7-induced ros formation and death in eoc13 microglia . a previous study demonstrated that the atp - induced death of murine raw264.7 macrophages was mediated by ros derived from nadph oxidase downstream of p2x7 activation . this contrasts with another study , which found that p2x7-induced ros formation , but not death , was attenuated in primary macrophages from nadph oxidase deficient mice . our data using the ros scavenger nac supports a role for ros formation in the p2x7-induced death of eoc13 microglia . the capacity of nac to prevent p2x7-induced eoc13 microglia death was dependent on the preincubation time with nac , as well as the total incubation time with atp , with only 4560 min preincubations with nac preventing cell death induced by transient 3045 min exposures to atp . in contrast , 24 h incubation with nac induced significant amounts of eoc13 microglia death , equivalent to that induced by atp alone . this cytotoxicity of nac may have occurred due to increased toxic metabolic byproducts such as reduced glutathione . alternatively , scavenging of ros by nac may indicate that low amounts of ros are important for eoc13 cell homeostasis . of note , the ros inhibitor dpi also induced the death of eoc13 microglia , albeit over a much faster time course . finally , it should be noted that nac inhibition of p2x7-induced death and ros formation in eoc13 microglia may have been partly due to direct inhibition of p2x7 . nac inhibited atp - induced pore formation by 30% compared to a 74 and 99% inhibition of atp - induced ros formation and cell death , respectively . this direct inhibition of p2x7 by nac was not due to an acidic ph , which is known to impair p2x7 function , as the nac - containing solutions were adjusted to ph 7.4 before each assay . thus , our results indicate that either cellular signalling involving ros may modulate p2x7 activation in eoc13 microglia or that nac may directly impair p2x7 at 40 mm . the concentration of nac used in these experiments ( 40 mm ) is 48-fold higher than that used in a number of similar studies ( e.g. , ) . the requirement for this high concentration of nac remains unknown but may reflect a reduced ability of nac to cross the plasma membrane or to be converted to glutathione in eoc13 cells . the presence of functional p2x7 on j774 macrophage cells was confirmed in the current study . p2x7 is present in this cell line , and activation of p2x7 leads to the release of mature il-1 , the formation of macrophage - derived multinucleated giant cells [ 5052 ] , and cell death . in this study , the potency of four p2x7 antagonists against 1 mm atp , the atp concentration most commonly used to study p2x7 , was determined . the ic50 values for bbg , a438079 , and az11645373 ( 1.8 , 7.9 , and 1.5 m , resp . ) were within one log range of those published for recombinant murine p2x7 [ 54 , 55 ] . in contrast , the ic50 value for az10606120 has not been reported for murine p2x7 , although this compound has been shown to specifically bind to and inhibit rat and human p2x7 . in the current study , this highly specific p2x7 antagonist also completely impaired atp - induced ethidium uptake , ros formation , and death of murine eoc13 cells . thus , az10606120 will be useful for future studies of murine p2x7 . in the cns , atp acts as a neurotransmitter and is released from neurons during synaptic transmission and from dying cells . under normal physiological conditions , extracellular atp concentrations in the cns are estimated to be in the nanomolar to micromolar range , depending on the balance between atp release and degradation , while intracellular microglial atp concentrations are in the millimolar range . after cns injury , however , extracellular atp concentrations increase and can reach as high as the millimolar range . furthermore , it is hypothesised that atp may act on microglial p2x7 at very close range where the concentration of atp may be quite high . activation of p2x7 on primary microglia and microglial cell lines leads to the release of proinflammatory il-1 and tumour necrosis factor- [ 33 , 58 ] and ros formation . although proinflammatory factors are important for immunity , prolonged or inappropriate release of such factors from chronically activated microglial can be highly toxic to neurons and can promote neuroinflammation and neurodegeneration . there are a number of diseases in the cns characterised by the presence of activated microglia , including alzheimer 's disease , prion infection , cerebral ischemia , multiple sclerosis , and amyotrophic lateral sclerosis . in such diseases , p2x7 has also been reported to be upregulated [ 25 , 37 , 59 , 60 ] . this raises questions of possible roles for p2x7 in mediating inappropriate microglial responses in cns disorders . activation of this receptor by atp resulted in organic cation uptake , ros formation , and death in these cells . moreover , the eoc13 cell line may be useful for investigating p2x7-mediated events in microglia and the role of this receptor in microglia - mediated inflammatory disorders .

the p2x7 purinergic receptor is a ligand - gated cation channel expressed on leukocytes including microglia . this study aimed to determine if p2x7 activation induces the uptake of organic cations , reactive oxygen species ( ros ) formation , and death in the murine microglial eoc13 cell line . using the murine macrophage j774 cell line as a positive control , rt - pcr , immunoblotting , and immunolabelling established the presence of p2x7 in eoc13 cells . a cytofluorometric assay demonstrated that the p2x7 agonists adenosine-5-triphosphate ( atp ) and 2(3)-o-(4-benzoylbenzoyl ) atp induced ethidium+ or yo - pro-12 + uptake into both cell lines . atp induced ethidium+ uptake into eoc13 cells in a concentration - dependent manner , with an ec50 of ~130 m . the p2x7 antagonists brilliant blue g , a438079 , az10606120 , and az11645373 inhibited atp - induced cation uptake into eoc13 cells by 75100% . a cytofluorometric assay demonstrated that p2x7 activation induced ros formation in eoc13 cells , via a mechanism independent of ca2 + influx and k+ efflux . cytofluorometric measurements of annexin - v binding and 7aad uptake demonstrated that p2x7 activation induced eoc13 cell death . the ros scavenger n - acetyl - l - cysteine impaired both p2x7-induced eoc13 ros formation and cell death , suggesting that ros mediate p2x7-induced eoc13 death . in conclusion , p2x7 activation induces the uptake of organic cations , ros formation , and death in eoc13 microglia .

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

these factors may be inflammatory , regulatory , or cytotoxic in nature and include reactive oxygen species ( ros ) , nitric oxide ( no ) , proinflammatory and anti - inflammatory cytokines , prostaglandins , and growth factors [ 4 , 5 ] . the p2x7 receptor is a trimeric ligand - gated cation channel belonging to the p2x family of purinergic receptors . in particular activation of p2x7 by extracellular adenosine-5-triphosphate ( atp ) , or the most potent p2x7 agonist , 2(3)-o-(4-benzoylbenzoyl ) atp ( bzatp ) , causes the passage of small cations including ca , na , and k across the plasma membrane , as well as organic cations , such as the fluorescent dyes ethidium and yo - pro-1 . using molecular , immunochemical , and pharmacological techniques , we demonstrate in the current study that the murine microglial eoc13 cell line expresses functional p2x7 . activation of p2x7 by atp in this cell line induces the uptake of organic cations , ros formation , and cell death . atp , bzatp , ethidium bromide , dimethyl sulfoxide ( dmso ) , glycerol gelatin , and the p2x7 antagonist brilliant blue g ( bbg ) were from sigma - aldrich ( st . the p2x7 antagonists a438079 , az10606120 , and az11645373 were from tocris bioscience ( ellisville , mo ) . the viability dye 7-aminoactinomycin d ( 7aad ) and the ros scavenger n - acetyl - l - cysteine ( nac ) were from enzo life sciences ( plymouth meeting , pa ) . cells preincubated with h2o soluble compounds ( bbg , a438079 , and az10606120 ) were compared to cells preincubated in the absence of each compound . the murine macrophage j774 cell line , the murine microglial eoc13 cell line , and the murine lymphoblast ladmac cell line , all originally obtained from the american type culture collection ( manassas , va ) , were kindly provided by jasmyn dunn ( university of queensland , brisbane , australia ) ( j774 ) and iain campbell ( university of sydney , sydney , australia ) ( eoc13 and ladmac ) . cells were then incubated with 25 m ethidium ( or 1 m yo - pro-1 where indicated ) in the absence or presence of the p2x7 agonists atp or bzatp ( as indicated ) for 5 min . in some experiments , atp - induced cation uptake was assessed with cells suspended in kcl medium ( 150 mm kcl , 5 mm glucose , and 10 mm hepes , ph 7.4 ) or in nacl medium containing 1 mm cacl2 or 100 m egta . in other experiments , cells were preincubated in the absence or presence of p2x7 antagonists or the ros scavenger nac ( as indicated ) for 15 and 30 min , respectively , prior to cation and atp addition . in some experiments , atp - induced ros formation was assessed in kcl medium , in nacl medium in the absence of 1 mm cacl2 or presence of 100 m egta , or in complete dmem medium in the absence or presence of 10 m az10606120 ( 15 min preincubation , prior to atp addition ) . in other experiments , cells were preincubated in the absence or presence of az10606120 , nac , or dpi ( as indicated ) for 15 , 30 , and 30 min , respectively , prior to atp addition . in other experiments , cells were preincubated in the absence or presence of 40 mm nac for 90 min , with 2 mm atp added in the final 1560 min , and then the medium replaced and cells incubated at 37c , 95% air/5% co2 for a further 24 h. following the 24 h incubations , cells were harvested from wells using 0.05% trypsin ( 5 min , 37c ) and washed once with annexin - v binding medium ( nacl medium containing 5 mm cacl2 ) . cells were then incubated with annexin - v binding medium containing annexin - v - fluorescein and 7aad at room temperature protected from light for 15 min . and the mfi of annexin - v - fluorescein and 7aad determined using flowjo software . quadrant markers were used to determine the percentage of annexin - v/7aad , annexin - v/7aad , and annexin - v/7aad cells . the murine macrophage j774 cell line is well known to express functional p2x7 . moreover , our group has demonstrated the presence of functional p2x7 in various cell types using a fixed - time fluorescent cation uptake assay ( e.g. therefore , this technique was used to confirm the presence of p2x7 in j774 cells and to validate the use of this cell line as a positive control . a number of highly specific p2x7 antagonists , including a438079 , az10606120 , and az11645373 , have recently become available . therefore , to determine the optimum concentrations of these antagonists required to inhibit murine p2x7 , j774 cells were preincubated in the absence or presence of varying concentrations of bbg , a438079 , az10606120 , and az11645373 and the atp - induced ethidium uptake assessed . each antagonist impaired 1 mm atp - induced ethidium uptake in a concentration - dependent manner , with ic50 values of 1.8 0.2 , 7.9 0.4 , 1.0 0.1 , and 1.5 0.1 m , respectively ( figure 1(c ) ) . to determine whether eoc13 microglial cells express p2x7 , a series of experiments using j774 cells as a positive control were performed . the presence of total p2x7 protein was determined by probing separated whole lysates of both cell lines with an anti - p2x7 ab . moreover , both cell lines were incubated with an anti - p2x7 mab and the presence of cell - surface p2x7 determined by flow cytometry . preincubation of the anti - p2x7 ab with blocking peptide completely abrogated the detection of p2x7 in both cell lines ( data not shown ) . to determine whether p2x7 was functional in eoc13 cells , the fixed - time ethidium uptake assay was performed . both atp and bzatp were found to induce significant ethidium uptake into eoc13 cells compared to cells incubated in the absence of nucleotide ( figure 3(a ) ) . atp induced ethidium uptake in a concentration - dependent manner , with maximal uptake obtained at an atp concentration of 1 mm and with an ec50 of 130 30 m ( figure 3(b ) ) . to determine if the observed atp - induced ethidium uptake into eoc13 cells was mediated by p2x7 , cells were preincubated in the absence or presence of p2x7 antagonists at inhibitory concentrations optimal for 1 mm atp - induced ethidium uptake in j774 cells , as demonstrated above ( figure 1(c ) ) . preincubation of eoc13 cells with 30 m bbg , 100 m a438079 , 10 m az10606120 , and 30 m az11645373 resulted in significant impairment of atp - induced ethidium uptake by 75 2 , 90 1 , 100 0 , and 99 1% , respectively ( figure 3(c ) ) . none of the p2x7 antagonists except az11645373 significantly altered the basal ethidium uptake into eoc13 cells . to determine if p2x7 activation could induce the uptake of a second organic cation into eoc13 cells , cells were preincubated in the absence or presence of az10606120 , and atp - induced yo - pro-1 uptake examined . similar to ethidium uptake , 1 mm atp induced significant yo - pro-1 uptake into eoc13 cells compared to cells incubated in the absence of atp ( figure 3(d ) ) . p2x7 activation has been reported to induce ros formation in a number of cell types , including primary microglia [ 24 , 25 ] . thus , atp - induced ros formation in the eoc13 cell line was investigated using the ros sensitive probe dcf . as extracellular ca has been reported to be important for p2x7-induced ros formation in a number of cell types [ 24 , 26 , 27 ] , assays were initially conducted in the presence of 1 mm ca . incubation with 2 mm atp induced significant ros formation in eoc13 cells compared to cells incubated in the absence of atp ( mfi of ros formation 16.3 0.6 and 5.18 0.06 , resp . furthermore , preincubation of cells with 10 m az10606120 resulted in complete inhibition of atp - induced ros formation ( figure 4(a ) ) , indicating that this process is mediated by p2x7 activation . p2x7 is a ligand - gated cation channel ; therefore the possible roles for cation fluxes in p2x7-induced ros formation were next investigated . thus , to determine if ca influx is required for p2x7-mediated ros formation in eoc13 cells , atp - induced ros formation in the absence and presence of ca was investigated . atp induced significant ros formation in both the absence and presence of 1 mm ca compared to similarly treated cells in the absence of atp ( figure 4(b ) ) . cells incubated in the absence of ca had significantly higher atp - induced ros formation compared to those incubated in the presence of ca . in contrast , atp - induced ethidium uptake ( p2x7 function ) was similar in cells incubated in the absence or presence of ca ( figure 4(b ) ) , indicating that the differences in atp - induced ros formation were not due to altered p2x7 function . thus , to further exclude a role for ca in p2x7-mediated ros formation in eoc13 cells , atp - induced ros formation was investigated in the absence and presence of the ca chelator egta . incubation with 1.4 mm atp induced significant but similar amounts of ros formation in both the absence and presence of 100 m egta compared to similarly treated cells in the absence of atp ( figure 4(c ) ) . again , atp - induced ethidium uptake was similar in cells incubated in the absence or presence of egta ( figure 4(c ) ) . finally , the role of k in p2x7-induced ros formation in eoc13 cells was investigated . thus , to determine if k efflux is involved in p2x7-mediated ros formation in eoc13 cells , atp - induced ros formation was compared with cells in nacl medium and kcl medium , which prevents the loss of intracellular k. incubation with 1.4 mm atp induced significant ros formation in both nacl and kcl media , with similar levels of ros formation in both media ( figure 4(d ) ) . likewise , atp - induced ethidium uptake was similar in nacl and kcl media ( figure 4(d ) ) , indicating that the inability of high extracellular k to impair atp - induced ros formation was not due to altered p2x7 function . to confirm that p2x7 activation induced ros formation in eoc13 microglia , dcf - loaded cells in nacl medium were preincubated in the absence or presence of the ros scavenger nac , before incubation in the absence or presence of atp . preincubation with 40 mm nac inhibited atp - induced ros formation by 73.7 0.3% ( figure 5(a ) ) . dcf - loaded cells were also preincubated in the absence or presence of the broad - spectrum ros inhibitor dpi and the atp - induced ros formation investigated . to further verify that p2x7 activation induces the formation of reactive species in eoc13 cells , atp - induced no formation was investigated using the no sensitive probe daf - fm da . incubation with 1.4 mm atp induced significant no formation in eoc13 cells compared to cells incubated in the absence of atp ( figure 6 ) . to determine whether atp induces the death of eoc13 microglia , cells in complete dmem medium were incubated in the absence or presence of atp for 24 h , and then the percentage of annexin - v/7aad , annexin - v/7aad , and annexin - v/7aad cells examined by flow cytometry ( figure 7(a ) ) . next , to determine if the atp - induced eoc13 death was mediated by p2x7 activation , cells were preincubated in the absence or presence of az10606120 and then incubated in the absence or presence of atp for 24 h. as above ( figure 7(a ) ) , 2 mm atp induced significant cell death , with higher percentages of total cell death compared to cells incubated in the absence of atp ( figure 7(b ) ) . preincubation with 10 m az10606120 completely inhibited atp - induced cell death ( figure 7(b ) ) , indicating that this process is mediated by p2x7 activation . to confirm that p2x7 induced ros formation under conditions used to induce cell death , dcf - loaded eoc13 cells in complete dmem medium were incubated in the absence or presence of atp , and then subsequent ros formation determined by flow cytometry . similar to atp - induced cell death ( figure 7(a ) ) , incubation with 2 or 3 but not 1 mm atp induced significant ros formation in eoc13 cells compared to cells incubated in the absence of atp ( figure 7(c ) ) . to confirm that this ros formation was mediated by p2x7 , cells were preincubated with az10606120 and the amounts of atp - induced ros formation determined . preincubation with 10 m az10606120 completely inhibited this atp - induced ros formation ( figure 7(d ) ) . eoc13 cells were preincubated in the absence or presence of nac followed by atp for 24 h. however , 24 h incubation with 40 mm nac in the absence of atp led to significant amounts of eoc13 cell death ( data not shown ) . the current study demonstrates for the first time that the murine microglial eoc13 cell line expresses functional p2x7 . firstly , the presence of p2x7 mrna and protein was established using rt - pcr and immunoblotting techniques . moreover , p2x7 on eoc13 microglia was functional , as the p2x7 agonists atp and bzatp induced significant ethidium or yo - pro-1 uptake into these cells . in these experiments , atp induced ethidium uptake with an ec50 value which falls within the typical range for atp - induced cation fluxes mediated by recombinant murine p2x7 . furthermore , pretreatment of cells with p2x7 antagonists inhibited atp - induced organic cation uptake . lastly , atp could induce ros formation in and the death of eoc13 cells , and both of these events , which are often associated with p2x7 activation [ 1012 ] , could be inhibited by the p2x7 antagonist az10606120 . the presence of functional p2x7 on eoc13 microglia is consistent with the presence of this receptor on primary microglia and microglial cell lines ( including n9 , n13 , bv-2 , and ntw8 cells ) [ 3235 ] . p2x7 activation induces the production of the ros , superoxide , in primary rat microglia , while this receptor is upregulated in a transgenic mouse model of alzheimer 's disease . nevertheless , our observation that p2x7 activation also induces the formation of no in eoc13 cells supports a role for this receptor in the formation of reactive species . however , in the absence of other markers of apoptosis and necrosis , this remains to be established , especially since p2x7 activation induces both apoptosis and necrosis in the microglial n13 cell line . nevertheless , our observations support previous studies in which p2x7 activation induced death in primary microglia and other microglial cell lines [ 41 , 42 ] . moreover , our data also showed that a transient incubation with atp of 3060 but not 15 min induced cell death in eoc13 microglia . the current study examined a potential link between p2x7-induced ros formation and death in eoc13 microglia . a previous study demonstrated that the atp - induced death of murine raw264.7 macrophages was mediated by ros derived from nadph oxidase downstream of p2x7 activation . our data using the ros scavenger nac supports a role for ros formation in the p2x7-induced death of eoc13 microglia . the capacity of nac to prevent p2x7-induced eoc13 microglia death was dependent on the preincubation time with nac , as well as the total incubation time with atp , with only 4560 min preincubations with nac preventing cell death induced by transient 3045 min exposures to atp . finally , it should be noted that nac inhibition of p2x7-induced death and ros formation in eoc13 microglia may have been partly due to direct inhibition of p2x7 . nac inhibited atp - induced pore formation by 30% compared to a 74 and 99% inhibition of atp - induced ros formation and cell death , respectively . p2x7 is present in this cell line , and activation of p2x7 leads to the release of mature il-1 , the formation of macrophage - derived multinucleated giant cells [ 5052 ] , and cell death . in the current study , this highly specific p2x7 antagonist also completely impaired atp - induced ethidium uptake , ros formation , and death of murine eoc13 cells . activation of this receptor by atp resulted in organic cation uptake , ros formation , and death in these cells .

this gram - positive anaerobe causes both histotoxic infections , such as gas gangrene ( clostridial myonecrosis ) , and enteric infections , such as human food poisoning . as typical amongst pathogenic clostridial spp . , the virulence of c. perfringens is largely attributable to its ability to produce an arsenal of potent protein toxins . production of four of these toxins ( alpha , beta , epsilon , and iota ) is used to classify c. perfringens strains into one of five types ( a e ) . less than 5% of all c. perfringens type a isolates produce another toxin named c. perfringens enterotoxin ( cpe ) that is biomedically important , although not used in the toxin typing classification system . after a brief introduction to this unique toxin , promising efforts to utilize cpe , or its derivatives , for cancer therapy will be described cpe - producing c. perfringens type a strains cause the second most common bacterial food poisoning in the usa , as well as many cases of human nonfoodborne gastrointestinal diseases , such as antibiotic - associated diarrhea . the food poisoning develops when foods contaminated with large numbers of cpe - producing type a strains are ingested ; the ingested bacteria then briefly grow in the small intestine before committing to sporulation . molecular koch 's postulates analyses have demonstrated that production of cpe is essential for cpe - positive type a human food poisoning or nonfoodborne gastrointestinal disease isolates to cause gastrointestinal effects in animal models . most , but not all , c. perfringens type a food poisoning strains carry their enterotoxin gene ( cpe ) on the chromosome . in contrast , the cpe gene of type a nonfoodborne human disease strains is located on large ( ~7075 kb ) plasmids . amongst cpe - positive type a strains there are two major families of cpe plasmids . these enterotoxin plasmids can be conjugative , presumably due to the presence of a tn916-like region named tcp that has been experimentally shown to mediate transfer of other c. perfringens conjugative plasmids . expression of the cpe gene is regulated by sporulation - associated alternative sigma factors [ 8 , 9 ] . specifically , one alternative sigma factor ( sigf ) controls expression of two other alternative sigma factors ( sigk and sige ) , which then direct transcription of cpe mrna from several sigk- or sige - dependent promoters located upstream of the cpe orf . exceptionally large amounts of cpe can be produced during sporulation ; for example , cpe can represent 20% of total protein in some sporulating cpe - positive c. perfringens type a strains . instead it accumulates in the cytoplasm of the mother cell until the completion of sporulation . when the mother cell then lyses to release the mature spore , cpe is released into the intestinal lumen , where it binds and acts as described in the following section . the in vivo outcome of natural cpe action during gastrointestinal disease is desquamation of the intestinal epithelium , intestinal necrosis , and the accumulation of luminal fluid . these effects account for the natural gastrointestinal symptoms of cpe - associated disease , which most commonly include diarrhea and abdominal cramps . typically , people are sickened with c. perfringens type a food poisoning for 1224 hours and then recover . however , this illness can be fatal in the elderly or in people suffering from medication - induced constipation . as shown in figure 1 , the current model of cpe action begins with binding of this toxin to claudin receptors ( described in detail below ) . this binding results in formation of a small ( ~90 kda ) sds - sensitive complex . besides cpe , the small complex also contains both claudin receptors and claudins incapable of binding cpe ( i.e. , nonreceptor claudins ) . presumably the presence of nonreceptor claudins in small complex is attributable to claudin : claudin interactions . six small complexes are then thought to oligomerize into an sds - resistant large complex named cpe hexamer-1 , or ch-1 . this hypothesis is based upon results of heteromer gel shift analyses , which identified the presence of six cpe molecules in each ch-1 complex . ch-1 is ~450 kda in size and contains , in addition to six cpe molecules , both receptor and nonreceptor claudins . ch-1 initially assembles as a prepore on the membrane surface ; however , at 37c this prepore then rapidly inserts into membranes to form an active pore . formation of the ch-1 pore results in calcium influx , which ( via calpain activation ) leads to cell death [ 13 , 14 ] . at moderate cpe doses , where modest cpe pore formation allows only limited calcium influx , cpe - treated cells die from a classical caspase 3-mediated apoptosis . at higher cpe doses , where large amounts of cpe pore formation results in a massive calcium influx , cells die from oncosis . cpe pore formation also leads to morphologic damage that exposes the basolateral surface of cells . this allows formation of a second bigger ( ~650 kda ) large complex named ch-2 . in addition to six copies of cpe and both receptor and nonreceptor claudins , ch-2 also contains another tight junction protein named occludin [ 11 , 15 ] . formation of ch-2 leads to internalization of occludin into the cytoplasm ; claudins are also internalized inside native cpe - treated cells although it is not clear if this is due to ch-1 formation , ch-2 formation , or to formation of both complexes . these effects likely help to explain the observed ability of native cpe to disrupt tight junctions . cpe consists of a 319 amino acid polypeptide ( mr 35,317 ) with a unique primary sequence . the cpe structure / function relationship has been extensively analyzed by combined genetic , biochemical , and structural biology approaches ( figure 2 ) . as this review this structure revealed that cpe is a three - domain protein , reminiscent of several other pore - forming toxins . the n - terminal 37 amino acids of native cpe lack a definable structure and are not necessary for toxicity . in fact , removing these amino acids by chymotrypsin , as may occur in the intestines during disease , produces a 23-fold more potent toxin . this proteolytic activation occurs because removal of the n - terminal cpe sequences exposes the cpe region between amino acids 47 to 51 , which reside in domain ii , to promote ch-1 formation . site - directed mutagenesis studies showed that particularly important domain ii residues for ch-1 formation are ( i ) the asp at cpe residue 48 and ( ii ) the ile at cpe residue 51 . cpe domain ii also contains a region spanning from residues 81 to 106 that appears to be a transmembrane stem . removal of these residues produces a cpe variant that can form ch-1 but is unable to kill cells or form pores . this effect is consistent with cpe residues 81 to 106 mediating the insertion of this transmembrane stem into membranes . membrane insertion of all six transmembrane stems , from the six cpe proteins present in ch-1 , results in -barrel pore formation . domain iii may undergo structural changes during the prepore to pore transition that facilitate insertion of the cpe transmembrane stem into lipid bilayers , facilitating pore formation . over 20 years ago it was shown that a recombinant cpe fragment corresponding to the c - terminal half of the native toxin retains full receptor - binding activity . however , ( as expected from the preceding section ) this c - terminal cpe fragment named c - cpe was nontoxic since it lacks the n - terminal regions necessary for ch-1 formation and insertion of ch-1 into membranes to form a pore . deletion mutagenesis and synthetic peptide approaches later localized most cpe receptor - binding activity to the 30 c - terminal amino acids ( figure 2 ) . more recently , site - directed mutagenesis studies demonstrated that three tyr residues , located at positions 306 , 310 , and 312 of the native toxin protein are important for receptor binding . c - cpe ( residues 194 to 319 of the native toxin ) approximately corresponds to domain i of the native cpe protein [ 17 , 18 ] . domain i consists of a nine beta strand sandwich that shares structural similarity with the receptor binding domains of some other bacterial toxins , including the large cry family of bacillus thuringiensis toxins . by correlating this c - cpe structure with the previous binding - activity mapping studies described above , it is apparent that the receptor - binding site of cpe resides on a large loop located between beta strands 8 and 9 . mammalian tight junctions act as both fences and gates , that is , they represent important barriers for an epithelium and also regulate paracellular permeability . studies conducted over the past 15 years have determined that the tight junction is comprised of several proteins , the most important of which are the claudins . the claudins are a 24-member family of ~2025 kda proteins that are predicted to consist of four transmembrane domains , two extracellular loops ( ecl-1 and ecl-2 ) , and a cytoplasmic tail that can mediate signaling cascades . , claudins are also overexpressed in many cancers . in 1997 , katahira et al . reported that when fibroblasts , which are naturally cpe- resistant , were transfected to express an ~22 kda vero cell protein , they gained cpe sensitivity . the vero cell protein expressed by these transfectants had properties of a cpe receptor since the fibroblast transfectants acquired the ability to bind significant levels of the toxin and to form high molecular weight complexes that are now recognized as ch-1 . this vero cell cpe receptor protein it was also determined that , at physiologic concentrations , the enterotoxin can bind to transfected fibroblasts expressing claudin-3 , -4 , -6 , -8 , or -14 . however , no cpe binding was detected to transfectants expressing claudins-1 , -2 , -5 , or -10 . a more recent study by robertson et al . demonstrated that cpe also interacts with claudins in naturally cpe - sensitive caco-2 cells , which are a human enterocyte - like cell line . using coimmunoprecipitation and electroelution approaches , this study showed both the cpe small complex and ch-1 large complex formed in caco-2 cells can contain , in addition to cpe , receptor claudins-3 and -4 , along with the nonreceptor claudin-1 . in addition to cpe , receptor claudins , and nonreceptor claudins , the ch-2 complex formed in caco-2 cells also contains occludin . the stoichiometry of claudins in ch-1 and ch-2 , or occludin in ch-2 , has not yet been determined . the structure of a claudin has not been solved at the time when this review is being prepared . however , as mentioned earlier ( and depicted in figure 2 ) , claudins are predicted to possess two extracellular loops named ecl-1 and ecl-2 . an early study using claudin chimeras consisting of the n - terminal half of cpe receptor claudin-4 fused with the c - terminal half of cpe nonreceptor claudin-1 suggested that cpe interacts with the putative ecl-2 region . this hypothesis was rigorously confirmed by a recent study using more specific chimeric claudins , which showed that substituting only the predicted ecl-2 sequence of claudin-4 into a claudin-1 backbone is sufficient to produce fibroblast transfectants that are very sensitive to cpe . the reverse was also true , that is , transfectants expressing a chimeric claudin , where only the claudin-1 ecl-2 had been specifically substituted into the claudin-4 backbone , were cpe - insensitive . several recent studies ( reviewed in ) have focused on understanding the specific ecl-2 residues of claudin receptors that mediate cpe binding . these studies have utilized a variety of approaches including arrays of immobilized ecl-2 synthetic peptides , solubilized claudins , or transfectants expressing claudin mutant . results from these studies suggested that ecl-2 possesses a helix - turn - helix motif that interacts with cpe . an asn residue in the middle of this turn appears to be important for cpe binding ; some evidence suggests that a leu residue located two residues from this asn may also participate in the binding of this toxin . since approximately 85% of malignant tumors are derived from epithelial cells , an epithelium - targeted therapeutic strategy has been the focus of cancer translational research . as mentioned earlier , claudins are the major components of paracellular tight junctions ( tjs ) , distribute at the most apical junctions between epithelial cells , and play an essential role in the control of paracellular transport . furthermore , claudin-3 and -4 have been identified as the specific receptors for cpe , which is of potential therapeutic significance since these two claudins are abundantly expressed in ovarian , breast , uterine , and pancreatic cancers . while cpe can trigger lysis of epithelial cells by binding to claudin-3 and claudin-4 , with resultant initiation of massive permeability changes , osmotic cell ballooning , and cytolysis within 515 min ( see previous sections of this review ) , cells lacking expression of the cpe receptors are completely unaffected by this enterotoxin . these observations have raised the possibility that cpe may be an innovative claudin - targeted therapy for malignant tumors . in fact , efforts have been made to use cpe in the treatment of claudin - overexpressing cancers during the past few years . approximately 90% of patients with advanced - stage ovarian cancer develop recurrence and inevitably die from the development of chemotherapy resistance . previous studies have demonstrated that claudin-3 and -4 were among the six most differentially upregulated genes in ovarian cancer cells , but their expression is undetectable in normal ovaries . of particular note , chemotherapy - resistant / recurrent ovarian cancers express claudin-3 and -4 at significantly higher levels than chemotherapy - sensitive cancers . these overexpressed claudins may represent promising targets for the use of cpe as a tumor - targeting therapy against this aggressive disease . indeed , santin and colleagues successfully used cpe to treat an animal model of chemotherapy - resistant human ovarian cancer . they found that all ovarian cancer cells , regardless of their resistance to chemotherapeutic agents , showed a dose - dependent cytotoxic response and died rapidly after 24 hours of exposure to cpe . furthermore , in this animal model employing chemotherapy - resistant human ovarian cancer xenografts , multiple intraperitoneal ( i.p . ) sublethal doses of cpe ranging from 5 to 8.5 g / ml significantly inhibited tumor growth and extended the survival of mice harboring a large tumor burden of chemotherapy - resistant ovarian cancer . therefore , cpe - based therapy may have potential as a novel treatment for chemotherapy - resistant ovarian cancer . breast cancer is one of the most common malignancies worldwide . despite tamoxifen and aromatase inhibitor having significantly improved long - term survival of patients diagnosed at the early stages , advanced breast cancer and metastatic breast cancer are still incurable diseases . claudin-3 and claudin-4 are overexpressed in most primary breast carcinomas and breast cancer brain metastases but undetectable in normal breast epithelial cells . in 2004 , kominsky et al . reported for the first time that intratumoral cpe treatment of t47d human breast cancer cell xenografts resulted in significant tumor suppression and necrosis in scid mice without any side effects . administration of the same dose of cpe was toxic and had no effect on tumor volume . cpe also damaged breast cancer cell lines in a claudin - dependent manner but did not affect cell lines lacking claudin-3 and -4 . in later studies , those investigators found that intracranial administration of cpe retarded tumor growth and increased survival in two murine models of breast cancer brain metastasis without any apparent systematic or cns toxicity ( figure 3 ) . these two studies suggest that the local administration of cpe may be useful in the treatment of breast cancer . overexpression of claudin-3 and -4 has been found in uterine serous papillary carcinoma ( uspc ) and correlates with a more aggressive phenotype and a worse prognosis . a study by santin et al . has demonstrated that cpe effectively and specifically triggers cytolysis of primary and metastatic uspc cell lines in a dose - dependent manner whereas normal cells lacking claudin-3 and -4 are unaffected by cpe treatment . in particular , multiple intratumoral injections of cpe in large subcutaneous uspc xenografts led to tumor necrosis and even tumor disappearance in 100% of animals . injection of sublethal doses of cpe significantly suppressed tumor growth and extended survival of animals harboring chemotherapy - resistant intra - abdominal uspc . the local / regional administrations of cpe were well tolerated without observable adverse events in animals . cpe has also been used to treat pancreatic and prostate cancers in nude mice or in vitro by different research groups . reported that intratumoral injections of cpe in pancreatic cancer xenografts resulted in apparent tumor suppression and necrosis in mice , and that cpe treatment also caused an acute dose - dependent cytotoxic effect in pancreatic cancer cells in vitro . in prostate cancer , long et al . showed that the prostate cancer metastatic cells from the bone marrow were sensitive to cpe - mediated cytolysis in vitro . these two preclinical studies suggest that cpe may have potential as a novel therapy for primary and metastatic malignancies expressing claudin-3 and -4 . although the specificity and rapidity of cpe - mediated cytolysis may increase anticancer efficacy and reduce opportunity for the development of drug resistance , its clinical application encounters some challenges . since claudin-3 and -4 are expressed in some normal tissues such as prostate , lung , and the gastrointestinal tract , systemic toxicity is an important concern for cpe therapy . in this context , many investigators have chosen the local administration of native cpe to treat cancers . this approach , however , does not appear to be a good option for some metastatic cancers such as lung metastasis or multiple metastases . moreover , some adverse events have sometimes been observed even following the local administration of cpe during several studies [ 30 , 34 ] . furthermore , cpe is recognized as a virulence factor responsible for the pathophysiological responses associated with a common food poisoning , proinflammatory cytokine response and other human diseases . since cpe - associated foodborne illness is so common , many people already have serum antibodies against this toxin . it is unclear whether these serum cpe antibodies include neutralizing antibodies but cpe does contain at least one neutralizing epitope , present in the cpe binding domain . other challenges include determining optimal dosage and regimen , development of drug resistance , and long - term safety concerns . although the clinical application of cpe is limited by its potentially significant toxic side effects , the c - terminal binding domain of cpe ( c - cpe ) overcomes this drawback of cpe and has recently emerged as a promising cancer therapeutic agent due to its unique properties . for example , c - cpe can disrupt the paracellular tj barrier by binding to claudin-3 and -4 in the epithelia and thus improve drug delivery in a noncytotoxic fashion . it also has a smaller molecular size that might provide less immunogenicity than cpe . the paracellular tjs are the primary barrier to the transport of solutes from the apical surface to the core of cells . agent uptake via the paracellular pathway in the epithelia is considered an attractive route for the absorption of chemotherapies . given that claudin-3 and -4 are overexpressed in several cancers and are major components of cell tjs , their downregulation by c - cpe may prove a novel strategy for enhancing conventional chemotherapy delivery into claudin - positive cancer cells . our group recently investigated the efficacy of a combination therapy using a chemotherapeutic agent with c - cpe as compared to the single - agent chemotherapeutic agent . using three - dimensional and monolayer culture models and a xenograft mouse model of human eoc cells , we found that c - cpe enhanced the chemosensitivities of eoc cell lines to taxol or carboplatin at low concentrations in a claudin - dependent fashion . administration of c - cpe in combination with taxol significantly suppressed large tumor burdens by about 59% compared with control or taxol alone and showed no apparent toxic drawback of cpe as encountered in previous studies ( figure 4 ) . our study suggests that , at relatively low concentrations , c - cpe may enhance the sensitivity of eoc cells and other claudin - sensitive tumor cells to conventional chemotherapy and thus alleviate systemic side effects of the agents . indeed , c - cpe could be useful not only as an anticancer drug enhancer but also as a carrier to deliver a variety of toxins leading to a spectrum of new anticancer drugs of high selectivity . tnf- has been demonstrated to be an attractive antitumor agent in a variety of animal models but clinical application has been limited due to its failure to concentrate at the site of tumors and the development of severe side effects . to solve the problem , yuan et al . engineered a c - cpe - tnf fusion toxin that was > 6.7-fold more cytotoxic than free tnf to ovarian cancer cells expressing claudin-3 and -4 ; whereas the tnf component in the fusion was 5-fold less potent than free tnf , suggesting that c - cpe - tnf fusion may prevent or decrease the systemic side effects caused by tnf . besides ovarian cancer , saeki et al . fused c - cpe to the protein synthesis inhibitory factor ( psif ) derived from pseudomonas aeruginosa exotoxin a for breast cancer that expresses high levels of claudin-3 and -4 . the c - cpe - psif fusion selectively damaged claudin - expressing breast cancer cell lines , and repeated intratumoral injections significantly suppressed tumor size by 36% without causing apparent side effects in mice . these two studies indicate that c - cpe may be a useful carrier for delivering toxins to claudin - sensitive malignancies with higher anticancer potency and less systemic side effects . the advantages of this claudin - targeted c - cpe - based cancer therapy are significant : tumor - targeted therapy , less immune response , and an improved therapeutic potency not achieved with previous treatments . there remain challenges to be overcome , however , before we can see any major medical advances in treating cancer with c - cpe : ( 1 ) determining safe dosages and schedules , ( 2 ) choosing optimal administration routes , ( 3 ) avoiding potential immunogenicity , and ( 4 ) improving effectiveness for preventing and/or treating cancer metastases . therefore , further studies and clinical trials are required to determine whether c - cpe could be developed as a claudin - targeted novel therapeutic agent for the treatment of cancer . in addition to cancer treatment , c - cpe has been utilized to treat other medical conditions . for example , with the intention of developing a potent mucosal vaccination approach , a nasal vaccine of c - cpe - fused antigen has been prepared and applied in mice without mucosal injury and side effects . in addition , the ability of c - cpe to enhance paracellular permeability has been exploited by using this protein to increase drug absorption from the intestines . one potential issue regarding the use of c - cpe for increasing intestinal drug absorption could be possible gastrointestinal side effects such as diarrhea , due to increased paracellular permeability . however , c - cpe does not increase luminal fluid levels in rabbit ileal loops , possibly arguing against this concern .

clostridium perfringens enterotoxin ( cpe ) causes the symptoms associated with several common gastrointestinal diseases . cpe is a 35 kda polypeptide consisting of three structured domains , that is , c - terminal domain i ( responsible for receptor binding ) , domain ii ( responsible for oligomerization and membrane insertion ) , and domain iii ( which may participate in physical changes when the cpe protein inserts into membranes ) . native cpe binds to claudin receptors , which are components of the tight junction . the bound toxin then assembles into a hexameric prepore on the membrane surface , prior to the insertion of this oligomer into membranes to form an active pore . the toxin is especially lethal for cells expressing large amounts of claudin-3 or -4 , which includes many cancer cells . initial studies suggest that native cpe has potential usefulness for treating several cancers where claudin cpe receptors are overexpressed . however , some challenges with immunogenicity , toxicity , and ( possibly ) the development of resistance may need to be overcome . an alternative approach now being explored is to utilize c - cpe , which corresponds approximately to receptor binding domain i , to enhance paracellular permeability and delivery of chemotherapeutic agents against cancer cells . alternatively , c - cpe fusion proteins may prove superior to use of native cpe for cancer treatment . finally , c - cpe may have application for other medical treatments , including vaccination or increasing drug absorption . the coming years should witness increasing exploitation of this otherwise formidable toxin .

1. Introduction to 2. The CPE Structure/Function Relationship 3. Claudins as CPE Receptors 4. CPE and Cancer Treatment 5. C-CPE and Cancer Treatment 6. Use of C-CPE for Other Medical Applications

this gram - positive anaerobe causes both histotoxic infections , such as gas gangrene ( clostridial myonecrosis ) , and enteric infections , such as human food poisoning . production of four of these toxins ( alpha , beta , epsilon , and iota ) is used to classify c. perfringens strains into one of five types ( a e ) . less than 5% of all c. perfringens type a isolates produce another toxin named c. perfringens enterotoxin ( cpe ) that is biomedically important , although not used in the toxin typing classification system . after a brief introduction to this unique toxin , promising efforts to utilize cpe , or its derivatives , for cancer therapy will be described cpe - producing c. perfringens type a strains cause the second most common bacterial food poisoning in the usa , as well as many cases of human nonfoodborne gastrointestinal diseases , such as antibiotic - associated diarrhea . molecular koch 's postulates analyses have demonstrated that production of cpe is essential for cpe - positive type a human food poisoning or nonfoodborne gastrointestinal disease isolates to cause gastrointestinal effects in animal models . most , but not all , c. perfringens type a food poisoning strains carry their enterotoxin gene ( cpe ) on the chromosome . in contrast , the cpe gene of type a nonfoodborne human disease strains is located on large ( ~7075 kb ) plasmids . these enterotoxin plasmids can be conjugative , presumably due to the presence of a tn916-like region named tcp that has been experimentally shown to mediate transfer of other c. perfringens conjugative plasmids . expression of the cpe gene is regulated by sporulation - associated alternative sigma factors [ 8 , 9 ] . specifically , one alternative sigma factor ( sigf ) controls expression of two other alternative sigma factors ( sigk and sige ) , which then direct transcription of cpe mrna from several sigk- or sige - dependent promoters located upstream of the cpe orf . exceptionally large amounts of cpe can be produced during sporulation ; for example , cpe can represent 20% of total protein in some sporulating cpe - positive c. perfringens type a strains . instead it accumulates in the cytoplasm of the mother cell until the completion of sporulation . when the mother cell then lyses to release the mature spore , cpe is released into the intestinal lumen , where it binds and acts as described in the following section . the in vivo outcome of natural cpe action during gastrointestinal disease is desquamation of the intestinal epithelium , intestinal necrosis , and the accumulation of luminal fluid . these effects account for the natural gastrointestinal symptoms of cpe - associated disease , which most commonly include diarrhea and abdominal cramps . however , this illness can be fatal in the elderly or in people suffering from medication - induced constipation . as shown in figure 1 , the current model of cpe action begins with binding of this toxin to claudin receptors ( described in detail below ) . besides cpe , the small complex also contains both claudin receptors and claudins incapable of binding cpe ( i.e. presumably the presence of nonreceptor claudins in small complex is attributable to claudin : claudin interactions . this hypothesis is based upon results of heteromer gel shift analyses , which identified the presence of six cpe molecules in each ch-1 complex . ch-1 initially assembles as a prepore on the membrane surface ; however , at 37c this prepore then rapidly inserts into membranes to form an active pore . formation of the ch-1 pore results in calcium influx , which ( via calpain activation ) leads to cell death [ 13 , 14 ] . at higher cpe doses , where large amounts of cpe pore formation results in a massive calcium influx , cells die from oncosis . in addition to six copies of cpe and both receptor and nonreceptor claudins , ch-2 also contains another tight junction protein named occludin [ 11 , 15 ] . formation of ch-2 leads to internalization of occludin into the cytoplasm ; claudins are also internalized inside native cpe - treated cells although it is not clear if this is due to ch-1 formation , ch-2 formation , or to formation of both complexes . these effects likely help to explain the observed ability of native cpe to disrupt tight junctions . the cpe structure / function relationship has been extensively analyzed by combined genetic , biochemical , and structural biology approaches ( figure 2 ) . as this review this structure revealed that cpe is a three - domain protein , reminiscent of several other pore - forming toxins . the n - terminal 37 amino acids of native cpe lack a definable structure and are not necessary for toxicity . this proteolytic activation occurs because removal of the n - terminal cpe sequences exposes the cpe region between amino acids 47 to 51 , which reside in domain ii , to promote ch-1 formation . site - directed mutagenesis studies showed that particularly important domain ii residues for ch-1 formation are ( i ) the asp at cpe residue 48 and ( ii ) the ile at cpe residue 51 . cpe domain ii also contains a region spanning from residues 81 to 106 that appears to be a transmembrane stem . this effect is consistent with cpe residues 81 to 106 mediating the insertion of this transmembrane stem into membranes . membrane insertion of all six transmembrane stems , from the six cpe proteins present in ch-1 , results in -barrel pore formation . domain iii may undergo structural changes during the prepore to pore transition that facilitate insertion of the cpe transmembrane stem into lipid bilayers , facilitating pore formation . over 20 years ago it was shown that a recombinant cpe fragment corresponding to the c - terminal half of the native toxin retains full receptor - binding activity . however , ( as expected from the preceding section ) this c - terminal cpe fragment named c - cpe was nontoxic since it lacks the n - terminal regions necessary for ch-1 formation and insertion of ch-1 into membranes to form a pore . deletion mutagenesis and synthetic peptide approaches later localized most cpe receptor - binding activity to the 30 c - terminal amino acids ( figure 2 ) . more recently , site - directed mutagenesis studies demonstrated that three tyr residues , located at positions 306 , 310 , and 312 of the native toxin protein are important for receptor binding . c - cpe ( residues 194 to 319 of the native toxin ) approximately corresponds to domain i of the native cpe protein [ 17 , 18 ] . domain i consists of a nine beta strand sandwich that shares structural similarity with the receptor binding domains of some other bacterial toxins , including the large cry family of bacillus thuringiensis toxins . by correlating this c - cpe structure with the previous binding - activity mapping studies described above , it is apparent that the receptor - binding site of cpe resides on a large loop located between beta strands 8 and 9 . mammalian tight junctions act as both fences and gates , that is , they represent important barriers for an epithelium and also regulate paracellular permeability . studies conducted over the past 15 years have determined that the tight junction is comprised of several proteins , the most important of which are the claudins . the claudins are a 24-member family of ~2025 kda proteins that are predicted to consist of four transmembrane domains , two extracellular loops ( ecl-1 and ecl-2 ) , and a cytoplasmic tail that can mediate signaling cascades . reported that when fibroblasts , which are naturally cpe- resistant , were transfected to express an ~22 kda vero cell protein , they gained cpe sensitivity . the vero cell protein expressed by these transfectants had properties of a cpe receptor since the fibroblast transfectants acquired the ability to bind significant levels of the toxin and to form high molecular weight complexes that are now recognized as ch-1 . this vero cell cpe receptor protein it was also determined that , at physiologic concentrations , the enterotoxin can bind to transfected fibroblasts expressing claudin-3 , -4 , -6 , -8 , or -14 . however , no cpe binding was detected to transfectants expressing claudins-1 , -2 , -5 , or -10 . demonstrated that cpe also interacts with claudins in naturally cpe - sensitive caco-2 cells , which are a human enterocyte - like cell line . using coimmunoprecipitation and electroelution approaches , this study showed both the cpe small complex and ch-1 large complex formed in caco-2 cells can contain , in addition to cpe , receptor claudins-3 and -4 , along with the nonreceptor claudin-1 . in addition to cpe , receptor claudins , and nonreceptor claudins , the ch-2 complex formed in caco-2 cells also contains occludin . however , as mentioned earlier ( and depicted in figure 2 ) , claudins are predicted to possess two extracellular loops named ecl-1 and ecl-2 . an early study using claudin chimeras consisting of the n - terminal half of cpe receptor claudin-4 fused with the c - terminal half of cpe nonreceptor claudin-1 suggested that cpe interacts with the putative ecl-2 region . this hypothesis was rigorously confirmed by a recent study using more specific chimeric claudins , which showed that substituting only the predicted ecl-2 sequence of claudin-4 into a claudin-1 backbone is sufficient to produce fibroblast transfectants that are very sensitive to cpe . the reverse was also true , that is , transfectants expressing a chimeric claudin , where only the claudin-1 ecl-2 had been specifically substituted into the claudin-4 backbone , were cpe - insensitive . several recent studies ( reviewed in ) have focused on understanding the specific ecl-2 residues of claudin receptors that mediate cpe binding . an asn residue in the middle of this turn appears to be important for cpe binding ; some evidence suggests that a leu residue located two residues from this asn may also participate in the binding of this toxin . as mentioned earlier , claudins are the major components of paracellular tight junctions ( tjs ) , distribute at the most apical junctions between epithelial cells , and play an essential role in the control of paracellular transport . furthermore , claudin-3 and -4 have been identified as the specific receptors for cpe , which is of potential therapeutic significance since these two claudins are abundantly expressed in ovarian , breast , uterine , and pancreatic cancers . while cpe can trigger lysis of epithelial cells by binding to claudin-3 and claudin-4 , with resultant initiation of massive permeability changes , osmotic cell ballooning , and cytolysis within 515 min ( see previous sections of this review ) , cells lacking expression of the cpe receptors are completely unaffected by this enterotoxin . these observations have raised the possibility that cpe may be an innovative claudin - targeted therapy for malignant tumors . in fact , efforts have been made to use cpe in the treatment of claudin - overexpressing cancers during the past few years . approximately 90% of patients with advanced - stage ovarian cancer develop recurrence and inevitably die from the development of chemotherapy resistance . previous studies have demonstrated that claudin-3 and -4 were among the six most differentially upregulated genes in ovarian cancer cells , but their expression is undetectable in normal ovaries . these overexpressed claudins may represent promising targets for the use of cpe as a tumor - targeting therapy against this aggressive disease . they found that all ovarian cancer cells , regardless of their resistance to chemotherapeutic agents , showed a dose - dependent cytotoxic response and died rapidly after 24 hours of exposure to cpe . therefore , cpe - based therapy may have potential as a novel treatment for chemotherapy - resistant ovarian cancer . breast cancer is one of the most common malignancies worldwide . claudin-3 and claudin-4 are overexpressed in most primary breast carcinomas and breast cancer brain metastases but undetectable in normal breast epithelial cells . administration of the same dose of cpe was toxic and had no effect on tumor volume . these two studies suggest that the local administration of cpe may be useful in the treatment of breast cancer . overexpression of claudin-3 and -4 has been found in uterine serous papillary carcinoma ( uspc ) and correlates with a more aggressive phenotype and a worse prognosis . the local / regional administrations of cpe were well tolerated without observable adverse events in animals . cpe has also been used to treat pancreatic and prostate cancers in nude mice or in vitro by different research groups . reported that intratumoral injections of cpe in pancreatic cancer xenografts resulted in apparent tumor suppression and necrosis in mice , and that cpe treatment also caused an acute dose - dependent cytotoxic effect in pancreatic cancer cells in vitro . these two preclinical studies suggest that cpe may have potential as a novel therapy for primary and metastatic malignancies expressing claudin-3 and -4 . although the specificity and rapidity of cpe - mediated cytolysis may increase anticancer efficacy and reduce opportunity for the development of drug resistance , its clinical application encounters some challenges . since claudin-3 and -4 are expressed in some normal tissues such as prostate , lung , and the gastrointestinal tract , systemic toxicity is an important concern for cpe therapy . in this context , many investigators have chosen the local administration of native cpe to treat cancers . this approach , however , does not appear to be a good option for some metastatic cancers such as lung metastasis or multiple metastases . moreover , some adverse events have sometimes been observed even following the local administration of cpe during several studies [ 30 , 34 ] . furthermore , cpe is recognized as a virulence factor responsible for the pathophysiological responses associated with a common food poisoning , proinflammatory cytokine response and other human diseases . it is unclear whether these serum cpe antibodies include neutralizing antibodies but cpe does contain at least one neutralizing epitope , present in the cpe binding domain . other challenges include determining optimal dosage and regimen , development of drug resistance , and long - term safety concerns . although the clinical application of cpe is limited by its potentially significant toxic side effects , the c - terminal binding domain of cpe ( c - cpe ) overcomes this drawback of cpe and has recently emerged as a promising cancer therapeutic agent due to its unique properties . for example , c - cpe can disrupt the paracellular tj barrier by binding to claudin-3 and -4 in the epithelia and thus improve drug delivery in a noncytotoxic fashion . the paracellular tjs are the primary barrier to the transport of solutes from the apical surface to the core of cells . given that claudin-3 and -4 are overexpressed in several cancers and are major components of cell tjs , their downregulation by c - cpe may prove a novel strategy for enhancing conventional chemotherapy delivery into claudin - positive cancer cells . our group recently investigated the efficacy of a combination therapy using a chemotherapeutic agent with c - cpe as compared to the single - agent chemotherapeutic agent . using three - dimensional and monolayer culture models and a xenograft mouse model of human eoc cells , we found that c - cpe enhanced the chemosensitivities of eoc cell lines to taxol or carboplatin at low concentrations in a claudin - dependent fashion . administration of c - cpe in combination with taxol significantly suppressed large tumor burdens by about 59% compared with control or taxol alone and showed no apparent toxic drawback of cpe as encountered in previous studies ( figure 4 ) . our study suggests that , at relatively low concentrations , c - cpe may enhance the sensitivity of eoc cells and other claudin - sensitive tumor cells to conventional chemotherapy and thus alleviate systemic side effects of the agents . indeed , c - cpe could be useful not only as an anticancer drug enhancer but also as a carrier to deliver a variety of toxins leading to a spectrum of new anticancer drugs of high selectivity . tnf- has been demonstrated to be an attractive antitumor agent in a variety of animal models but clinical application has been limited due to its failure to concentrate at the site of tumors and the development of severe side effects . engineered a c - cpe - tnf fusion toxin that was > 6.7-fold more cytotoxic than free tnf to ovarian cancer cells expressing claudin-3 and -4 ; whereas the tnf component in the fusion was 5-fold less potent than free tnf , suggesting that c - cpe - tnf fusion may prevent or decrease the systemic side effects caused by tnf . fused c - cpe to the protein synthesis inhibitory factor ( psif ) derived from pseudomonas aeruginosa exotoxin a for breast cancer that expresses high levels of claudin-3 and -4 . the c - cpe - psif fusion selectively damaged claudin - expressing breast cancer cell lines , and repeated intratumoral injections significantly suppressed tumor size by 36% without causing apparent side effects in mice . these two studies indicate that c - cpe may be a useful carrier for delivering toxins to claudin - sensitive malignancies with higher anticancer potency and less systemic side effects . the advantages of this claudin - targeted c - cpe - based cancer therapy are significant : tumor - targeted therapy , less immune response , and an improved therapeutic potency not achieved with previous treatments . there remain challenges to be overcome , however , before we can see any major medical advances in treating cancer with c - cpe : ( 1 ) determining safe dosages and schedules , ( 2 ) choosing optimal administration routes , ( 3 ) avoiding potential immunogenicity , and ( 4 ) improving effectiveness for preventing and/or treating cancer metastases . therefore , further studies and clinical trials are required to determine whether c - cpe could be developed as a claudin - targeted novel therapeutic agent for the treatment of cancer . in addition to cancer treatment , c - cpe has been utilized to treat other medical conditions . for example , with the intention of developing a potent mucosal vaccination approach , a nasal vaccine of c - cpe - fused antigen has been prepared and applied in mice without mucosal injury and side effects . in addition , the ability of c - cpe to enhance paracellular permeability has been exploited by using this protein to increase drug absorption from the intestines . one potential issue regarding the use of c - cpe for increasing intestinal drug absorption could be possible gastrointestinal side effects such as diarrhea , due to increased paracellular permeability . however , c - cpe does not increase luminal fluid levels in rabbit ileal loops , possibly arguing against this concern .

human prolactin ( prl ) is a hormone secreted by the anterior pituitary lactotropic cells . like any other anterior pituitary hormone , secretion of prl also falls under hypothalamic control . prl is unique amongst the adeno - hypophyseal hormones , in that the primary control of its secretion is inhibitory rather than stimulatory . dopamine is believed to be the principal prolactin inhibiting factor ( pif ) that regulates prl secretion ; -aminobutyric acid ( gaba ) can also inhibit prl release , but thyroid releasing hormone ( trh ) tends to stimulate its secretion . when a preprolactin molecule is cleaved , the resulting prl poly - peptide has a molecular weight of 23 kda . this monomeric form of prl is the major circulatory form and is also known as little prl and it is known to be both biologically and immuno - logically active . human prl is , however , found to be heterogenous in molecular size the other forms mainly include the big prl with a molecular weight of approximately 50 kda and the tetrameric big - big form with a molecular weight greater than 150 kda ( 1 , 2 ) . first described an interesting case of hyperprolactinemia with predominant big - big prl on gel chromatography . the patient showed no clinical symptoms related to hyperprolactinemia , such as amenorrhea or galactorrhea . despite high prl levels , anderson et al . also demonstrated the pre - dominance of the highest molecular weight prolactin in a woman complaining of infertility who conceived subsequently . they demonstrated the bioactivity of macroprolactin component in vitro and suggested that the absence of in vivo bioactivity might be the result of the high molecular mass of the complex preventing passage through the capillary endothelium to its target cells ( 4 ) . ( 5 ) first used the term macroprolactinemia for such patients with marked hyperprolactinemia whose prl mainly consisted of big - big prl . this review aims to discuss the etiology of hyperprolactinemia with a special emphasis on macroprolactinemia , its diagnostic strategies , its clinical implications and the importance of its detection in clinical settings . whittaker et al . first described an interesting case of hyperprolactinemia with predominant big - big prl on gel chromatography . the patient showed no clinical symptoms related to hyperprolactinemia , such as amenorrhea or galactorrhea . despite high prl levels , anderson et al . also demonstrated the pre - dominance of the highest molecular weight prolactin in a woman complaining of infertility who conceived subsequently . they demonstrated the bioactivity of macroprolactin component in vitro and suggested that the absence of in vivo bioactivity might be the result of the high molecular mass of the complex preventing passage through the capillary endothelium to its target cells ( 4 ) . later in1985 , jackson et al . ( 5 ) first used the term macroprolactinemia for such patients with marked hyperprolactinemia whose prl mainly consisted of big - big prl . this review aims to discuss the etiology of hyperprolactinemia with a special emphasis on macroprolactinemia , its diagnostic strategies , its clinical implications and the importance of its detection in clinical settings . a comprehensive literature search was conducted on the websites of the national library of medicine ( http://www.ncbl.nlm.nih.gov ) and pubmed central , the us national library of medicine 's digital archive of life sciences literature ( http://www.pubmedcentral.nih.gov/ ) . however , in some cases the high levels of prl can not be explained even after an extensive clinical , hormonal and neuro - radiological work - up ( 6 ) . some of these patients may have radiologically undetected microprolactinoma , however , some may present with macroprolactinemia . macroprolactinemia can be a significant cause of hyperprolactinemia and should not be overlooked while making a differential diagnosis for hyperprolactinemia . the common causes of hyperprolactinemia may be broadly grouped into physiological and pathological causes as described below : physiological causes includepregnancystresspain statesexcessive physical training physiological causes include excessive physical training repetitive mechanical stimulation of breastchest wall traumahepatorenal diseaseprimary hypothyroidismpituitary adenomaintracranial tumors compressing the pituitary stalk or hypothalamusempty sella syndromeprl stimulating drugs : dopaminergic blocking agentsdopaminergic depleting agentsnon - catecholamine dependent agentsh2 receptor blocking agentstricyclic antidepressantsidiopathic : ( unknown causes ) which may be due to macroprolactin repetitive mechanical stimulation of breast primary hypothyroidism intracranial tumors compressing the pituitary stalk or hypothalamus prl stimulating drugs : dopaminergic blocking agents dopaminergic depleting agents non - catecholamine dependent agents h2 receptor blocking agents tricyclic antidepressants idiopathic : ( unknown causes ) which may be due to macroprolactin the condition is characterized by the predominance of circulating high molecular mass prl forms which have coupled with anti - prl immunoglobulins . these autoantibodies have been found to be immunoglobin g ( igg ) isotypes with low receptor affinity ( 711 ) . the other evidence supporting the igg nature of the autoantibodies is the presence of macroprolactin in the fetal cord blood from a mother with macroprolactinemia ( 12 ) , suggesting the passive transfer of igg - bound prolactin from mother to fetus . a positive correlation has been demonstrated with anti - prl antibody titers and the serum prl concentrations indicating autoantibodies as a possible cause of hyperprolactinemia in such cases ( 10 ) . macroprolactinemia occurs when more than 30 - 60% of the patients prolactin is in the form of macroprolactin ( 13 ) . despite the high prevalence of macroprolactinemia , a speculation on the source of these antibodies suggests that posttranslational modifications ( glycosylation and phosphorylation ) of some proteins may create neo - epitopes for the production of autoantibodies ( 14 , 15 ) . posttranslational modifications of prl have also been reported : glycosylation in most species ; phosphorylation in rats , bovines and birds ; deamidation in rats , mice , sheep and humans and sulfation in ovine or sheep and buffalo ( 16 ) . furthermore , hattori et al . recently showed that human pituitary prl was phosphorylated at serine residues and it existed in partially dephosphorylated form in the blood ( 17 ) . if pituitary phosphorylated forms of prl are intolerant to the immune system , leakage of such forms of prl upon hypophysitis or lack of dephosphorylation may cause an autoimmune response leading to the presence of autoantibodies associated with macroprolactin . monomeric prolactin is bioactive , whereas macroprolactin is considered biologically inactive , although it retains its immunoreactivity properties ( 7 , 10 , 18 , 19 ) . a recent study on the characterization of macroprolactin confirmed that it mainly contained an igg molecule/ fragment with a prl molecule ( 20 ) . evaluated the relevant autoimmune markers in such cases _ sera were assayed for antithyroid antibodies , antinuclear antibodies , c - reactive protein ( crp ) and cd5 positive b cells . normal , hyperprolactinemic and macroprolactinemic seras were compared and the macroprolactinemic sera did not yield any evidence related to the increase of autoimmunity markers when compared with the other two , suggesting no probable association between macroprolactin autoimmunity ( 20 ) . the bound form of the molecule is unable to bind to its receptors resulting in the failure of hypothalamic negative feed - back mechanism leading to hyperprolactinemia . at the same time , macroprolactin is not readily cleared by the kidneys which lead to its increased concentration in the body . the clearance study done in rats has revealed that igg - bound prl is cleared from the circulation more slowly as compared to free prl ( 10 ) . a recent study has also demonstrated that anti - prl autoantibodies are stable for at least 5 weeks , suggesting that macroprolactinemia is a chronic condition in humans ( 21 ) . a great cause of concern lies in the fact that macroprolactinemia is often neglected in the differential diagnosis of hyperprolactinemia . consequently , patients may need to undergo unnecessary and costly diagnostic investigations , inappropriate treatments and unnecessary followups . the most common reasons for this could be the lack of awareness amongst specialists and also partly due to the lack of proper and cost - effective diagnostic methods . macroprolactin is found to interfere with most commercially available immunoassays used for prolactin . as a result , false high prolactin values ( apparent hyperprolactinemia ) are obtained and these values depend on the assay method employed ( 11 ) . immunoassays used for the measurement of prl have been subdivided into three classes according to their reactivity with macroprolactin : low- , medium- , and high - reading methods ( 22 ) . in the past , there are a few studies indicating the use of prolactin suppressive and stimulatory tests ( using bromocriptine , dopamine and trh ) in patients with macroprolactinemia but with conflicting results ( 10 , 23 ) . the gel filtration chromatography ( gfc ) is known to be the gold standard or the reference assay for detecting macroprolactin ( 24 ) , but it is a time - consuming and labor intensive method which discourages clinicians for the work - up of an unclear finding . however , in recent years poly - ethylene - glycol ( peg ) precipitation method has offered a simple , cheap and a rapid method for the detection of macroprolactin ( 25 ) . it is a highly suitable method for screening macroprolactin and is 27 times cheaper compared to gel filtration chromatography ( 26 ) . a concentration of 25% peg is added to an aliquot of serum specimen and the peg - treated sample is incubated for a short period and then centrifuged to precipitate out macroprolactin . after centrifugation , the supernatant containing the unprecipitated prolactin is tested along with an unprecipitated aliquot of the serum specimen . recovery of less than 40% after peg is considered a reliable diagnostic criteria for macroprolactin ; recovery values around 40 - 50% should ideally be taken for chromatographic work - up and values > 50% rule out macroprolactinemia ( 2527 ) . more recent studies have also confirmed peg precipitation method as a reliable and cost effective method for diagnosing macroprolactin ( 28 , 29 ) . ( 30 ) have found that monomeric prl is co - precipitated with serum globulins by peg and the increased serum globulin concentrations can increase the amount of monomeric prl precipitated by peg giving a false estimate of the monomeric prl and a false impression of macroprolactin presence . the results of peg precipitation test should , therefore , be interpreted with caution in patients with elevated serum globulin concentrations , such as in patients with igg myeloma and polyclonal hypergammaglobulinaemia due to human immunodeficiency virus [ hiv ] infection . pituitary imaging by computerized axial tomography ( ct ) or by magnetic resonance imaging ( mri ) are usually negative in patients with macroprolactinemia . however , in some cases , radiographic abnormalities appear , but the frequency is much less as compared to that seen in patients with hyperprolactinemia due to other causes . due to the unexpected high frequency of pituitary abnormalities observed in one of the studies , the author suggested that the diagnostic algorithm of hyperprolactinemic states should include both polyethylene glycol ( peg ) precipitation test and mri imaging ( 31 ) . however , in some cases the high levels of prl can not be explained even after an extensive clinical , hormonal and neuro - radiological work - up ( 6 ) . some of these patients may have radiologically undetected microprolactinoma , however , some may present with macroprolactinemia . macroprolactinemia can be a significant cause of hyperprolactinemia and should not be overlooked while making a differential diagnosis for hyperprolactinemia . the common causes of hyperprolactinemia may be broadly grouped into physiological and pathological causes as described below : physiological causes includepregnancystresspain statesexcessive physical training physiological causes include excessive physical training repetitive mechanical stimulation of breastchest wall traumahepatorenal diseaseprimary hypothyroidismpituitary adenomaintracranial tumors compressing the pituitary stalk or hypothalamusempty sella syndromeprl stimulating drugs : dopaminergic blocking agentsdopaminergic depleting agentsnon - catecholamine dependent agentsh2 receptor blocking agentstricyclic antidepressantsidiopathic : ( unknown causes ) which may be due to macroprolactin repetitive mechanical stimulation of breast primary hypothyroidism intracranial tumors compressing the pituitary stalk or hypothalamus prl stimulating drugs : dopaminergic blocking agents dopaminergic depleting agents non - catecholamine dependent agents h2 receptor blocking agents tricyclic antidepressants idiopathic : ( unknown causes ) which may be due to macroprolactin the condition is characterized by the predominance of circulating high molecular mass prl forms which have coupled with anti - prl immunoglobulins . these autoantibodies have been found to be immunoglobin g ( igg ) isotypes with low receptor affinity ( 711 ) . the other evidence supporting the igg nature of the autoantibodies is the presence of macroprolactin in the fetal cord blood from a mother with macroprolactinemia ( 12 ) , suggesting the passive transfer of igg - bound prolactin from mother to fetus . a positive correlation has been demonstrated with anti - prl antibody titers and the serum prl concentrations indicating autoantibodies as a possible cause of hyperprolactinemia in such cases ( 10 ) . macroprolactinemia occurs when more than 30 - 60% of the patients prolactin is in the form of macroprolactin ( 13 ) . despite the high prevalence of macroprolactinemia , a speculation on the source of these antibodies suggests that posttranslational modifications ( glycosylation and phosphorylation ) of some proteins may create neo - epitopes for the production of autoantibodies ( 14 , 15 ) . posttranslational modifications of prl have also been reported : glycosylation in most species ; phosphorylation in rats , bovines and birds ; deamidation in rats , mice , sheep and humans and sulfation in ovine or sheep and buffalo ( 16 ) . furthermore , hattori et al . recently showed that human pituitary prl was phosphorylated at serine residues and it existed in partially dephosphorylated form in the blood ( 17 ) . if pituitary phosphorylated forms of prl are intolerant to the immune system , leakage of such forms of prl upon hypophysitis or lack of dephosphorylation may cause an autoimmune response leading to the presence of autoantibodies associated with macroprolactin . monomeric prolactin is bioactive , whereas macroprolactin is considered biologically inactive , although it retains its immunoreactivity properties ( 7 , 10 , 18 , 19 ) . a recent study on the characterization of macroprolactin confirmed that it mainly contained an igg molecule/ fragment with a prl molecule ( 20 ) . evaluated the relevant autoimmune markers in such cases _ sera were assayed for antithyroid antibodies , antinuclear antibodies , c - reactive protein ( crp ) and cd5 positive b cells . normal , hyperprolactinemic and macroprolactinemic seras were compared and the macroprolactinemic sera did not yield any evidence related to the increase of autoimmunity markers when compared with the other two , suggesting no probable association between macroprolactin autoimmunity ( 20 ) . the bound form of the molecule is unable to bind to its receptors resulting in the failure of hypothalamic negative feed - back mechanism leading to hyperprolactinemia . at the same time , macroprolactin is not readily cleared by the kidneys which lead to its increased concentration in the body . the clearance study done in rats has revealed that igg - bound prl is cleared from the circulation more slowly as compared to free prl ( 10 ) . a recent study has also demonstrated that anti - prl autoantibodies are stable for at least 5 weeks , suggesting that macroprolactinemia is a chronic condition in humans ( 21 ) . a great cause of concern lies in the fact that macroprolactinemia is often neglected in the differential diagnosis of hyperprolactinemia . consequently , patients may need to undergo unnecessary and costly diagnostic investigations , inappropriate treatments and unnecessary followups . the most common reasons for this could be the lack of awareness amongst specialists and also partly due to the lack of proper and cost - effective diagnostic methods . macroprolactin is found to interfere with most commercially available immunoassays used for prolactin . as a result , false high prolactin values ( apparent hyperprolactinemia ) are obtained and these values depend on the assay method employed ( 11 ) . immunoassays used for the measurement of prl have been subdivided into three classes according to their reactivity with macroprolactin : low- , medium- , and high - reading methods ( 22 ) . in the past , there are a few studies indicating the use of prolactin suppressive and stimulatory tests ( using bromocriptine , dopamine and trh ) in patients with macroprolactinemia but with conflicting results ( 10 , 23 ) . the gel filtration chromatography ( gfc ) is known to be the gold standard or the reference assay for detecting macroprolactin ( 24 ) , but it is a time - consuming and labor intensive method which discourages clinicians for the work - up of an unclear finding . however , in recent years poly - ethylene - glycol ( peg ) precipitation method has offered a simple , cheap and a rapid method for the detection of macroprolactin ( 25 ) . it is a highly suitable method for screening macroprolactin and is 27 times cheaper compared to gel filtration chromatography ( 26 ) . a concentration of 25% peg is added to an aliquot of serum specimen and the peg - treated sample is incubated for a short period and then centrifuged to precipitate out macroprolactin . after centrifugation , the supernatant containing the unprecipitated prolactin is tested along with an unprecipitated aliquot of the serum specimen . recovery of less than 40% after peg is considered a reliable diagnostic criteria for macroprolactin ; recovery values around 40 - 50% should ideally be taken for chromatographic work - up and values > 50% rule out macroprolactinemia ( 2527 ) . more recent studies have also confirmed peg precipitation method as a reliable and cost effective method for diagnosing macroprolactin ( 28 , 29 ) . ( 30 ) have found that monomeric prl is co - precipitated with serum globulins by peg and the increased serum globulin concentrations can increase the amount of monomeric prl precipitated by peg giving a false estimate of the monomeric prl and a false impression of macroprolactin presence . the results of peg precipitation test should , therefore , be interpreted with caution in patients with elevated serum globulin concentrations , such as in patients with igg myeloma and polyclonal hypergammaglobulinaemia due to human immunodeficiency virus [ hiv ] infection . pituitary imaging by computerized axial tomography ( ct ) or by magnetic resonance imaging ( mri ) are usually negative in patients with macroprolactinemia . however , in some cases , radiographic abnormalities appear , but the frequency is much less as compared to that seen in patients with hyperprolactinemia due to other causes . due to the unexpected high frequency of pituitary abnormalities observed in one of the studies , the author suggested that the diagnostic algorithm of hyperprolactinemic states should include both polyethylene glycol ( peg ) precipitation test and mri imaging ( 31 ) . patients with macroprolactinemia usually have normal menstrual cycles , minimal galactor - rhea and spontaneous conception . however , some patients may present with clinical symptoms of hyperprolactinemia , if high levels of both macroprolactin and little prl are present ( 10 ) . in the study by hittori n. et al . , 15 women with macroprolactinemia were evaluated and it was found that 11 had normal menses , two had oligomenorrhea and two were in postmenopausal state ( 10 ) . two of these patients also complained of galactorrhea . in another study by vallette - kasic et al . , out of 106 patients with macroprolactinemia , 61% had normal menstruation and 54% did not have galactorrhea ( 24 ) . in another study undertaken in 14- to 40-year - old hyperandrogenic women with hyperprolactinemia , presence of macroprolactin was demonstrated in 55% of the patients ( 32 ) . taghavi m. et al . investigated 17 infertile women with hyperprolactinemia for macroprolactin using peg . galactor - rhea was present in 81.8% of women with true hyperprolactinemia and 33.3% of women with macroprolactinemia , whereas oligomenorrhea was found to be present in 90.9% and 16.6% of the women in the respective groups ( 33 ) . however , the pituitary images were normal in 45.5% of the women with true hyperprolactinemia and 100% of women with macroprolactinemia . the possible reasons for asymptomatic presentation of macroprolactinemia include binding of antibodies to the epitopes for prl receptors , thus , reducing the bioactivity ; while others may bind to the epitopes unrelated to receptor binding . hattori et al . recently reported that epitopes of anti - prl autoantibodies in patients with macroprolactinemia were located near the binding site 1 of human prl receptors ( 34 ) , suggesting a possibility that anti - prl autoantibodies may compete with the prl molecule for binding to its receptors , resulting in reduced in vivo bioactivity . the second reason for the absence of symptoms can probably be related to the limited activity of macroprolactin . there have been studies indicating lack of macroprolactin in the pituitary tissue and in the extravascular space ( cerebro - spinal fluid ) ( 35 ) . the culture medium of pituitary tissue also has shown no macroprolactin in cases of hyperprolactinemia attributable to macroprolactin ( 24 ) . this absence of macroprolactin in extravascular spaces and pituitary might be explained by the large molecular size/ changes in the net charges of the macroprolactin molecules , which can not cross the endothelium and thus remain in the intravascular compartment preventing its access to prl receptors at all . on the other hand the rise in the levels of little prl leads to the onset of clinical symptoms of hyperprolactinemia , suggesting that the binding of prl to the receptor and the post - receptor mechanisms are intact . however , recent studies have debated that macroprolactin may have some in vivo bioactivity ; therefore , it could be regarded as a circulating store of potentially bioactive prl . it is possible that intermittent dissociation from the low affinity , high capacity igg antibody in macroprolactin may lead to increased bioavailability of monomeric prl ( 36 ) . thus , there arises the need to differentiate between the apparent benign clinical condition of macroprolactinemia , that hyperprolactinemia is entirely explained by the presence of macroprolactin and the true hyperprolactinemia , which is due to increased levels of monomeric prl and requires therapy . macroprolactinemia is not known to require specific treatment ( 10 , 25 ) and no response to the antiprolactinemic therapy has been discovered in the studies carried out for this purpose ( 10 , 19 ) . however , one study showed that treatment with dopamine agonists lowers the serum macroprolactin levels ( 37 ) . the question on the clinical importance of high - molecular prolactin isoforms is stressed upon in modern scientific literature . for several years , macroprolactinaemia did not attract much attention as the identity of the large forms was unknown and their identification by gel filtration was difficult and expensive . however , over the last few years , the subject has been studied , especially the introduction of peg method for the diagnosis of macroprolactin . literature reveals that failure to identify macroprolactinaemia leads to unnecessary investigation , incorrect diagnosis and inappropriate treatment . the presence of macroprolactin should always be suspected when a patient 's clinical history and/ or radiographic data are incompatible with his/ her prl values . awareness amongst the medical laboratories is very important as not many laboratories take into account the interference of macroprolactin in prl assays . routine screening of patients with high prl values is strongly recommended so as to reduce the use of imaging and dopamine agonist treatment and to save time and money . however , it is recommended that laboratories should validate their method by comparison with gfc ( using their own prolactin assay ) before using the cost effective peg method . more studies characterizing the molecular aspects and the pathophysiology of macroprolactin are required to understand the clinical relevance and significance of macroprolactinemia .

introductionmacroprolactin is a significant cause of misdiagnosis , unnecessary investigation , and inappropriate treatment in patients with hyperprolactinemia . its frequency has not been clearly established due to technical difficulties in identifying it . most laboratories and clinicians are unaware of macroprolactin interferences in prolactin assays.materials and methodsa comprehensive literature search was conducted on the websites of the national library of medicine ( http://www.ncbl.nlm.nih.gov ) and pubmed central , the us national library of medicine 's digital archive of life sciences literature ( http://www.pubmedcentral.nih.gov/ ) . the data were also looked for in relevant books and journal.resultsmacroprolactin is a non - bioactive prolactin isoform usually composed of a prolactin monomer and an igg molecule having a prolonged clearance rate similar to that of immunoglobulins . this isoform is clinically non - reactive but it interferes with immunological assays used for the detection of prolactin.conclusionthere is a need to understand and explore the recent progress in the diagnosis and pathophysiology of macroprolactinemia for improving patient care .

Introduction Historical Significance Materials and Methods Results Etiology of Hyperprolactinemia Causes of Hyperprolactinemia Pathological causes Pathophysiology of Macroprolactinemia Laboratory Investigation for Macroprolactin Radiological Evaluations in Macroprolactinemia Discussion Conclusion

human prolactin ( prl ) is a hormone secreted by the anterior pituitary lactotropic cells . when a preprolactin molecule is cleaved , the resulting prl poly - peptide has a molecular weight of 23 kda . also demonstrated the pre - dominance of the highest molecular weight prolactin in a woman complaining of infertility who conceived subsequently . they demonstrated the bioactivity of macroprolactin component in vitro and suggested that the absence of in vivo bioactivity might be the result of the high molecular mass of the complex preventing passage through the capillary endothelium to its target cells ( 4 ) . ( 5 ) first used the term macroprolactinemia for such patients with marked hyperprolactinemia whose prl mainly consisted of big - big prl . also demonstrated the pre - dominance of the highest molecular weight prolactin in a woman complaining of infertility who conceived subsequently . they demonstrated the bioactivity of macroprolactin component in vitro and suggested that the absence of in vivo bioactivity might be the result of the high molecular mass of the complex preventing passage through the capillary endothelium to its target cells ( 4 ) . ( 5 ) first used the term macroprolactinemia for such patients with marked hyperprolactinemia whose prl mainly consisted of big - big prl . a comprehensive literature search was conducted on the websites of the national library of medicine ( http://www.ncbl.nlm.nih.gov ) and pubmed central , the us national library of medicine 's digital archive of life sciences literature ( http://www.pubmedcentral.nih.gov/ ) . macroprolactinemia can be a significant cause of hyperprolactinemia and should not be overlooked while making a differential diagnosis for hyperprolactinemia . the common causes of hyperprolactinemia may be broadly grouped into physiological and pathological causes as described below : physiological causes includepregnancystresspain statesexcessive physical training physiological causes include excessive physical training repetitive mechanical stimulation of breastchest wall traumahepatorenal diseaseprimary hypothyroidismpituitary adenomaintracranial tumors compressing the pituitary stalk or hypothalamusempty sella syndromeprl stimulating drugs : dopaminergic blocking agentsdopaminergic depleting agentsnon - catecholamine dependent agentsh2 receptor blocking agentstricyclic antidepressantsidiopathic : ( unknown causes ) which may be due to macroprolactin repetitive mechanical stimulation of breast primary hypothyroidism intracranial tumors compressing the pituitary stalk or hypothalamus prl stimulating drugs : dopaminergic blocking agents dopaminergic depleting agents non - catecholamine dependent agents h2 receptor blocking agents tricyclic antidepressants idiopathic : ( unknown causes ) which may be due to macroprolactin the condition is characterized by the predominance of circulating high molecular mass prl forms which have coupled with anti - prl immunoglobulins . the other evidence supporting the igg nature of the autoantibodies is the presence of macroprolactin in the fetal cord blood from a mother with macroprolactinemia ( 12 ) , suggesting the passive transfer of igg - bound prolactin from mother to fetus . a positive correlation has been demonstrated with anti - prl antibody titers and the serum prl concentrations indicating autoantibodies as a possible cause of hyperprolactinemia in such cases ( 10 ) . macroprolactinemia occurs when more than 30 - 60% of the patients prolactin is in the form of macroprolactin ( 13 ) . despite the high prevalence of macroprolactinemia , a speculation on the source of these antibodies suggests that posttranslational modifications ( glycosylation and phosphorylation ) of some proteins may create neo - epitopes for the production of autoantibodies ( 14 , 15 ) . recently showed that human pituitary prl was phosphorylated at serine residues and it existed in partially dephosphorylated form in the blood ( 17 ) . a recent study on the characterization of macroprolactin confirmed that it mainly contained an igg molecule/ fragment with a prl molecule ( 20 ) . evaluated the relevant autoimmune markers in such cases _ sera were assayed for antithyroid antibodies , antinuclear antibodies , c - reactive protein ( crp ) and cd5 positive b cells . the bound form of the molecule is unable to bind to its receptors resulting in the failure of hypothalamic negative feed - back mechanism leading to hyperprolactinemia . at the same time , macroprolactin is not readily cleared by the kidneys which lead to its increased concentration in the body . a recent study has also demonstrated that anti - prl autoantibodies are stable for at least 5 weeks , suggesting that macroprolactinemia is a chronic condition in humans ( 21 ) . a great cause of concern lies in the fact that macroprolactinemia is often neglected in the differential diagnosis of hyperprolactinemia . consequently , patients may need to undergo unnecessary and costly diagnostic investigations , inappropriate treatments and unnecessary followups . the most common reasons for this could be the lack of awareness amongst specialists and also partly due to the lack of proper and cost - effective diagnostic methods . macroprolactin is found to interfere with most commercially available immunoassays used for prolactin . as a result , false high prolactin values ( apparent hyperprolactinemia ) are obtained and these values depend on the assay method employed ( 11 ) . immunoassays used for the measurement of prl have been subdivided into three classes according to their reactivity with macroprolactin : low- , medium- , and high - reading methods ( 22 ) . in the past , there are a few studies indicating the use of prolactin suppressive and stimulatory tests ( using bromocriptine , dopamine and trh ) in patients with macroprolactinemia but with conflicting results ( 10 , 23 ) . the gel filtration chromatography ( gfc ) is known to be the gold standard or the reference assay for detecting macroprolactin ( 24 ) , but it is a time - consuming and labor intensive method which discourages clinicians for the work - up of an unclear finding . however , in recent years poly - ethylene - glycol ( peg ) precipitation method has offered a simple , cheap and a rapid method for the detection of macroprolactin ( 25 ) . it is a highly suitable method for screening macroprolactin and is 27 times cheaper compared to gel filtration chromatography ( 26 ) . after centrifugation , the supernatant containing the unprecipitated prolactin is tested along with an unprecipitated aliquot of the serum specimen . ( 30 ) have found that monomeric prl is co - precipitated with serum globulins by peg and the increased serum globulin concentrations can increase the amount of monomeric prl precipitated by peg giving a false estimate of the monomeric prl and a false impression of macroprolactin presence . the results of peg precipitation test should , therefore , be interpreted with caution in patients with elevated serum globulin concentrations , such as in patients with igg myeloma and polyclonal hypergammaglobulinaemia due to human immunodeficiency virus [ hiv ] infection . pituitary imaging by computerized axial tomography ( ct ) or by magnetic resonance imaging ( mri ) are usually negative in patients with macroprolactinemia . however , in some cases , radiographic abnormalities appear , but the frequency is much less as compared to that seen in patients with hyperprolactinemia due to other causes . due to the unexpected high frequency of pituitary abnormalities observed in one of the studies , the author suggested that the diagnostic algorithm of hyperprolactinemic states should include both polyethylene glycol ( peg ) precipitation test and mri imaging ( 31 ) . macroprolactinemia can be a significant cause of hyperprolactinemia and should not be overlooked while making a differential diagnosis for hyperprolactinemia . the common causes of hyperprolactinemia may be broadly grouped into physiological and pathological causes as described below : physiological causes includepregnancystresspain statesexcessive physical training physiological causes include excessive physical training repetitive mechanical stimulation of breastchest wall traumahepatorenal diseaseprimary hypothyroidismpituitary adenomaintracranial tumors compressing the pituitary stalk or hypothalamusempty sella syndromeprl stimulating drugs : dopaminergic blocking agentsdopaminergic depleting agentsnon - catecholamine dependent agentsh2 receptor blocking agentstricyclic antidepressantsidiopathic : ( unknown causes ) which may be due to macroprolactin repetitive mechanical stimulation of breast primary hypothyroidism intracranial tumors compressing the pituitary stalk or hypothalamus prl stimulating drugs : dopaminergic blocking agents dopaminergic depleting agents non - catecholamine dependent agents h2 receptor blocking agents tricyclic antidepressants idiopathic : ( unknown causes ) which may be due to macroprolactin the condition is characterized by the predominance of circulating high molecular mass prl forms which have coupled with anti - prl immunoglobulins . the other evidence supporting the igg nature of the autoantibodies is the presence of macroprolactin in the fetal cord blood from a mother with macroprolactinemia ( 12 ) , suggesting the passive transfer of igg - bound prolactin from mother to fetus . a positive correlation has been demonstrated with anti - prl antibody titers and the serum prl concentrations indicating autoantibodies as a possible cause of hyperprolactinemia in such cases ( 10 ) . macroprolactinemia occurs when more than 30 - 60% of the patients prolactin is in the form of macroprolactin ( 13 ) . despite the high prevalence of macroprolactinemia , a speculation on the source of these antibodies suggests that posttranslational modifications ( glycosylation and phosphorylation ) of some proteins may create neo - epitopes for the production of autoantibodies ( 14 , 15 ) . recently showed that human pituitary prl was phosphorylated at serine residues and it existed in partially dephosphorylated form in the blood ( 17 ) . monomeric prolactin is bioactive , whereas macroprolactin is considered biologically inactive , although it retains its immunoreactivity properties ( 7 , 10 , 18 , 19 ) . a recent study on the characterization of macroprolactin confirmed that it mainly contained an igg molecule/ fragment with a prl molecule ( 20 ) . evaluated the relevant autoimmune markers in such cases _ sera were assayed for antithyroid antibodies , antinuclear antibodies , c - reactive protein ( crp ) and cd5 positive b cells . the bound form of the molecule is unable to bind to its receptors resulting in the failure of hypothalamic negative feed - back mechanism leading to hyperprolactinemia . at the same time , macroprolactin is not readily cleared by the kidneys which lead to its increased concentration in the body . a recent study has also demonstrated that anti - prl autoantibodies are stable for at least 5 weeks , suggesting that macroprolactinemia is a chronic condition in humans ( 21 ) . a great cause of concern lies in the fact that macroprolactinemia is often neglected in the differential diagnosis of hyperprolactinemia . consequently , patients may need to undergo unnecessary and costly diagnostic investigations , inappropriate treatments and unnecessary followups . the most common reasons for this could be the lack of awareness amongst specialists and also partly due to the lack of proper and cost - effective diagnostic methods . macroprolactin is found to interfere with most commercially available immunoassays used for prolactin . as a result , false high prolactin values ( apparent hyperprolactinemia ) are obtained and these values depend on the assay method employed ( 11 ) . immunoassays used for the measurement of prl have been subdivided into three classes according to their reactivity with macroprolactin : low- , medium- , and high - reading methods ( 22 ) . in the past , there are a few studies indicating the use of prolactin suppressive and stimulatory tests ( using bromocriptine , dopamine and trh ) in patients with macroprolactinemia but with conflicting results ( 10 , 23 ) . the gel filtration chromatography ( gfc ) is known to be the gold standard or the reference assay for detecting macroprolactin ( 24 ) , but it is a time - consuming and labor intensive method which discourages clinicians for the work - up of an unclear finding . however , in recent years poly - ethylene - glycol ( peg ) precipitation method has offered a simple , cheap and a rapid method for the detection of macroprolactin ( 25 ) . it is a highly suitable method for screening macroprolactin and is 27 times cheaper compared to gel filtration chromatography ( 26 ) . after centrifugation , the supernatant containing the unprecipitated prolactin is tested along with an unprecipitated aliquot of the serum specimen . ( 30 ) have found that monomeric prl is co - precipitated with serum globulins by peg and the increased serum globulin concentrations can increase the amount of monomeric prl precipitated by peg giving a false estimate of the monomeric prl and a false impression of macroprolactin presence . the results of peg precipitation test should , therefore , be interpreted with caution in patients with elevated serum globulin concentrations , such as in patients with igg myeloma and polyclonal hypergammaglobulinaemia due to human immunodeficiency virus [ hiv ] infection . pituitary imaging by computerized axial tomography ( ct ) or by magnetic resonance imaging ( mri ) are usually negative in patients with macroprolactinemia . however , in some cases , radiographic abnormalities appear , but the frequency is much less as compared to that seen in patients with hyperprolactinemia due to other causes . due to the unexpected high frequency of pituitary abnormalities observed in one of the studies , the author suggested that the diagnostic algorithm of hyperprolactinemic states should include both polyethylene glycol ( peg ) precipitation test and mri imaging ( 31 ) . patients with macroprolactinemia usually have normal menstrual cycles , minimal galactor - rhea and spontaneous conception . in the study by hittori n. et al . , 15 women with macroprolactinemia were evaluated and it was found that 11 had normal menses , two had oligomenorrhea and two were in postmenopausal state ( 10 ) . two of these patients also complained of galactorrhea . , out of 106 patients with macroprolactinemia , 61% had normal menstruation and 54% did not have galactorrhea ( 24 ) . in another study undertaken in 14- to 40-year - old hyperandrogenic women with hyperprolactinemia , presence of macroprolactin was demonstrated in 55% of the patients ( 32 ) . investigated 17 infertile women with hyperprolactinemia for macroprolactin using peg . galactor - rhea was present in 81.8% of women with true hyperprolactinemia and 33.3% of women with macroprolactinemia , whereas oligomenorrhea was found to be present in 90.9% and 16.6% of the women in the respective groups ( 33 ) . however , the pituitary images were normal in 45.5% of the women with true hyperprolactinemia and 100% of women with macroprolactinemia . the possible reasons for asymptomatic presentation of macroprolactinemia include binding of antibodies to the epitopes for prl receptors , thus , reducing the bioactivity ; while others may bind to the epitopes unrelated to receptor binding . recently reported that epitopes of anti - prl autoantibodies in patients with macroprolactinemia were located near the binding site 1 of human prl receptors ( 34 ) , suggesting a possibility that anti - prl autoantibodies may compete with the prl molecule for binding to its receptors , resulting in reduced in vivo bioactivity . the second reason for the absence of symptoms can probably be related to the limited activity of macroprolactin . there have been studies indicating lack of macroprolactin in the pituitary tissue and in the extravascular space ( cerebro - spinal fluid ) ( 35 ) . the culture medium of pituitary tissue also has shown no macroprolactin in cases of hyperprolactinemia attributable to macroprolactin ( 24 ) . this absence of macroprolactin in extravascular spaces and pituitary might be explained by the large molecular size/ changes in the net charges of the macroprolactin molecules , which can not cross the endothelium and thus remain in the intravascular compartment preventing its access to prl receptors at all . on the other hand the rise in the levels of little prl leads to the onset of clinical symptoms of hyperprolactinemia , suggesting that the binding of prl to the receptor and the post - receptor mechanisms are intact . thus , there arises the need to differentiate between the apparent benign clinical condition of macroprolactinemia , that hyperprolactinemia is entirely explained by the presence of macroprolactin and the true hyperprolactinemia , which is due to increased levels of monomeric prl and requires therapy . macroprolactinemia is not known to require specific treatment ( 10 , 25 ) and no response to the antiprolactinemic therapy has been discovered in the studies carried out for this purpose ( 10 , 19 ) . the question on the clinical importance of high - molecular prolactin isoforms is stressed upon in modern scientific literature . for several years , macroprolactinaemia did not attract much attention as the identity of the large forms was unknown and their identification by gel filtration was difficult and expensive . however , over the last few years , the subject has been studied , especially the introduction of peg method for the diagnosis of macroprolactin . literature reveals that failure to identify macroprolactinaemia leads to unnecessary investigation , incorrect diagnosis and inappropriate treatment . the presence of macroprolactin should always be suspected when a patient 's clinical history and/ or radiographic data are incompatible with his/ her prl values . awareness amongst the medical laboratories is very important as not many laboratories take into account the interference of macroprolactin in prl assays . routine screening of patients with high prl values is strongly recommended so as to reduce the use of imaging and dopamine agonist treatment and to save time and money . more studies characterizing the molecular aspects and the pathophysiology of macroprolactin are required to understand the clinical relevance and significance of macroprolactinemia .

hypertension is a major independent risk factor for coronary artery disease , stroke , and renal failure . reducing blood pressure ( bp ) below the target goal is important to prevent cardiovascular and cerebrovascular events.1 various kinds of antihypertensive drugs such as diuretics , calcium channel blockers , beta blockers , angiotensin - converting enzyme ( ace ) inhibitors , and angiotensin - receptor blockers ( arbs ) have been used to lower bp effectively . fimasartan is a new antihypertensive drug that lowers bp by blocking the angiotensin ii type 1 receptor.2 the efficacy of fimasartan in reducing office - measured bp was shown to be greater than that of losartan.3 maintenance of 24-hour bp reduction by fimasartan was comparable to or slightly better than by valsartan.4 the safety profile of fimasartan was also similar to losartan and valsartan.3,4 despite the availability of various antihypertensive drugs , achieving target bp is difficult in the majority of patients with hypertension , although the control rate is improving.5,6 most patients require a combination of two or more drugs to achieve their target bp because effective bp reduction is difficult with monotherapy.7 the drug commonly used in combination with arbs is hydrochlorothiazide ( hctz ) . angiotensin aldosterone system ( raas ) inhibitor and hctz compared to either treatment alone.810 because administration of hctz alone reduces plasma volume and activates the raas,11 the addition of a raas inhibitor to hctz may offset the diuretic - induced increase in plasma renin activity and could theoretically attenuate the metabolic effects of hctz . the primary purpose of the study reported here was to compare the bp - lowering efficacy of fimasartan alone with that of fimasartan / hctz combination treatment in patients whose bp goal was not achieved after 4 weeks of treatment with once - daily fimasartan 60 mg . male and female patients aged 18 years and above were enrolled in the study if they met the following criteria : on the screening visit , mean values of two sitting diastolic blood pressure ( sidbp ) readings had to be < 110 mmhg if the patient were on antihypertensive medication ; if the patient were antihypertensive nave , the mean values of two sidbp readings had to be 90 mmhg and < 120 mmhg . patients were excluded if they had : a mean sitting systolic blood pressure ( sisbp ) 200 mmhg at the screening visit ; a difference of sisbp 20 mmhg or sidbp 10 mmhg between arms ; secondary hypertension ( ie , renovascular hypertension , endocrinologic disease , and use of hormonal contraceptives or drugs affecting bp ) ; hepatic ( aspartate transaminase and alanine transaminase 2.0 upper limit of normal ) or renal impairment ( serum creatinine 1.5 upper limit of normal ) ; active hepatitis b or c ( including carriers ) ; positive status for hiv ; known allergy to the study drugs ; a sodium ( < 133 mmol / l or 145 mmol / l ) or potassium ( < 3.5 mmol / l or 5.5 mmol / l ) electrolyte imbalance ; insulin - dependent or uncontrolled diabetes mellitus ( glycated hemoglobin [ hba1c ] > 9% ) ; retinal hemorrhage or exudates within the previous 6 months ; drug or alcohol dependency ; heart or cerebrovascular disease within the previous 6 months ( coronary heart disease , heart failure , significant valvular heart disease , cerebral infarction , or cerebral hemorrhage ) ; active inflammatory gastrointestinal disease in the preceding 12 months or a history of gastrointestinal surgery or disease that could interfere with drug absorption ; or the presence of a wasting disorder , autoimmune disease , or connective tissue disease . women who were pregnant , breastfeeding , or those with child - bearing potential who were not sterilized and had no intention of using a contraceptive were also excluded . the study was conducted at 18 institutions in korea as a multicenter , randomized , active - controlled , double - blind , parallel - group , dose - titration trial . the study design was approved by the korea food and drug administration and the institutional review board of each site . after screening , patients who met the eligibility criteria were given once - daily fimasartan 60 mg for 4 weeks . patients already receiving antihypertensive therapy discontinued taking their previously prescribed drugs and were directly rolled into once - daily fimasartan 60 mg without a washout period . after 4 weeks of treatment with once - daily fimasartan 60 mg , patients with sidbp 90 mmhg were randomly assigned to receive either once - daily fimasartan 60 mg / hctz 12.5 mg or fimasartan 60 mg for 4 weeks at a 2:1 ratio by using sealed envelopes with the randomization number . the dose of the study drug was increased to fimasartan 120 mg / hctz 12.5 mg or fimasartan 120 mg then maintained for another 4 weeks if the sidbp was still > 90 mmhg after 4 weeks of treatment with fimasartan 60 mg / hctz 12.5 mg and fimasartan 60 mg . the study - drug dose was maintained in patients whose sidbp was < 90 mmhg . all patients were instructed to take the study drug once daily between 6 am and 10 am for the study duration . patients were instructed to fast for 12 hours prior to the scheduled visit and to refrain from taking the study medication in the morning before trough bp measurement . at each visit , after at least 5 minutes of rest in a sitting position , sisbp , sidbp , and pulse rate were measured twice with a 1-minute interval between measurements in the same arm using a semiautomated sphygmomanometer ( hem-7080it [ equivalent to 705it ] , omron corporation , kyoto , japan).12 the average of the two sitting bp measurements was used . this study was designed to compare the antihypertensive efficacy of fimasartan 60 mg / hctz 12.5 mg combination treatment to that of treatment with fimasartan 60 mg alone in patients who did not achieve the target bp after 4 weeks of treatment with fimasartan 60 mg . the primary goal of this study was to compare the changes in mean sidbp from the baseline to week 4 of treatment with the study drug ( fimasartan 60 mg / hctz 12.5 mg ) and the control drug ( fimasartan 60 mg ) in patients who did not achieve target sidbp after 4 weeks of prior treatment with once - daily fimasartan 60 mg . the secondary efficacy points were : ( 1 ) change of mean sisbp from baseline at 4 and 8 weeks ; ( 2 ) bp control rate ( a proportion of patients who achieved mean sidbp < 90 mmhg ) and response rate ( a proportion of patients who achieved a reduction of sidbp 10 mmhg from baseline and/or a mean sidbp < 90 mmhg ) at 4 and 8 weeks ; and ( 3 ) change of mean sidbp from baseline at 8 weeks . safety and tolerability were assessed at each visit by physical examination , direct questioning , and clinical laboratory test . blood and urine samples were collected for laboratory tests at baseline , week 4 , and week 8 . all adverse events that occurred during the study and details of their nature , occurrence , and elimination date , duration , severity , significance , and the relationship with the study drug were recorded . to identify the primary hypothesis , we assumed a potential difference of 3 mmhg in mean change of sidbp with a standard deviation of 7.5 mmhg between the two therapies.3,1319 with a randomization ratio of 2:1 between patients assigned fimasartan 60 mg / hctz 12.5 mg and those assigned fimasartan 60 mg , significance level of 5% , and statistical power of 80% , the total number of required subjects was 225 ( 150 for fimasartan 60 mg / hctz 12.5 mg and 75 for fimasartan 60 mg ) . allowing for a drop - out rate of 10% , the total number of subjects to be enrolled was estimated to be 250 ( 167 for the study drug and 83 for the control drug ) . the number of subjects required for screening , assuming a nonresponder rate of 50% , was estimated to be approximately 500 . for efficacy analysis , the intention - to - treat ( itt ) analysis set was used for the main analysis while the per - protocol ( pp ) analysis set was additional . the itt analysis set included all subjects with efficacy assessment variables for at least one time after randomization . among the itt population , the pp analysis set included subjects who completed the study without major or serious protocol violations . if any efficacy assessment variables were missed , they were imputed by the last - observation - carried - forward method . for safety analysis , we included all subjects who received the investigational drug at least once . subjects who had been enrolled in the study but dropped out before drug administration were excluded . the changes from baseline in sidbp and sisbp of the two groups at the end of 4 and 8 weeks of treatment were compared by two - sample t - test . in addition , we conducted analysis of covariance for changes of sidbp with baseline sidbp and investigation centers as covariates , and the two treatment groups as the factor . descriptive statistics for the rates of the responders ( sidbp < 90 mmhg or sidbp reduction from the baseline 10 mmhg ) and control subjects ( sidbp < 90 mmhg ) at weeks 4 and 8 were calculated and the differences between the two groups were analyzed by pearson s chi - square . adverse events were coded using the medical dictionary for regulatory activities ( meddra ; v 13.0 ) and the percentage of the subjects who experienced any adverse events was determined . demographic characteristics were compared between the two groups using the t - test or wilcoxon rank - sum test for continuous variables and the chi - square test or fisher s exact test for categorical variables . all data were analyzed using sas software ( v 9.2 ; sas institute , cary , nc , usa ) and all tests were done at a significance level of 5% using a two - sided test . male and female patients aged 18 years and above were enrolled in the study if they met the following criteria : on the screening visit , mean values of two sitting diastolic blood pressure ( sidbp ) readings had to be < 110 mmhg if the patient were on antihypertensive medication ; if the patient were antihypertensive nave , the mean values of two sidbp readings had to be 90 mmhg and < 120 mmhg . patients were excluded if they had : a mean sitting systolic blood pressure ( sisbp ) 200 mmhg at the screening visit ; a difference of sisbp 20 mmhg or sidbp 10 mmhg between arms ; secondary hypertension ( ie , renovascular hypertension , endocrinologic disease , and use of hormonal contraceptives or drugs affecting bp ) ; hepatic ( aspartate transaminase and alanine transaminase 2.0 upper limit of normal ) or renal impairment ( serum creatinine 1.5 upper limit of normal ) ; active hepatitis b or c ( including carriers ) ; positive status for hiv ; known allergy to the study drugs ; a sodium ( < 133 mmol / l or 145 mmol / l ) or potassium ( < 3.5 mmol / l or 5.5 mmol / l ) electrolyte imbalance ; insulin - dependent or uncontrolled diabetes mellitus ( glycated hemoglobin [ hba1c ] > 9% ) ; retinal hemorrhage or exudates within the previous 6 months ; drug or alcohol dependency ; heart or cerebrovascular disease within the previous 6 months ( coronary heart disease , heart failure , significant valvular heart disease , cerebral infarction , or cerebral hemorrhage ) ; active inflammatory gastrointestinal disease in the preceding 12 months or a history of gastrointestinal surgery or disease that could interfere with drug absorption ; or the presence of a wasting disorder , autoimmune disease , or connective tissue disease . women who were pregnant , breastfeeding , or those with child - bearing potential who were not sterilized and had no intention of using a contraceptive were also excluded . the study was conducted at 18 institutions in korea as a multicenter , randomized , active - controlled , double - blind , parallel - group , dose - titration trial . the study design was approved by the korea food and drug administration and the institutional review board of each site . after screening , patients who met the eligibility criteria were given once - daily fimasartan 60 mg for 4 weeks . patients already receiving antihypertensive therapy discontinued taking their previously prescribed drugs and were directly rolled into once - daily fimasartan 60 mg without a washout period . after 4 weeks of treatment with once - daily fimasartan 60 mg , patients with sidbp 90 mmhg were randomly assigned to receive either once - daily fimasartan 60 mg / hctz 12.5 mg or fimasartan 60 mg for 4 weeks at a 2:1 ratio by using sealed envelopes with the randomization number . the dose of the study drug was increased to fimasartan 120 mg / hctz 12.5 mg or fimasartan 120 mg then maintained for another 4 weeks if the sidbp was still > 90 mmhg after 4 weeks of treatment with fimasartan 60 mg / hctz 12.5 mg and fimasartan 60 mg . the study - drug dose was maintained in patients whose sidbp was < 90 mmhg . all patients were instructed to take the study drug once daily between 6 am and 10 am for the study duration . patients were instructed to fast for 12 hours prior to the scheduled visit and to refrain from taking the study medication in the morning before trough bp measurement . at each visit , after at least 5 minutes of rest in a sitting position , sisbp , sidbp , and pulse rate were measured twice with a 1-minute interval between measurements in the same arm using a semiautomated sphygmomanometer ( hem-7080it [ equivalent to 705it ] , omron corporation , kyoto , japan).12 the average of the two sitting bp measurements was used . this study was designed to compare the antihypertensive efficacy of fimasartan 60 mg / hctz 12.5 mg combination treatment to that of treatment with fimasartan 60 mg alone in patients who did not achieve the target bp after 4 weeks of treatment with fimasartan 60 mg . the primary goal of this study was to compare the changes in mean sidbp from the baseline to week 4 of treatment with the study drug ( fimasartan 60 mg / hctz 12.5 mg ) and the control drug ( fimasartan 60 mg ) in patients who did not achieve target sidbp after 4 weeks of prior treatment with once - daily fimasartan 60 mg . the secondary efficacy points were : ( 1 ) change of mean sisbp from baseline at 4 and 8 weeks ; ( 2 ) bp control rate ( a proportion of patients who achieved mean sidbp < 90 mmhg ) and response rate ( a proportion of patients who achieved a reduction of sidbp 10 mmhg from baseline and/or a mean sidbp < 90 mmhg ) at 4 and 8 weeks ; and ( 3 ) change of mean sidbp from baseline at 8 weeks . safety and tolerability were assessed at each visit by physical examination , direct questioning , and clinical laboratory test . blood and urine samples were collected for laboratory tests at baseline , week 4 , and week 8 . all adverse events that occurred during the study and details of their nature , occurrence , and elimination date , duration , severity , significance , and the relationship with the study drug were recorded . to identify the primary hypothesis , we assumed a potential difference of 3 mmhg in mean change of sidbp with a standard deviation of 7.5 mmhg between the two therapies.3,1319 with a randomization ratio of 2:1 between patients assigned fimasartan 60 mg / hctz 12.5 mg and those assigned fimasartan 60 mg , significance level of 5% , and statistical power of 80% , the total number of required subjects was 225 ( 150 for fimasartan 60 mg / hctz 12.5 mg and 75 for fimasartan 60 mg ) . allowing for a drop - out rate of 10% , the total number of subjects to be enrolled was estimated to be 250 ( 167 for the study drug and 83 for the control drug ) . the number of subjects required for screening , assuming a nonresponder rate of 50% , was estimated to be approximately 500 . for efficacy analysis , the intention - to - treat ( itt ) analysis set was used for the main analysis while the per - protocol ( pp ) analysis set was additional . the itt analysis set included all subjects with efficacy assessment variables for at least one time after randomization . among the itt population , the pp analysis set included subjects who completed the study without major or serious protocol violations . if any efficacy assessment variables were missed , they were imputed by the last - observation - carried - forward method . for safety analysis subjects who had been enrolled in the study but dropped out before drug administration were excluded . the changes from baseline in sidbp and sisbp of the two groups at the end of 4 and 8 weeks of treatment were compared by two - sample t - test . in addition , we conducted analysis of covariance for changes of sidbp with baseline sidbp and investigation centers as covariates , and the two treatment groups as the factor . descriptive statistics for the rates of the responders ( sidbp < 90 mmhg or sidbp reduction from the baseline 10 mmhg ) and control subjects ( sidbp < 90 mmhg ) at weeks 4 and 8 were calculated and the differences between the two groups were analyzed by pearson s chi - square . adverse events were coded using the medical dictionary for regulatory activities ( meddra ; v 13.0 ) and the percentage of the subjects who experienced any adverse events was determined . demographic characteristics were compared between the two groups using the t - test or wilcoxon rank - sum test for continuous variables and the chi - square test or fisher s exact test for categorical variables . all data were analyzed using sas software ( v 9.2 ; sas institute , cary , nc , usa ) and all tests were done at a significance level of 5% using a two - sided test . among 654 patients screened , 263 were matched to eligible randomization criteria after 4 weeks of treatment with once - daily fimasartan 60 mg . of the 263 patients , 175 were assigned to 4 weeks of fimasartan / htcz treatment and 88 were assigned to 4 weeks of fimasartan treatment ( figure 1 ) . among the remaining 391 patients who were not randomized , 233 ( 59.6% ) had a diastolic bp < 90 mmhg meeting the exclusion criterion of randomization . of these , six discontinued because of inclusion / exclusion deviation , 14 for consent withdrawal , five for adverse events , two for unsatisfactory responses , and three for other reasons . of the 263 patients , 256 were included in the primary efficacy analysis and seven were excluded because of missing efficacy data . there were no significant differences in baseline characteristics between the groups with the exception of the number of smokers ( table 1 ) . the number of smokers was higher in the fimasartan / hctz group than in the fimasartan group . the majority of patients were men ( 76.6% ) . among the prior medication taken by the patients , one had been on a cardiac drug ( nicorandil ) , and another took a peripheral vasodilator ( nicametate citrate ) . the baseline sidbp and sisbp were not different between the treatment groups ( p=0.7032 and 0.4015 , respectively ) . the means of trough sidbp and sisbp at baseline , week 4 , and week 8 are presented in table 2 . the reduction of sidbp and sisbp was greater in the fimasartan / hctz group than in the fimasartan group . the reduction of sidbp was 6.888.10 mmhg in the fimasartan / hctz group and 3.387.33 mmhg in the fimasartan group at week 4 ( p=0.0008 ) . the difference in sidbp reduction between the treatment groups was 3.35 mmhg ( 95% confidence interval : 5.39 to 1.32 , p=0.0013 by analysis of covariance ) . at week 8 , the reduction in sidbp was 8.679.39 mmhg in the fimasartan / hctz group and 5.028.27 mmhg in the fimasartan group ( p=0.0023 ) . the reduction in sisbp was 10.5013.76 mmhg in the fimasartan / hctz group and 5.7512.18 mmhg in the fimasartan group at week 4 ( p=0.0069 ) . at week 8 , the reduction in sisbp was 13.4515.15 mmhg in the fimasartan / hctz group and 6.8413.57 mmhg in the fimasartan group ( p=0.0007 ) . when the fimasartan dose of both treatment groups was increased to 120 mg in patients who did not achieve the target sidbp ( 90 mmhg ) at week 4 , although there was no statistical significance , the fimasartan / hctz group showed greater reduction of sidbp and sisbp compared to the fimasartan group . reduction in sidbp was 5.729.34 mmhg in the fimasartan / hctz group and 3.646.97 mmhg in the fimasartan group ( p=0.1922 ) . reduction in sisbp was 7.8615.40 mmhg in the fimasartan / hctz group and 4.4511.45 mmhg in the fimasartan group ( p=0.1943 ) . the response rate at week 4 was 53.6% ( 90/168 ) in the fimasartan / hctz group and 39.8% ( 35/88 ) in the fimasartan group ( p=0.0359 ) . at week 8 , the response rate was 63.1% ( 106/168 ) in the fimasartan / hctz group and 51.1% ( 45/88 ) in the fimasartan group , which was not statistically different ( p=0.0647 ) . in the pp analysis , the response rate of the fimasartan / htcz treatment group was higher than that of the fimasartan treatment group ( at week 4 , 55.7% vs 40.2% , p=0.0245 and , at week 8 , 67.1% vs 52.4% , p=0.0280 ) . although there was no statistically significant difference , the fimasartan / hctz group had a higher control rate at weeks 4 and 8 . in the safety - analysis population , the incidence of adverse events considered to be at least partly related to the study drugs was 11.79% ( table 4 ) . there was no statistically significant difference in the incidence of headache and dizziness between the treatment groups ( p=0.0698 and 0.6568 , respectively ) . among the other adverse events with an incidence of < 1% patients in the fimasartan / hctz group reported chest discomfort , asthenia , pruritus , erectile dysfunction , and flushing sensations . the incidence of significant changes in laboratory parameters was small . in both treatment groups , the elevation of aspartate transaminase or alanine transaminase in three patients was noted to be treatment related . the level of serum potassium increased to 5.3 mmol / l at week 8 from 4.7 mmol / l at baseline and 4.3 mmol / l at week 4 in a patient from the fimasartan / hctz treatment group . the level of serum sodium decreased to 124 mmol / l at week 8 , from 135 mmol / l at baseline , and 127 mmol / l at week 4 in a patient from the fimasartan / hctz treatment group . among 654 patients screened , 263 were matched to eligible randomization criteria after 4 weeks of treatment with once - daily fimasartan 60 mg . of the 263 patients , 175 were assigned to 4 weeks of fimasartan / htcz treatment and 88 were assigned to 4 weeks of fimasartan treatment ( figure 1 ) . among the remaining 391 patients who were not randomized , 233 ( 59.6% ) had a diastolic bp < 90 mmhg meeting the exclusion criterion of randomization . of these , six discontinued because of inclusion / exclusion deviation , 14 for consent withdrawal , five for adverse events , two for unsatisfactory responses , and three for other reasons . of the 263 patients , 256 were included in the primary efficacy analysis and seven were excluded because of missing efficacy data . there were no significant differences in baseline characteristics between the groups with the exception of the number of smokers ( table 1 ) . the number of smokers was higher in the fimasartan / hctz group than in the fimasartan group . the majority of patients were men ( 76.6% ) . among the prior medication taken by the patients , one had been on a cardiac drug ( nicorandil ) , and another took a peripheral vasodilator ( nicametate citrate ) . the baseline sidbp and sisbp were not different between the treatment groups ( p=0.7032 and 0.4015 , respectively ) . the means of trough sidbp and sisbp at baseline , week 4 , and week 8 are presented in table 2 . the reduction of sidbp and sisbp was greater in the fimasartan / hctz group than in the fimasartan group . the reduction of sidbp was 6.888.10 mmhg in the fimasartan / hctz group and 3.387.33 mmhg in the fimasartan group at week 4 ( p=0.0008 ) . the difference in sidbp reduction between the treatment groups was 3.35 mmhg ( 95% confidence interval : 5.39 to 1.32 , p=0.0013 by analysis of covariance ) . at week 8 , the reduction in sidbp was 8.679.39 mmhg in the fimasartan / hctz group and 5.028.27 mmhg in the fimasartan group ( p=0.0023 ) . the reduction in sisbp was 10.5013.76 mmhg in the fimasartan / hctz group and 5.7512.18 mmhg in the fimasartan group at week 4 ( p=0.0069 ) . at week 8 , the reduction in sisbp was 13.4515.15 mmhg in the fimasartan / hctz group and 6.8413.57 mmhg in the fimasartan group ( p=0.0007 ) . when the fimasartan dose of both treatment groups was increased to 120 mg in patients who did not achieve the target sidbp ( 90 mmhg ) at week 4 , sidbp and sisbp decreased significantly by week 8 ( table 3 ) . although there was no statistical significance , the fimasartan / hctz group showed greater reduction of sidbp and sisbp compared to the fimasartan group . reduction in sidbp was 5.729.34 mmhg in the fimasartan / hctz group and 3.646.97 mmhg in the fimasartan group ( p=0.1922 ) . reduction in sisbp was 7.8615.40 mmhg in the fimasartan / hctz group and 4.4511.45 mmhg in the fimasartan group ( p=0.1943 ) . the response rate at week 4 was 53.6% ( 90/168 ) in the fimasartan / hctz group and 39.8% ( 35/88 ) in the fimasartan group ( p=0.0359 ) . at week 8 , the response rate was 63.1% ( 106/168 ) in the fimasartan / hctz group and 51.1% ( 45/88 ) in the fimasartan group , which was not statistically different ( p=0.0647 ) . in the pp analysis , the response rate of the fimasartan / htcz treatment group was higher than that of the fimasartan treatment group ( at week 4 , 55.7% vs 40.2% , p=0.0245 and , at week 8 , 67.1% vs 52.4% , p=0.0280 ) . although there was no statistically significant difference , the fimasartan / hctz group had a higher control rate at weeks 4 and 8 . in the safety - analysis population , the incidence of adverse events considered to be at least partly related to the study drugs was 11.79% ( table 4 ) . there was no statistically significant difference in the incidence of headache and dizziness between the treatment groups ( p=0.0698 and 0.6568 , respectively ) . among the other adverse events with an incidence of < 1% patients in the fimasartan / hctz group reported chest discomfort , asthenia , pruritus , erectile dysfunction , and flushing sensations . the incidence of significant changes in laboratory parameters was small . in both treatment groups , the elevation of aspartate transaminase or alanine transaminase in three patients was noted to be treatment related . the level of serum potassium increased to 5.3 mmol / l at week 8 from 4.7 mmol / l at baseline and 4.3 mmol / l at week 4 in a patient from the fimasartan / hctz treatment group . the level of serum sodium decreased to 124 mmol / l at week 8 , from 135 mmol / l at baseline , and 127 mmol / l at week 4 in a patient from the fimasartan / hctz treatment group . as far as we are aware , this is the first study to have demonstrated that combination treatment with fimasartan and hctz is effective in the treatment of patients with essential hypertension who respond poorly to fimasartan monotherapy . similar to the studies that evaluated combination arb and hctz treatment,8,10,20,21 the combination treatment of fimasartan and hctz had better efficacy than fimasartan monotherapy . after 4 weeks of treatment , the combination of fimasartan and hctz was more effective in reducing sidbp and sisbp than fimasartan monotherapy . the difference in reduction of sidbp and sisbp between combination treatment and monotherapy was statistically significant ( p<0.05 ) . the enhanced bp - lowering effect of combination therapy persisted over 8 weeks of treatment with optional dose escalation . although not significant , the control rate of the combination treatment was higher than that of the monotherapy . the magnitude of bp lowering was comparable to other combination treatments of arbs and hctz although they can not be compared directly due to differences in study design.9,10,22 the difference in bp lowering between 8 weeks treatment of valsartan 160 mg plus htcz 12.5 mg and valsartan 160 mg was approximately 2.0 mmhg in sidbp and 3.7 mmhg in sisbp.22 combination olmasartan and hctz treatment also showed similar bp lowering compared to monotherapy ( sidbp : ~3.4 mmhg , sisbp : ~5.3 mmhg).10 in the current study , the difference in bp lowering after 4 weeks treatment was 3.50 mmhg in sidbp and 4.75 mmhg in sisbp . the incidence did not differ between the treatment groups , and was also comparable to that reported in the previous phase ii and iii studies of fimasartan.3,23 in terms of biochemical abnormalities , hypokalemia is a commonly associated biochemical abnormality of hctz.24,25 in the current study , no patient exhibited hypokalemia . it may be the addition of arb which resulted in favorable effects regarding the biochemical abnormalities associated with hctz . another biochemical abnormality associated with hctz is increase in triglyceride,26 which appeared in only one patient in the current study . three patients experienced treatment - related elevation of liver enzyme after escalation of the fimasartan dose to 120 mg / day . the elevated liver enzyme level in two patients returned to normal at the follow - up examination without intervention . the persistent elevation of liver enzyme in the remaining patient at the follow - up examination was concluded to be related to drinking alcohol . the elevation of liver enzyme at the higher dose is consistent with previous studies3,4,23 and improved without intervention . a limitation of the current study is that a dose - dependent reduction in bp was not presented , because the study design did not aim at evaluating the dose response relation . despite this limitation , dose escalation of fimasartan / hctz to 120 mg/12.5 mg or fimasartan 120 mg in patients who did not achieve the target sidbp at week 4 resulted in significant reductions in sidbp and sisbp at week 8 , indicating a dose - dependent bp - lowering effect . another limitation is that the effect of combination treatment with hctz 25 mg was not evaluated . although several studies have shown that 25 mg of hctz is more effective in lowering bp , we did not evaluate hctz at that dose because of potential biochemical abnormalities such as potassium depletion or uric acid elevation.8,9 the results of the current study suggest that combination treatment with fimasartan and hctz is effective in patients with hypertension which is not adequately controlled with fimasartan alone . the safety and tolerability of combination fimasartan and hctz treatment are comparable to those of fimasartan monotherapy .

backgroundthe study reported here compared the blood pressure ( bp)-lowering efficacy of fimasartan alone with that of fimasartan / hydrochlorothiazide ( hctz ) combination in patients whose bp goal was not achieved after 4 weeks of treatment with once - daily fimasartan 60 mg.methodspatients with sitting diastolic blood pressure ( sidbp ) 90 mmhg with 4 weeks of once - daily fimasartan 60 mg were randomly assigned to receive either once - daily fimasartan 60 mg / hctz 12.5 mg or fimasartan 60 mg for 4 weeks . after 4 weeks , the dose was increased from fimasartan 60 mg / hctz 12.5 mg to fimasartan 120 mg / hctz 12.5 mg or from fimasartan 60 mg to fimasartan 120 mg if sidbp was 90 mmhg.resultsof the 263 randomized patients , 256 patients who had available efficacy data were analyzed . the fimasartan / hctz treatment group showed a greater reduction of sidbp compared to the fimasartan treatment group at week 4 ( 6.888.10 mmhg vs 3.387.33 , p=0.0008 ) , and the effect persisted at week 8 ( 8.679.39 mmhg vs 5.028.27 mmhg , p=0.0023 ) . reduction of sitting systolic bp in the fimasartan / hctz treatment group was also greater than that in the fimasartan treatment group ( at week 4 , 10.5013.76 mmhg vs 5.7512.18 mmhg , p=0.0069 and , at week 8 , 13.4515.15 mmhg vs 6.8413.57 mmhg , p=0.0007 ) . the proportion of patients who achieved a reduction of sidbp 10 mmhg from baseline and/or a mean sidbp < 90 mmhg after 4 weeks of treatment was higher in the fimasartan / hctz treatment group than in the fimasartan treatment group ( 53.6% vs 39.8% , p=0.0359 ) . the overall incidence of adverse drug reaction was 11.79% with no significant difference between the treatment groups.conclusionthe combination treatment of fimasartan and hctz achieved better bp control than fimasartan monotherapy , and had comparable safety and tolerance to fimasartan monotherapy .

Introduction Methods Patients Study design Efficacy evaluation Safety evaluation Sample size Statistical analyses Results Patients disposition Efficacy Safety and tolerability Discussion Limitations Conclusion

fimasartan is a new antihypertensive drug that lowers bp by blocking the angiotensin ii type 1 receptor.2 the efficacy of fimasartan in reducing office - measured bp was shown to be greater than that of losartan.3 maintenance of 24-hour bp reduction by fimasartan was comparable to or slightly better than by valsartan.4 the safety profile of fimasartan was also similar to losartan and valsartan.3,4 despite the availability of various antihypertensive drugs , achieving target bp is difficult in the majority of patients with hypertension , although the control rate is improving.5,6 most patients require a combination of two or more drugs to achieve their target bp because effective bp reduction is difficult with monotherapy.7 the drug commonly used in combination with arbs is hydrochlorothiazide ( hctz ) . the primary purpose of the study reported here was to compare the bp - lowering efficacy of fimasartan alone with that of fimasartan / hctz combination treatment in patients whose bp goal was not achieved after 4 weeks of treatment with once - daily fimasartan 60 mg . male and female patients aged 18 years and above were enrolled in the study if they met the following criteria : on the screening visit , mean values of two sitting diastolic blood pressure ( sidbp ) readings had to be < 110 mmhg if the patient were on antihypertensive medication ; if the patient were antihypertensive nave , the mean values of two sidbp readings had to be 90 mmhg and < 120 mmhg . patients were excluded if they had : a mean sitting systolic blood pressure ( sisbp ) 200 mmhg at the screening visit ; a difference of sisbp 20 mmhg or sidbp 10 mmhg between arms ; secondary hypertension ( ie , renovascular hypertension , endocrinologic disease , and use of hormonal contraceptives or drugs affecting bp ) ; hepatic ( aspartate transaminase and alanine transaminase 2.0 upper limit of normal ) or renal impairment ( serum creatinine 1.5 upper limit of normal ) ; active hepatitis b or c ( including carriers ) ; positive status for hiv ; known allergy to the study drugs ; a sodium ( < 133 mmol / l or 145 mmol / l ) or potassium ( < 3.5 mmol / l or 5.5 mmol / l ) electrolyte imbalance ; insulin - dependent or uncontrolled diabetes mellitus ( glycated hemoglobin [ hba1c ] > 9% ) ; retinal hemorrhage or exudates within the previous 6 months ; drug or alcohol dependency ; heart or cerebrovascular disease within the previous 6 months ( coronary heart disease , heart failure , significant valvular heart disease , cerebral infarction , or cerebral hemorrhage ) ; active inflammatory gastrointestinal disease in the preceding 12 months or a history of gastrointestinal surgery or disease that could interfere with drug absorption ; or the presence of a wasting disorder , autoimmune disease , or connective tissue disease . after screening , patients who met the eligibility criteria were given once - daily fimasartan 60 mg for 4 weeks . after 4 weeks of treatment with once - daily fimasartan 60 mg , patients with sidbp 90 mmhg were randomly assigned to receive either once - daily fimasartan 60 mg / hctz 12.5 mg or fimasartan 60 mg for 4 weeks at a 2:1 ratio by using sealed envelopes with the randomization number . the dose of the study drug was increased to fimasartan 120 mg / hctz 12.5 mg or fimasartan 120 mg then maintained for another 4 weeks if the sidbp was still > 90 mmhg after 4 weeks of treatment with fimasartan 60 mg / hctz 12.5 mg and fimasartan 60 mg . this study was designed to compare the antihypertensive efficacy of fimasartan 60 mg / hctz 12.5 mg combination treatment to that of treatment with fimasartan 60 mg alone in patients who did not achieve the target bp after 4 weeks of treatment with fimasartan 60 mg . the primary goal of this study was to compare the changes in mean sidbp from the baseline to week 4 of treatment with the study drug ( fimasartan 60 mg / hctz 12.5 mg ) and the control drug ( fimasartan 60 mg ) in patients who did not achieve target sidbp after 4 weeks of prior treatment with once - daily fimasartan 60 mg . the secondary efficacy points were : ( 1 ) change of mean sisbp from baseline at 4 and 8 weeks ; ( 2 ) bp control rate ( a proportion of patients who achieved mean sidbp < 90 mmhg ) and response rate ( a proportion of patients who achieved a reduction of sidbp 10 mmhg from baseline and/or a mean sidbp < 90 mmhg ) at 4 and 8 weeks ; and ( 3 ) change of mean sidbp from baseline at 8 weeks . to identify the primary hypothesis , we assumed a potential difference of 3 mmhg in mean change of sidbp with a standard deviation of 7.5 mmhg between the two therapies.3,1319 with a randomization ratio of 2:1 between patients assigned fimasartan 60 mg / hctz 12.5 mg and those assigned fimasartan 60 mg , significance level of 5% , and statistical power of 80% , the total number of required subjects was 225 ( 150 for fimasartan 60 mg / hctz 12.5 mg and 75 for fimasartan 60 mg ) . descriptive statistics for the rates of the responders ( sidbp < 90 mmhg or sidbp reduction from the baseline 10 mmhg ) and control subjects ( sidbp < 90 mmhg ) at weeks 4 and 8 were calculated and the differences between the two groups were analyzed by pearson s chi - square . male and female patients aged 18 years and above were enrolled in the study if they met the following criteria : on the screening visit , mean values of two sitting diastolic blood pressure ( sidbp ) readings had to be < 110 mmhg if the patient were on antihypertensive medication ; if the patient were antihypertensive nave , the mean values of two sidbp readings had to be 90 mmhg and < 120 mmhg . patients were excluded if they had : a mean sitting systolic blood pressure ( sisbp ) 200 mmhg at the screening visit ; a difference of sisbp 20 mmhg or sidbp 10 mmhg between arms ; secondary hypertension ( ie , renovascular hypertension , endocrinologic disease , and use of hormonal contraceptives or drugs affecting bp ) ; hepatic ( aspartate transaminase and alanine transaminase 2.0 upper limit of normal ) or renal impairment ( serum creatinine 1.5 upper limit of normal ) ; active hepatitis b or c ( including carriers ) ; positive status for hiv ; known allergy to the study drugs ; a sodium ( < 133 mmol / l or 145 mmol / l ) or potassium ( < 3.5 mmol / l or 5.5 mmol / l ) electrolyte imbalance ; insulin - dependent or uncontrolled diabetes mellitus ( glycated hemoglobin [ hba1c ] > 9% ) ; retinal hemorrhage or exudates within the previous 6 months ; drug or alcohol dependency ; heart or cerebrovascular disease within the previous 6 months ( coronary heart disease , heart failure , significant valvular heart disease , cerebral infarction , or cerebral hemorrhage ) ; active inflammatory gastrointestinal disease in the preceding 12 months or a history of gastrointestinal surgery or disease that could interfere with drug absorption ; or the presence of a wasting disorder , autoimmune disease , or connective tissue disease . after screening , patients who met the eligibility criteria were given once - daily fimasartan 60 mg for 4 weeks . after 4 weeks of treatment with once - daily fimasartan 60 mg , patients with sidbp 90 mmhg were randomly assigned to receive either once - daily fimasartan 60 mg / hctz 12.5 mg or fimasartan 60 mg for 4 weeks at a 2:1 ratio by using sealed envelopes with the randomization number . the dose of the study drug was increased to fimasartan 120 mg / hctz 12.5 mg or fimasartan 120 mg then maintained for another 4 weeks if the sidbp was still > 90 mmhg after 4 weeks of treatment with fimasartan 60 mg / hctz 12.5 mg and fimasartan 60 mg . this study was designed to compare the antihypertensive efficacy of fimasartan 60 mg / hctz 12.5 mg combination treatment to that of treatment with fimasartan 60 mg alone in patients who did not achieve the target bp after 4 weeks of treatment with fimasartan 60 mg . the primary goal of this study was to compare the changes in mean sidbp from the baseline to week 4 of treatment with the study drug ( fimasartan 60 mg / hctz 12.5 mg ) and the control drug ( fimasartan 60 mg ) in patients who did not achieve target sidbp after 4 weeks of prior treatment with once - daily fimasartan 60 mg . the secondary efficacy points were : ( 1 ) change of mean sisbp from baseline at 4 and 8 weeks ; ( 2 ) bp control rate ( a proportion of patients who achieved mean sidbp < 90 mmhg ) and response rate ( a proportion of patients who achieved a reduction of sidbp 10 mmhg from baseline and/or a mean sidbp < 90 mmhg ) at 4 and 8 weeks ; and ( 3 ) change of mean sidbp from baseline at 8 weeks . to identify the primary hypothesis , we assumed a potential difference of 3 mmhg in mean change of sidbp with a standard deviation of 7.5 mmhg between the two therapies.3,1319 with a randomization ratio of 2:1 between patients assigned fimasartan 60 mg / hctz 12.5 mg and those assigned fimasartan 60 mg , significance level of 5% , and statistical power of 80% , the total number of required subjects was 225 ( 150 for fimasartan 60 mg / hctz 12.5 mg and 75 for fimasartan 60 mg ) . descriptive statistics for the rates of the responders ( sidbp < 90 mmhg or sidbp reduction from the baseline 10 mmhg ) and control subjects ( sidbp < 90 mmhg ) at weeks 4 and 8 were calculated and the differences between the two groups were analyzed by pearson s chi - square . among 654 patients screened , 263 were matched to eligible randomization criteria after 4 weeks of treatment with once - daily fimasartan 60 mg . the reduction of sidbp and sisbp was greater in the fimasartan / hctz group than in the fimasartan group . the reduction of sidbp was 6.888.10 mmhg in the fimasartan / hctz group and 3.387.33 mmhg in the fimasartan group at week 4 ( p=0.0008 ) . at week 8 , the reduction in sidbp was 8.679.39 mmhg in the fimasartan / hctz group and 5.028.27 mmhg in the fimasartan group ( p=0.0023 ) . the reduction in sisbp was 10.5013.76 mmhg in the fimasartan / hctz group and 5.7512.18 mmhg in the fimasartan group at week 4 ( p=0.0069 ) . at week 8 , the reduction in sisbp was 13.4515.15 mmhg in the fimasartan / hctz group and 6.8413.57 mmhg in the fimasartan group ( p=0.0007 ) . when the fimasartan dose of both treatment groups was increased to 120 mg in patients who did not achieve the target sidbp ( 90 mmhg ) at week 4 , although there was no statistical significance , the fimasartan / hctz group showed greater reduction of sidbp and sisbp compared to the fimasartan group . the response rate at week 4 was 53.6% ( 90/168 ) in the fimasartan / hctz group and 39.8% ( 35/88 ) in the fimasartan group ( p=0.0359 ) . at week 8 , the response rate was 63.1% ( 106/168 ) in the fimasartan / hctz group and 51.1% ( 45/88 ) in the fimasartan group , which was not statistically different ( p=0.0647 ) . in the pp analysis , the response rate of the fimasartan / htcz treatment group was higher than that of the fimasartan treatment group ( at week 4 , 55.7% vs 40.2% , p=0.0245 and , at week 8 , 67.1% vs 52.4% , p=0.0280 ) . the level of serum potassium increased to 5.3 mmol / l at week 8 from 4.7 mmol / l at baseline and 4.3 mmol / l at week 4 in a patient from the fimasartan / hctz treatment group . the level of serum sodium decreased to 124 mmol / l at week 8 , from 135 mmol / l at baseline , and 127 mmol / l at week 4 in a patient from the fimasartan / hctz treatment group . among 654 patients screened , 263 were matched to eligible randomization criteria after 4 weeks of treatment with once - daily fimasartan 60 mg . of the 263 patients , 175 were assigned to 4 weeks of fimasartan / htcz treatment and 88 were assigned to 4 weeks of fimasartan treatment ( figure 1 ) . the number of smokers was higher in the fimasartan / hctz group than in the fimasartan group . the reduction of sidbp and sisbp was greater in the fimasartan / hctz group than in the fimasartan group . the reduction of sidbp was 6.888.10 mmhg in the fimasartan / hctz group and 3.387.33 mmhg in the fimasartan group at week 4 ( p=0.0008 ) . at week 8 , the reduction in sidbp was 8.679.39 mmhg in the fimasartan / hctz group and 5.028.27 mmhg in the fimasartan group ( p=0.0023 ) . the reduction in sisbp was 10.5013.76 mmhg in the fimasartan / hctz group and 5.7512.18 mmhg in the fimasartan group at week 4 ( p=0.0069 ) . at week 8 , the reduction in sisbp was 13.4515.15 mmhg in the fimasartan / hctz group and 6.8413.57 mmhg in the fimasartan group ( p=0.0007 ) . when the fimasartan dose of both treatment groups was increased to 120 mg in patients who did not achieve the target sidbp ( 90 mmhg ) at week 4 , sidbp and sisbp decreased significantly by week 8 ( table 3 ) . although there was no statistical significance , the fimasartan / hctz group showed greater reduction of sidbp and sisbp compared to the fimasartan group . the response rate at week 4 was 53.6% ( 90/168 ) in the fimasartan / hctz group and 39.8% ( 35/88 ) in the fimasartan group ( p=0.0359 ) . at week 8 , the response rate was 63.1% ( 106/168 ) in the fimasartan / hctz group and 51.1% ( 45/88 ) in the fimasartan group , which was not statistically different ( p=0.0647 ) . in the pp analysis , the response rate of the fimasartan / htcz treatment group was higher than that of the fimasartan treatment group ( at week 4 , 55.7% vs 40.2% , p=0.0245 and , at week 8 , 67.1% vs 52.4% , p=0.0280 ) . the level of serum potassium increased to 5.3 mmol / l at week 8 from 4.7 mmol / l at baseline and 4.3 mmol / l at week 4 in a patient from the fimasartan / hctz treatment group . the level of serum sodium decreased to 124 mmol / l at week 8 , from 135 mmol / l at baseline , and 127 mmol / l at week 4 in a patient from the fimasartan / hctz treatment group . as far as we are aware , this is the first study to have demonstrated that combination treatment with fimasartan and hctz is effective in the treatment of patients with essential hypertension who respond poorly to fimasartan monotherapy . similar to the studies that evaluated combination arb and hctz treatment,8,10,20,21 the combination treatment of fimasartan and hctz had better efficacy than fimasartan monotherapy . after 4 weeks of treatment , the combination of fimasartan and hctz was more effective in reducing sidbp and sisbp than fimasartan monotherapy . the magnitude of bp lowering was comparable to other combination treatments of arbs and hctz although they can not be compared directly due to differences in study design.9,10,22 the difference in bp lowering between 8 weeks treatment of valsartan 160 mg plus htcz 12.5 mg and valsartan 160 mg was approximately 2.0 mmhg in sidbp and 3.7 mmhg in sisbp.22 combination olmasartan and hctz treatment also showed similar bp lowering compared to monotherapy ( sidbp : ~3.4 mmhg , sisbp : ~5.3 mmhg).10 in the current study , the difference in bp lowering after 4 weeks treatment was 3.50 mmhg in sidbp and 4.75 mmhg in sisbp . despite this limitation , dose escalation of fimasartan / hctz to 120 mg/12.5 mg or fimasartan 120 mg in patients who did not achieve the target sidbp at week 4 resulted in significant reductions in sidbp and sisbp at week 8 , indicating a dose - dependent bp - lowering effect .

with its densely populated and highly developed coastline , new york city faces significant risks from flooding , especially during coastal storms . due to sea level rise caused by climate change , flooding associated with coastal storms and hurricanes intense hurricanes may also become more frequent . in october 2012 , superstorm sandy brought these vulnerabilities into stark relief , causing a record storm surge , extensive flooding , loss of life , injury , widespread power outages , and widespread damage to property and coastal neighborhoods in queens , manhattan , staten island , brooklyn , and throughout the region . as of november 26 , 2012 , nyc had estimated that public and private losses in the city totaled at least $ 19 billion . many people living near the coasts may be vulnerable . from 1970 to 2010 , the population in coastal us areas has increased by 39% , and population density along the coasts is expected to continue to increase . many residents are older adults , a group that is particularly vulnerable to the effects of storms and flooding . people living in coastal areas will need to prepare for a wide variety of potential health impacts . many factors can influence the health impact of storms , including the severity and other characteristics of the storm , the exact timing and location of landfall , and the unique geographic and topographic characteristics of the affected area . for instance , although superstorm sandy was officially classified as a post - tropical storm by noaa when it made landfall , it had several characteristics that produced a record storm surge of over 13 feet at battery park in lower manhattan including making landfall during high tide and combining with a midlatitude trough system that increased the power and size of the storm . local housing characteristics and infrastructure , the existence and execution of evacuation and other emergency plans , and underlying population health and resilience also affect the impacts of storms . lessons learned from sandy , hurricane katrina , and other devastating storms , however , shed light on what adverse outcomes are possible and what factors may make people and neighborhoods more or less vulnerable to their impacts . preparing for a range of anticipated health impacts of coastal storms and floods could help reduce the health burden from these events . to this end , we conducted a broad review of the health impacts of us coastal storms , with a focus on outcomes relevant to new york city and urban coastal areas . we also identified population - level indicators that may be useful in identifying vulnerable neighborhoods . vulnerability mapping can help planners and communities better understand the baseline health status of neighborhoods in the evacuation zones and some of the factors that may make residents more vulnerable to a range of health effects during and after a major storm . recent literature about the health impacts of coastal storms and floods , such as injuries , depression , anxiety , and poor physical health , was reviewed . the intent was not to be exhaustive but to describe the range of potential health effects that could occur in nyc and other urban areas and describe likely and/or potentially severe outcomes . using the us national library of medicine 's pubmed database , we searched a set of general terms relating to storms and flooding to capture a broad range of health outcomes : ( cyclonic storms or floods or hurricane ) and ( mental health or health or injury or morbidity ) and united states . ( cyclonic storms or floods or hurricane ) and ( mental health or health or injury or morbidity ) and united states . a total of 70 published studies , which covered a wide range of potential exposures and adverse health outcomes , were compiled and reviewed in detail . this review was largely completed prior to superstorm sandy , and the lessons learned and health impacts from that incident are still under study . nonetheless , some initial lessons learned from the response to superstorm sandy were qualitatively considered in this review . based on vulnerable subgroups identified in the literature , potential indicators of population vulnerability for which data are available were identified and mapped within the 42 nyc united hospital fund ( uhf ) neighborhoods located within any nyc hurricane evacuation zone . uhf neighborhoods are zip code - aggregated areas within all five boroughs . for each indicator health outcomes can occur through multiple pathways ( see figure 1 ) including ( 1 ) hazards from exposure to storm impact ; ( 2 ) evacuation ; ( 3 ) post - storm hazards from utility outages and sheltering in place in inadequate housing ; ( 4 ) exposure to secondary hazards including contaminated drinking water , contact with contaminated floodwaters , and mold and moisture in housing ; ( 5 ) population displacement and disruption of services ; ( 6 ) mental health effects from traumatic or stressful experiences during and after the storms and ( 7 ) health and safety risks from clean - up and recovery activities . the most severe acute effect of hurricane landfall is death from drowning , electrocutions , or physical trauma [ 69 ] . nearly 85% of people killed during and in the immediate aftermath of hurricane katrina were aged 51 and older , and almost half were older than 75 years of age . as with other natural disasters , low - income populations may be particularly vulnerable . the causes and age pattern of deaths during the impact phase of superstorm sandy were generally consistent with prior storms . in the acute phase , sandy caused 43 deaths in nyc . death was caused most frequently by drowning associated with the storm surge ( n = 34 , 79% ) . other deaths were caused by falling trees , falls , electrocution , and other trauma . nearly half of fatalities occurred among adults aged 65 or older ( n = 20 , 47% ) , and more than half of deaths occurred on staten island ( n = 23 , 53% ) . epidemiologic studies will be needed in the coming months and years , however , to assess the storm 's full impact on excess mortality from accidental and natural causes , as well as other health impacts , in impacted communities . heavy precipitation weather events can also cause flash flooding , which occurs when water from heavy rains collects in a relatively short time and runoff is accelerated in mountainous or narrow valley terrain . nationally , flash floods are the most common cause of flood deaths via drowning [ 12 , 13 ] . flash floods occur in nyc but historically have rarely been life threatening because of local topography . however , much of nyc 's infrastructure , especially in low - lying or poor drainage areas , can not cope with more than one inch per hour of rainfall . hurricane landfall can also result in a range of non - fatal injuries , including blunt trauma , puncture wounds , lacerations , sprains / strains , motor vehicle crashes , animal bites , and electrocution [ 1518 ] . falls , traffic accidents , and other injuries can also occur in period immediately before a major storm hits , particularly among the elderly , as people try to evacuate and prepare their residences . those who do not evacuate prior to a storm and shelter in place , by choice or necessity , may risk injury or death during a coastal storm . poor and minority populations , and elderly nursing home residents , are more likely to lack transportation during disasters . these populations often have a high prevalence of chronic health problems , which increases their vulnerability to other storm - related hazards . researchers have also observed a 3-fold increase in the incidence of acute myocardial infarction among tulane health sciences center hospital patients two years after hurricane katrina related to emotional stress . while evacuations of health care facilities are undertaken to avoid health risks that result from major destruction such as flooding , power outages , and interruptions in medical care if health facilities are rendered inoperable , safe evacuations require advance planning and technical expertise . in the evacuation phase , frail or medically incapacitated people may need assistance getting to trains or buses or may require other modes of transport . for municipal health and transit staff , as well as private agencies serving this population , this represents a large undertaking , requiring organization , training , and adequate staffing , which could be difficult to ensure during an emergency . furthermore , receiving facilities located outside the storm surge zone will require additional supplies and patient care capacity . delayed ( post - storm ) evacuation of health care facilities can compound logistical problems and risks because of loss of power and damage to communications and transportation infrastructure , making it more difficult to transport and track patients who may already be compromised by failure of medical equipment or exposure to heat or cold . a survey of twenty gulf state nursing homes identified significant logistical and response problems during hurricane katrina related to transportation , staffing , maintaining complex medication regimens , insufficient emergency provisions , and failure to follow emergency plans . all facilities encountered problems , but there were slightly more negative effects ( including dehydration , depression , and skin tears ) reported by administrators from facilities that evacuated rather than sheltered in place . nursing home administrators reported little support from state and local emergency responders in evacuation decisions and implementation . three of them nyu langone medical center , bellevue hospital , and coney island hospital had to evacuate patients after the storm hit because of flood damage to critical equipment located in low - lying parts of the hospitals . many nursing and long - term care facilities also lost power and had to evacuate patients . local or widespread power outages could result from flood damage to underground and low - lying electrical infrastructure , as well as damage to utility poles and aboveground wires from high winds and downed trees . following widespread power outages , carbon monoxide ( co ) poisoning is a major health hazard . deaths and illness can occur when portable generators , cooking appliances , and other fuel burning equipments are used indoors or improperly [ 2434 ] . a study of the august 14 - 15 , 2003 , northeast blackout that affected nyc found increased mortality from both accidental and natural causes that resulted in approximately 90 excess deaths ( an increase of 28% ) . researchers theorized that increased mortality could be related to more physical demands on vulnerable people due to non - functioning elevators and subways , increased call times for ambulances , closed stores and pharmacies , and potentially the effects of increased exposure to air pollution and ambient air temperatures . power losses may lead to increased emergency medical services ( ems ) calls and emergency department ( ed ) visits from patients who rely on electrically powered medical equipments like ventilators and oxygen [ 24 , 27 , 32 , 34 , 3639 ] . frail residents of nursing homes and other health care facilities that shelter in place can be especially vulnerable to power outages in facilities without backup generators or with backups located in flood - vulnerable places such as basements . risks include the failure of medical equipment and exposure to hot or cold ambient temperatures , possibly compounded by a lack of supplies or adequate staff in facilities that are not well prepared [ 22 , 40 ] . incarcerated populations are also at potentially high risk . in new orleans during hurricane katrina , a lack of emergency preparedness and planning by corrections officials led to chaos during the storm at the over - crowded orleans parish prison . prisoners locked in cells were left alone without power , food , water , or even sufficient ventilation during the storm . prisoners described lack of access to medical care and interruptions in care for serious chronic illness during and after the storm . prison populations often have higher rates of mental and physical illness and substance use disorders than the general population and are at risk for exacerbations . after superstorm sandy made landfall , hundreds of thousands of nyc residents initially lost power . however , even after the electric grid had been largely restored , many residential buildings in storm - inundated areas still lacked electric power , heat , or running water , often because of salt water flood damage to buildings electrical and heating systems . many people who did not evacuate in advance of the storm sheltered in place in housing conditions that lacked one or more of these essential services . exposure to hot or cold ambient temperatures from lack of climate control could result in heat- or cold - related illness , including heat stroke or hypothermia , as well as exacerbation of respiratory , cardiovascular , and other chronic diseases [ 41 , 44 , 45 ] . in the days following sandy , health department surveillance data reflected the impact of people living without power or heat and , in some cases , trying to provide power or heat in unsafe ways . from the storm impact until november 9 ( 10 days ) , co - related emergency department visits and poison center ( pcc ) calls for co exposure were elevated for the time of year ; pcc data frequently identified storm - related sources of exposure including charcoal grills and household cooking appliances used for heating , as well as portable generators . counts of cold illness syndrome emergency department visits ( including hypothermia ) were also elevated through november 9 . while the potential health impacts resulting from sheltering in inadequate housing after a storm have not yet been formally evaluated , exacerbation of chronic health conditions , including physical , mental , and substance use disorders , could occur from a disruption of care due to lack of light , telecommunications , and elevator service that makes it difficult for people to access outside care , obtain medications , maintain self - care , and receive home - based care services . there may also be stress - related exacerbation of chronic physical and mental health problems related to isolation . injuries could include fire risks among those using stoves for heat or candles for light and risk of falls from inadequate lighting in dwellings , hallways , and stairwells . inability to access food and fresh water , particularly for people living in high - rise apartments where water delivery is dependent on electric pumps , could lead to infectious disease risk because of inability to properly wash hands or food , bathe , or flush toilets . people living in residential buildings without electricity may be more vulnerable to foodborne disease because they can not refrigerate food . after a 2003 summer power outage in nyc , increases in diarrheal illness caused by the consumption of spoiled foods , especially meat and seafood , were observed . furthermore , unless active steps are taken to remove spoiled food from residences and restaurants ( which typically pay private contractors to collect waste ) during prolonged outages , pest populations could increase . it is possible that proliferation of pests , including rodents and roaches , because of difficulty with cleaning and removing trash may exacerbate allergies , asthma , and other respiratory conditions . older adults , young children , individuals with pre - existing health conditions , and those living on the upper floors of high - rise buildings or those who are disabled may be especially vulnerable to health effects resulting from power and utility outages . safety concerns stemming from lighting outages and disabled safety systems in hallways and stairwells may deter this population from seeking or receiving assistance . in addition , it is possible that this population may be at risk for mental health outcomes observed in populations who have experienced long - term displacement , including post - traumatic stress and other mental health problems . exposure to mold in flood - damaged buildings could worsen allergic and asthmatic symptoms among those with pre - existing allergic sensitization and respiratory infections . although increased concentrations of outdoor and indoor mold were detected after hurricane katrina in areas that had experienced flooding [ 49 , 50 ] , the impact of the mold on health following katrina has not been well characterized , possibly because of under - reporting or under - detection of health problems , or by population displacement that reduced exposure . coastal storms can cause release of untreated sewage through direct damage and flooding of treatment facilities , power outage , or combined sewage overflows ( cso ) . csos are caused when heavy rains overwhelm combined systems of collecting storm and precipitation water runoff causing the discharge of untreated sewage into rivers . secondary exposure to sewage - contaminated floodwaters and wastewater , along with impaired access to potable water and flushable toilets , may lead to gastrointestinal infections , acute respiratory infections , skin infections , and insect bites [ 15 , 16 , 51 , 52 ] . there may also be a risk of increases in vectorborne diseases like west nile virus because sites where water collects after heavy rains could become breeding grounds for mosquitoes . rainfall , wind , and runoff in the watershed area can contribute to high turbidity levels , which can interfere with the disinfection process of drinking water . in nyc , a coastal storm will not necessarily impact the drinking water supply because the watershed and reservoirs are located up to 125 miles from the city . however , an increasing number of heavy precipitation events in the watershed may lead to more instances of high turbidity levels . the nyc department of environmental protection monitors turbidity at over 1,400 locations in the watershed and distribution system and activates the ashokan waste channel to reduce turbidity levels . storm damage may compromise sites storing toxic waste , and flood waters may move hazardous substances to new areas . following hurricane katrina , hazardous substances such as volatile organic compounds ( vocs ) , lead , and arsenic were detected in the air , soil , and sediment samples . no health effects have been directly observed in a storm - specific context [ 5459 ] . however , the potential for a toxic release of hazardous substances after a storm exists [ 5557 ] . following sandy , initial testing of two superfund sites indicated that contact with contaminated water from these areas was not a major health threat . displacement led to a host of adverse effects following hurricane katrina and major storms in other areas . health risks were often related to living in congregate shelters , disruption of access to health care , or some combination of both . the spread of infectious diseases , such as norovirus , was documented among residents of temporary shelters or evacuation centers in the wake of hurricane katrina . waterborne illnesses and infectious disease spread in shelters have been more frequently observed among young children and infants with nave immune systems [ 61 , 62 ] . contributing factors to disruption of care after katrina , hurricane ike , and other natural disasters included evacuees without medical history information , medications or knowledge of medication names and doses , and access to medical records [ 63 , 64 ] . following hurricane katrina , health care providers and focus groups also reported that chronic disease treatment interruptions especially among patients with cancer , hypertension , end stage renal disease , cardiovascular disease , and respiratory illnesses were problems . red cross shelter residents in the weeks immediately following katrina , slightly more than half of the residents had chronic medical conditions including hypertension , hypercholesterolemia , diabetes , and lung diseases . about a after hurricane katrina , there was also a need for urgent medical supplies , such as oxygen tanks . emergency responders may also need access to sufficient quantities of vaccines ( and facilities with proper storage capabilities ) , medications , and other preventive medical supplies for displaced populations . women who are displaced may have trouble accessing contraceptives and other reproductive health services . following hurricane ike in texas , racial disparities in access to contraceptives disruptions in prenatal care for pregnant women , including folate supplementation , could potentially lead to adverse birth outcomes [ 67 , 68 ] . displacement at 12 months following katrina was associated with increased risk of hip fracture among seniors , particularly among women , those with co - morbidities and a history of hip fracture . among nursing home residents in the gulf coast evacuated before a recent hurricane , 30- and 90-day mortality and hospitalization rates were higher compared with rates during non - hurricane control years . experiencing a hurricane or major natural disaster may exacerbate existing mental health conditions or contribute to new mental health and interpersonal problems [ 7074 ] . particularly in the months immediately following exposure to a natural disaster , increases in levels of post - traumatic stress disorder ( ptsd ) , as well as other mental health problems have been observed . more than a year after hurricane katrina , anxiety and mood disorders in the new orleans metro area were substantially elevated , and mental health conditions were broadly distributed in the population . serious mental illness was typically accompanied by ptsd , and important predictors of mental health problems were storm - related physical illness or injury , physical adversity , and property loss . in addition , two years after katrina , the prevalence of self - reported psychological and physical intimate partner violence increased among mississippi residents affected by the hurricane . self - reported poor physical and mental health before and after a storm has also been correlated with self - reported poor mental health after the storm . the duration of mental health problems may depend on the nature of exposure to the storm and on ongoing stressors related to the storm . one study of mental health conditions after hurricane ike in 2008 showed that prevalence of storm - related ptsd decreased within 18 months . however , elevated levels of ptsd and psychological distress among vulnerable populations have also been observed up to five years after a hurricane . following hurricane katrina , researchers have suggested that slow government responses may have exacerbated mental health problems and argued that an efficient emergency response can also help to minimize the mental health impacts of natural disasters . for those who have experienced displacement , short- and long - term mental health effects evacuees at the red cross shelter in austin , tx , usa , following katrina , were at increased risk of short - term acute stress disorder , while populations who were displaced or who experienced or witnessed traumatic events were at increased risk of long - term mental health effects , including ptsd , depression , anxiety , and suicidal ideation [ 70 , 77 , 78 , 81 , 82 , 84 , 85 ] . women , african - americans , and those with prior psychiatric history , poor physical health , and weak social networks have been identified as particularly vulnerable [ 75 , 78 , 79 , 81 , 82 , 85 ] . katrina evacuees living in houston were also found to be at risk for increased substance use [ 80 , 86 ] . many studies on mental health conditions were cross - sectional , and pre - storm depression and ptsd levels could not always be ascertained , thereby limiting conclusions that could be drawn from the data about the cause - effect relationship between storms and subsequent mental health outcomes . nevertheless , preventing the long - term mental health effects following a storm through ongoing mental health surveillance , appropriate intervention , and adaptation strategies should remain a priority . the clean - up and recovery period after a major storm may also present significant health risks . one study found that , at median , occupational deaths occurred 36.5 days after a storm event and were most often associated with clean - up ( 44% ) , restorative construction ( 26% ) , public utilities restoration ( 8% ) , and security / policing ( 6% ) . residents and volunteers trying to clean affected homes could suffer non - fatal injuries ( including cuts , wounds , sprains , and strains ) and other health risks during the course of removing debris or during minor and major home repairs ( for instance , removing wet building materials or wet wall insulation ) . indoor dust created during cleaning , exposure to mold , fumes from temporary heating sources , and the use of strong cleaning products can also irritate the eyes , throat , and lungs . the delivery of and access to health care and other basic services as well as efforts to respond in storm- or flood - damaged areas could be impeded if workers and volunteers have difficulty traveling to and within the areas . large - scale displacement could increase demands on the transportation infrastructure , and there may be occupational health concerns for municipal workers deployed to address flooding in transportation and communications infrastructure . during recovery , air quality may be negatively affected by dust from the home clean - up , debris movement , emissions from truck traffic , and the use of outdoor temporary boilers and emergency generators . following sandy , routine monitoring at rooftop air monitors in new york city showed that the levels of fine particles ( pm2.5)the pollutant most likely to be associated with combustion of fuels , dust from streets , and debris were not significantly elevated compared to levels typical for the season . similarly , two - week average pm2.5 concentrations at street level monitors near storm- impacted areas in the four weeks after the storm showed that levels were typical for the time of year and similar to those elsewhere in the city . , supplemental continuous air monitors were placed in flood - impacted neighborhoods with ongoing recovery operations . for the most part , 24-hour average pm concentrations in these locations through the winter tracked with levels at regional monitors elsewhere in the city , although on several days concentrations were somewhat higher near areas with concentrations of temporary generators and boilers or reconstruction activity . table 1 summarizes examples of sub - populations identified in prior studies as having increased susceptibility to adverse health effects from different exposures resulting from coastal storms . the health impacts and vulnerable subgroups listed depict a range of potential outcomes but are not exhaustive . for the most part , these studies characterize vulnerability to longer term environmental hazards and stressors from the aftermath of coastal storms rather than the risk of injury or death during the impact phase of a storm . vulnerable sub - populations vary somewhat by study and specific storm hazard . for the most part , however , groups most vulnerable to adverse storms - related outcomes include one or more of the following : older adults ; young children ; women ; those with pre - existing physical , mental health problems , or substance use disorders ; those living in low - income households ; members of disadvantaged racial / ethnic groups ; and those with weak social networks . based on the literature and data available at the neighborhood level , we identified 18 indicators of vulnerability in the domains of mental health , physical health , socioeconomic status , and housing ( table 2 ) . indicators were mapped within any 2012 evacuation zone . in nyc , many neighborhoods with high poverty levels also had relatively high levels of other vulnerability indicators . for instance , the percent of people living below the federal poverty level by neighborhood was highly correlated with indicators of population - level prevalence of disability frequent mental distress , social isolation , and poor housing quality , and moderately correlated with prevalence of chronic physical health conditions and lack of health insurance ( table 2 ) . the literature review process was not intended to be exhaustive but was rather a means to capture the majority of known health outcomes associated with flooding and coastal storms , and some subjective judgments were used to identify potential health effects for which there are no published studies . however , there may be health effects that are under - represented , not adequately characterized , or not described in the literature . population vulnerability may differ according to geographic region for a variety of reasons , so indicators based on studies from other areas may not adequately describe vulnerability in nyc or other urban areas . analyses of correlation between indicators and neighborhood poverty do not take variation in survey estimates or potential spatial auto - correlation into account . in addition , we mapped vulnerability indicators within relatively large neighborhood areas . studies of vulnerability in smaller areas will be important in helping planners and communities better identify and prepare for the potential health impact of storms , especially in assisting in the development and implementation of neighborhood - based interventions . it is also important to note that areas outside of evacuation zones are also not without risk . power outages related to wind damage or damage to utility substations can occur outside of the evacuation zones . hurricane evacuation zone designations will also likely expand in many places as rising sea levels are factored into the projected storm surge zone . furthermore , nyc indicators that were available and used for mapping are imperfect proxies for vulnerable subgroups identified in the literature . vulnerability maps based on these indicators do not represent an exact geospatial representation of vulnerability but rather a proxy measure of vulnerability for research and adaptation purposes [ 89 , 90 ] . with predicted warming of the climate , nyc and other coastal cities may be increasingly vulnerable to flooding during hurricanes and other severe storms . these events can have a devastating toll , as witnessed in 2005 with hurricane katrina in the gulf coast and in 2012 with superstorm sandy in the northeast . a wide range of potential acute and long - term health impacts were identified in the literature , from injury and death resulting from a failure to evacuate safely , physical and mental health problems in displaced populations because of disruption of care or stress , and injury and illness risk during repair and recovery , as well as a range of potential health impacts from exposures in damaged housing and from sheltering in place . mental health problems were some of the most frequently cited health consequences of major storms . for several dimensions of public health vulnerability to coastal storms , nyc neighborhoods with elevated poverty levels may be at increased risk for lasting impacts . while adaptation planning must be tailored to the needs of local health jurisdictions , public health protection depends to a great extent on minimizing critical infrastructure vulnerabilities , including enhancing the resilience of power delivery networks ( for instance , ensuring that key power infrastructure is located above anticipated high - water lines ) , buildings , transportation , and health care systems . it is also clear from experiences described in the literature that emergency preparedness for coastal storms will need to include preparation for both short - term and long - term needs of the most vulnerable populations , especially physical and mental health care and access to other essential services . increasing public health resilience to coastal storms also requires community engagement in climate - readiness strategies and an interdisciplinary approach to adaptation planning .

coastal storms can take a devastating toll on the public 's health . urban areas like new york city ( nyc ) may be particularly at risk , given their dense population , reliance on transportation , energy infrastructure that is vulnerable to flood damage , and high - rise residential housing , which may be hard - hit by power and utility outages . climate change will exacerbate these risks in the coming decades . sea levels are rising due to global warming , which will intensify storm surge . these projections make preparing for the health impacts of storms even more important . we conducted a broad review of the health impacts of us coastal storms to inform climate adaptation planning efforts , with a focus on outcomes relevant to nyc and urban coastal areas , and incorporated some lessons learned from recent experience with superstorm sandy . based on the literature , indicators of health vulnerability were selected and mapped within nyc neighborhoods . preparing for the broad range of anticipated effects of coastal storms and floods may help reduce the public health burden from these events .

1. Introduction 2. Materials and Methods 3. Results and Discussion 4. Summary of Neighborhood Vulnerability 5. Limitations 6. Conclusions

with its densely populated and highly developed coastline , new york city faces significant risks from flooding , especially during coastal storms . due to sea level rise caused by climate change , flooding associated with coastal storms and hurricanes intense hurricanes may also become more frequent . in october 2012 , superstorm sandy brought these vulnerabilities into stark relief , causing a record storm surge , extensive flooding , loss of life , injury , widespread power outages , and widespread damage to property and coastal neighborhoods in queens , manhattan , staten island , brooklyn , and throughout the region . many people living near the coasts may be vulnerable . many residents are older adults , a group that is particularly vulnerable to the effects of storms and flooding . people living in coastal areas will need to prepare for a wide variety of potential health impacts . many factors can influence the health impact of storms , including the severity and other characteristics of the storm , the exact timing and location of landfall , and the unique geographic and topographic characteristics of the affected area . for instance , although superstorm sandy was officially classified as a post - tropical storm by noaa when it made landfall , it had several characteristics that produced a record storm surge of over 13 feet at battery park in lower manhattan including making landfall during high tide and combining with a midlatitude trough system that increased the power and size of the storm . local housing characteristics and infrastructure , the existence and execution of evacuation and other emergency plans , and underlying population health and resilience also affect the impacts of storms . lessons learned from sandy , hurricane katrina , and other devastating storms , however , shed light on what adverse outcomes are possible and what factors may make people and neighborhoods more or less vulnerable to their impacts . preparing for a range of anticipated health impacts of coastal storms and floods could help reduce the health burden from these events . to this end , we conducted a broad review of the health impacts of us coastal storms , with a focus on outcomes relevant to new york city and urban coastal areas . vulnerability mapping can help planners and communities better understand the baseline health status of neighborhoods in the evacuation zones and some of the factors that may make residents more vulnerable to a range of health effects during and after a major storm . recent literature about the health impacts of coastal storms and floods , such as injuries , depression , anxiety , and poor physical health , was reviewed . the intent was not to be exhaustive but to describe the range of potential health effects that could occur in nyc and other urban areas and describe likely and/or potentially severe outcomes . using the us national library of medicine 's pubmed database , we searched a set of general terms relating to storms and flooding to capture a broad range of health outcomes : ( cyclonic storms or floods or hurricane ) and ( mental health or health or injury or morbidity ) and united states . a total of 70 published studies , which covered a wide range of potential exposures and adverse health outcomes , were compiled and reviewed in detail . this review was largely completed prior to superstorm sandy , and the lessons learned and health impacts from that incident are still under study . nonetheless , some initial lessons learned from the response to superstorm sandy were qualitatively considered in this review . based on vulnerable subgroups identified in the literature , potential indicators of population vulnerability for which data are available were identified and mapped within the 42 nyc united hospital fund ( uhf ) neighborhoods located within any nyc hurricane evacuation zone . for each indicator health outcomes can occur through multiple pathways ( see figure 1 ) including ( 1 ) hazards from exposure to storm impact ; ( 2 ) evacuation ; ( 3 ) post - storm hazards from utility outages and sheltering in place in inadequate housing ; ( 4 ) exposure to secondary hazards including contaminated drinking water , contact with contaminated floodwaters , and mold and moisture in housing ; ( 5 ) population displacement and disruption of services ; ( 6 ) mental health effects from traumatic or stressful experiences during and after the storms and ( 7 ) health and safety risks from clean - up and recovery activities . nearly 85% of people killed during and in the immediate aftermath of hurricane katrina were aged 51 and older , and almost half were older than 75 years of age . as with other natural disasters , low - income populations may be particularly vulnerable . the causes and age pattern of deaths during the impact phase of superstorm sandy were generally consistent with prior storms . in the acute phase , sandy caused 43 deaths in nyc . death was caused most frequently by drowning associated with the storm surge ( n = 34 , 79% ) . other deaths were caused by falling trees , falls , electrocution , and other trauma . nearly half of fatalities occurred among adults aged 65 or older ( n = 20 , 47% ) , and more than half of deaths occurred on staten island ( n = 23 , 53% ) . epidemiologic studies will be needed in the coming months and years , however , to assess the storm 's full impact on excess mortality from accidental and natural causes , as well as other health impacts , in impacted communities . heavy precipitation weather events can also cause flash flooding , which occurs when water from heavy rains collects in a relatively short time and runoff is accelerated in mountainous or narrow valley terrain . however , much of nyc 's infrastructure , especially in low - lying or poor drainage areas , can not cope with more than one inch per hour of rainfall . hurricane landfall can also result in a range of non - fatal injuries , including blunt trauma , puncture wounds , lacerations , sprains / strains , motor vehicle crashes , animal bites , and electrocution [ 1518 ] . poor and minority populations , and elderly nursing home residents , are more likely to lack transportation during disasters . these populations often have a high prevalence of chronic health problems , which increases their vulnerability to other storm - related hazards . researchers have also observed a 3-fold increase in the incidence of acute myocardial infarction among tulane health sciences center hospital patients two years after hurricane katrina related to emotional stress . while evacuations of health care facilities are undertaken to avoid health risks that result from major destruction such as flooding , power outages , and interruptions in medical care if health facilities are rendered inoperable , safe evacuations require advance planning and technical expertise . in the evacuation phase , frail or medically incapacitated people may need assistance getting to trains or buses or may require other modes of transport . for municipal health and transit staff , as well as private agencies serving this population , this represents a large undertaking , requiring organization , training , and adequate staffing , which could be difficult to ensure during an emergency . furthermore , receiving facilities located outside the storm surge zone will require additional supplies and patient care capacity . delayed ( post - storm ) evacuation of health care facilities can compound logistical problems and risks because of loss of power and damage to communications and transportation infrastructure , making it more difficult to transport and track patients who may already be compromised by failure of medical equipment or exposure to heat or cold . a survey of twenty gulf state nursing homes identified significant logistical and response problems during hurricane katrina related to transportation , staffing , maintaining complex medication regimens , insufficient emergency provisions , and failure to follow emergency plans . all facilities encountered problems , but there were slightly more negative effects ( including dehydration , depression , and skin tears ) reported by administrators from facilities that evacuated rather than sheltered in place . three of them nyu langone medical center , bellevue hospital , and coney island hospital had to evacuate patients after the storm hit because of flood damage to critical equipment located in low - lying parts of the hospitals . many nursing and long - term care facilities also lost power and had to evacuate patients . local or widespread power outages could result from flood damage to underground and low - lying electrical infrastructure , as well as damage to utility poles and aboveground wires from high winds and downed trees . deaths and illness can occur when portable generators , cooking appliances , and other fuel burning equipments are used indoors or improperly [ 2434 ] . researchers theorized that increased mortality could be related to more physical demands on vulnerable people due to non - functioning elevators and subways , increased call times for ambulances , closed stores and pharmacies , and potentially the effects of increased exposure to air pollution and ambient air temperatures . frail residents of nursing homes and other health care facilities that shelter in place can be especially vulnerable to power outages in facilities without backup generators or with backups located in flood - vulnerable places such as basements . prison populations often have higher rates of mental and physical illness and substance use disorders than the general population and are at risk for exacerbations . after superstorm sandy made landfall , hundreds of thousands of nyc residents initially lost power . however , even after the electric grid had been largely restored , many residential buildings in storm - inundated areas still lacked electric power , heat , or running water , often because of salt water flood damage to buildings electrical and heating systems . exposure to hot or cold ambient temperatures from lack of climate control could result in heat- or cold - related illness , including heat stroke or hypothermia , as well as exacerbation of respiratory , cardiovascular , and other chronic diseases [ 41 , 44 , 45 ] . from the storm impact until november 9 ( 10 days ) , co - related emergency department visits and poison center ( pcc ) calls for co exposure were elevated for the time of year ; pcc data frequently identified storm - related sources of exposure including charcoal grills and household cooking appliances used for heating , as well as portable generators . while the potential health impacts resulting from sheltering in inadequate housing after a storm have not yet been formally evaluated , exacerbation of chronic health conditions , including physical , mental , and substance use disorders , could occur from a disruption of care due to lack of light , telecommunications , and elevator service that makes it difficult for people to access outside care , obtain medications , maintain self - care , and receive home - based care services . inability to access food and fresh water , particularly for people living in high - rise apartments where water delivery is dependent on electric pumps , could lead to infectious disease risk because of inability to properly wash hands or food , bathe , or flush toilets . people living in residential buildings without electricity may be more vulnerable to foodborne disease because they can not refrigerate food . it is possible that proliferation of pests , including rodents and roaches , because of difficulty with cleaning and removing trash may exacerbate allergies , asthma , and other respiratory conditions . older adults , young children , individuals with pre - existing health conditions , and those living on the upper floors of high - rise buildings or those who are disabled may be especially vulnerable to health effects resulting from power and utility outages . in addition , it is possible that this population may be at risk for mental health outcomes observed in populations who have experienced long - term displacement , including post - traumatic stress and other mental health problems . although increased concentrations of outdoor and indoor mold were detected after hurricane katrina in areas that had experienced flooding [ 49 , 50 ] , the impact of the mold on health following katrina has not been well characterized , possibly because of under - reporting or under - detection of health problems , or by population displacement that reduced exposure . coastal storms can cause release of untreated sewage through direct damage and flooding of treatment facilities , power outage , or combined sewage overflows ( cso ) . secondary exposure to sewage - contaminated floodwaters and wastewater , along with impaired access to potable water and flushable toilets , may lead to gastrointestinal infections , acute respiratory infections , skin infections , and insect bites [ 15 , 16 , 51 , 52 ] . rainfall , wind , and runoff in the watershed area can contribute to high turbidity levels , which can interfere with the disinfection process of drinking water . however , an increasing number of heavy precipitation events in the watershed may lead to more instances of high turbidity levels . the nyc department of environmental protection monitors turbidity at over 1,400 locations in the watershed and distribution system and activates the ashokan waste channel to reduce turbidity levels . storm damage may compromise sites storing toxic waste , and flood waters may move hazardous substances to new areas . following hurricane katrina , hazardous substances such as volatile organic compounds ( vocs ) , lead , and arsenic were detected in the air , soil , and sediment samples . following sandy , initial testing of two superfund sites indicated that contact with contaminated water from these areas was not a major health threat . following hurricane katrina , health care providers and focus groups also reported that chronic disease treatment interruptions especially among patients with cancer , hypertension , end stage renal disease , cardiovascular disease , and respiratory illnesses were problems . red cross shelter residents in the weeks immediately following katrina , slightly more than half of the residents had chronic medical conditions including hypertension , hypercholesterolemia , diabetes , and lung diseases . emergency responders may also need access to sufficient quantities of vaccines ( and facilities with proper storage capabilities ) , medications , and other preventive medical supplies for displaced populations . particularly in the months immediately following exposure to a natural disaster , increases in levels of post - traumatic stress disorder ( ptsd ) , as well as other mental health problems have been observed . more than a year after hurricane katrina , anxiety and mood disorders in the new orleans metro area were substantially elevated , and mental health conditions were broadly distributed in the population . serious mental illness was typically accompanied by ptsd , and important predictors of mental health problems were storm - related physical illness or injury , physical adversity , and property loss . following hurricane katrina , researchers have suggested that slow government responses may have exacerbated mental health problems and argued that an efficient emergency response can also help to minimize the mental health impacts of natural disasters . women , african - americans , and those with prior psychiatric history , poor physical health , and weak social networks have been identified as particularly vulnerable [ 75 , 78 , 79 , 81 , 82 , 85 ] . many studies on mental health conditions were cross - sectional , and pre - storm depression and ptsd levels could not always be ascertained , thereby limiting conclusions that could be drawn from the data about the cause - effect relationship between storms and subsequent mental health outcomes . nevertheless , preventing the long - term mental health effects following a storm through ongoing mental health surveillance , appropriate intervention , and adaptation strategies should remain a priority . residents and volunteers trying to clean affected homes could suffer non - fatal injuries ( including cuts , wounds , sprains , and strains ) and other health risks during the course of removing debris or during minor and major home repairs ( for instance , removing wet building materials or wet wall insulation ) . indoor dust created during cleaning , exposure to mold , fumes from temporary heating sources , and the use of strong cleaning products can also irritate the eyes , throat , and lungs . large - scale displacement could increase demands on the transportation infrastructure , and there may be occupational health concerns for municipal workers deployed to address flooding in transportation and communications infrastructure . during recovery , air quality may be negatively affected by dust from the home clean - up , debris movement , emissions from truck traffic , and the use of outdoor temporary boilers and emergency generators . following sandy , routine monitoring at rooftop air monitors in new york city showed that the levels of fine particles ( pm2.5)the pollutant most likely to be associated with combustion of fuels , dust from streets , and debris were not significantly elevated compared to levels typical for the season . similarly , two - week average pm2.5 concentrations at street level monitors near storm- impacted areas in the four weeks after the storm showed that levels were typical for the time of year and similar to those elsewhere in the city . for the most part , 24-hour average pm concentrations in these locations through the winter tracked with levels at regional monitors elsewhere in the city , although on several days concentrations were somewhat higher near areas with concentrations of temporary generators and boilers or reconstruction activity . table 1 summarizes examples of sub - populations identified in prior studies as having increased susceptibility to adverse health effects from different exposures resulting from coastal storms . the health impacts and vulnerable subgroups listed depict a range of potential outcomes but are not exhaustive . for the most part , these studies characterize vulnerability to longer term environmental hazards and stressors from the aftermath of coastal storms rather than the risk of injury or death during the impact phase of a storm . for the most part , however , groups most vulnerable to adverse storms - related outcomes include one or more of the following : older adults ; young children ; women ; those with pre - existing physical , mental health problems , or substance use disorders ; those living in low - income households ; members of disadvantaged racial / ethnic groups ; and those with weak social networks . based on the literature and data available at the neighborhood level , we identified 18 indicators of vulnerability in the domains of mental health , physical health , socioeconomic status , and housing ( table 2 ) . indicators were mapped within any 2012 evacuation zone . for instance , the percent of people living below the federal poverty level by neighborhood was highly correlated with indicators of population - level prevalence of disability frequent mental distress , social isolation , and poor housing quality , and moderately correlated with prevalence of chronic physical health conditions and lack of health insurance ( table 2 ) . the literature review process was not intended to be exhaustive but was rather a means to capture the majority of known health outcomes associated with flooding and coastal storms , and some subjective judgments were used to identify potential health effects for which there are no published studies . however , there may be health effects that are under - represented , not adequately characterized , or not described in the literature . population vulnerability may differ according to geographic region for a variety of reasons , so indicators based on studies from other areas may not adequately describe vulnerability in nyc or other urban areas . studies of vulnerability in smaller areas will be important in helping planners and communities better identify and prepare for the potential health impact of storms , especially in assisting in the development and implementation of neighborhood - based interventions . power outages related to wind damage or damage to utility substations can occur outside of the evacuation zones . hurricane evacuation zone designations will also likely expand in many places as rising sea levels are factored into the projected storm surge zone . furthermore , nyc indicators that were available and used for mapping are imperfect proxies for vulnerable subgroups identified in the literature . vulnerability maps based on these indicators do not represent an exact geospatial representation of vulnerability but rather a proxy measure of vulnerability for research and adaptation purposes [ 89 , 90 ] . with predicted warming of the climate , nyc and other coastal cities may be increasingly vulnerable to flooding during hurricanes and other severe storms . these events can have a devastating toll , as witnessed in 2005 with hurricane katrina in the gulf coast and in 2012 with superstorm sandy in the northeast . a wide range of potential acute and long - term health impacts were identified in the literature , from injury and death resulting from a failure to evacuate safely , physical and mental health problems in displaced populations because of disruption of care or stress , and injury and illness risk during repair and recovery , as well as a range of potential health impacts from exposures in damaged housing and from sheltering in place . for several dimensions of public health vulnerability to coastal storms , nyc neighborhoods with elevated poverty levels may be at increased risk for lasting impacts . while adaptation planning must be tailored to the needs of local health jurisdictions , public health protection depends to a great extent on minimizing critical infrastructure vulnerabilities , including enhancing the resilience of power delivery networks ( for instance , ensuring that key power infrastructure is located above anticipated high - water lines ) , buildings , transportation , and health care systems . it is also clear from experiences described in the literature that emergency preparedness for coastal storms will need to include preparation for both short - term and long - term needs of the most vulnerable populations , especially physical and mental health care and access to other essential services . increasing public health resilience to coastal storms also requires community engagement in climate - readiness strategies and an interdisciplinary approach to adaptation planning .

despite efforts in prevention and improvements of care during the past decades , traumatic brain injuries ( tbis ) still present a significant medical and public health problem globally . the main drivers of increasing concerns are large increases of tbi incidence caused by traffic accidents in developing countries ( related to substantially increasing traffic density ) and aging of the population ( especially in countries with high economy level ) and the related high incidence of tbi caused by falls . a recent systematic review in europe reported the pooled incidence of hospital - admitted tbi at 262/100,000 person years while another systematic review reported crude incidence rates derived from 22 population - based studies , ranging from 47.3 to 694 per 100,000 . along with the high rates of hospital admissions , tbi is associated with population mortality ranging from 9 to 28.1 per 100,000 . adding to the overall public health burden of tbi is the fact that the life expectancy of persons after tbi is significantly lower when compared to the general population : an analysis of two large cohorts of patients after tbi reported a standardized mortality ratio of 2.4 and 3.9 , respectively . prognosis of outcome after tbi is an important tool in the assessment of quality of care , policy - making , clinical research , and practice and thus can help improving treatment and outcome . progress in quality and validity of available tbi prognostic tools was made possible by advancements in statistical modeling techniques that were applied to large datasets of patients with tbi . a systematic review in 2006 identified 102 various prognostic models for patients with tbi although many of these were of poor quality . more recent models , such as the impact and the crash models , have been developed on large datasets of patients , adhering to strict development criteria , and were externally validated for populations other than those used for their development , which proves their overall validity and generalizability . one of the drawbacks of multivariable prognostic models is that they provide prognosis based on a large and strictly defined set of predictors which are not always available in clinical or research settings , thus making predictions impossible . alternatively to multivariable prognostic models , simpler and more readily available scores are often used to describe the severity and extent of the injury and prognosis after tbi ( for simplicity , we refer to them as simple scores throughout the paper ) . somewhat simpler when compared to multivariable models are scoring systems that are based on computed tomographic ( ct ) scans of the head and brain . two major scoring systems have been developed and established as valid tools in research and clinical practice . the marshall - ct score ( m - ct ) was introduced in 1991 and it groups patients based on their ct abnormalities into six groups . the rotterdam ct score ( r - ct ) provides a score on a 16 scale that is based on the type and severity of lesions on the ct scan . both , the m - ct and the r - ct , have been proven as valid tools for prognosis of patients after tbi . the glasgow coma scale ( gcs ) , gcs motor score ( gcsm ) , and abbreviated injury scale ( ais ) provide yet even simpler and widely used measures of injury severity . the gcs , introduced by teasdale and jennett in 1974 , is used to assess the level of consciousness based on motoric , eye , and verbal responses of the patient . of the three components of the gcs , the motoric response was identified as containing virtually all the prognostic information carried by the summary score and had been used in multivariable prognostic models , such as the impact models . the ais has been developed by the ama committee on medical aspects of automotive safety to provide researchers with a comprehensive rating system to tissue damage in regions of the body including the region of head and neck ( ais for the head region [ ais - h ] ) . ais served as a basis for the development of the injury severity score a summary measure that describes the severity of the overall injury based on the assessment of the injury in each body part using the ais . the summary gcs , gcsm , and ais have all been previously used in prognostic models and have become standard measures of injury severity in patients after tbi . as they are routinely used and relatively easy to obtain , they potentially present an easily available prognostic tool . in this study , we sought to find out whether simple scores ( gcs , gcsm , ais - h , m - ct , or r - ct ) could be used for prognosis of outcome in patients after tbi alternatively to more complex multivariable models and how big is the difference between their predictive performances . the aim of this study was to perform a side - by - side comparison of the prognostic value of a set of simple injury severity measures ( gcs , gcsm , ais - h , m - ct , and r - ct ) between each other and against a gold standard presented by the impact - extended ( imp - e ) multivariable prognostic models . for this study , data from two observational clinical studies conducted in austrian hospitals were used . the first study : austrian tbi project focused on the introduction of evidence - based treatment of tbi in austria , ran from 2002 to 2005 , and collected data on 415 patients in five major austrian hospitals . all patients with a gcs of 8 or less and admitted to the hospital alive were included . the second study : early treatment of patients with severe and moderate tbi in austria , focused on evidence - based prehospital care and collected data on 778 patients with moderate or severe tbi ( gcs 12 or less ) in 14 austrian centers , in 20092012 . in both cases , compatible databases were used for data collection which allowed merging the two datasets in one , with a total of 1192 patient records . the study has been approved by the respective ethical committees of all participating centers where the data used in the paper were collected , in full accordance with the declaration of helsinki . the following data were collected for all patients : demographic characteristics ( age , sex ) ; data on injury type and severity ( including total gcs , its components , ais for the regions of head / neck , face , thorax , extremities , abdomen , and external ) ; data on ct scan ( allowing to categorize patients using the marshall and r - ct scores ) ; data on prehospital and intensive care unit ( icu ) treatment ; and data on outcome at icu discharge , hospital discharge , and 6 months postinjury . outcomes at icu discharge and hospital discharge were recorded as survival / death and the outcomes at 6 months were assessed using the glasgow outcome scale ( gos ) ; for this study , the gos at 6 months postinjury was categorized ( 1 ) as survival / death and ( 2 ) as favorable ( gos 45)/unfavorable ( gos 13 ) . the main line of analysis in this study is a side - by - side comparison of the prognostic value of the selected simple scores ( gcs , gcsm , ais - h , m - ct , and r - ct ) for the outcomes at icu discharge ( mortality ) , at hospital discharge ( mortality ) , and at 6 months postinjury ( mortality and favorable vs. unfavorable outcome ) . second , all scores are compared to the imp - e multivariable prognostic model selected as the gold standard . this model was selected as it showed good prognostic performance when externally validated for the prediction of outcomes in patients with tbi in austria . overview and characteristics of the scores that were analyzed for their prognostic value in predicting outcomes after traumatic brain injury binary logistic regression models were fit using the binary outcome ( either alive / dead or favorable / unfavorable ) as response variables , and the respective scores ( as single predictors ) or the set of variables defined in the imp - e model were used as predictors . to allow cross - comparisons of the size of the effect of the predicting variables on the outcome in each model , odds ratios with 95% confidence intervals are presented . to evaluate the prognostic value , the area under the receiver operating characteristics ( roc ) curve ( auc ) and nagelkerke 's r were used . the auc is a value ranging between 0.5 and 1 referring to the discrimination ability of the model ( discrimination between the two outcomes defined in the binary response variable ) : auc = 1 means perfect discrimination and auc = 0.5 means discrimination no better than by chance . nagelkerke 's r is a measure of the predictive ability ( ranging between 0 and 1 ) , indicating better performance when approaching 1 . model overfitting may eventually result in too optimistic expected performance on subjects other than those used for the development of the model . to correct for such optimism in the model predictions , the presented auc and nagelkerke 's r values are after such correction . to allow comparison of the performance of all models to the gold standard , the values of auc and nagelkerke 's r of each model were subtracted from the values calculated for the imp - e model . roc curves are presented to allow graphical assessment of the differences in aucs between the models . for this study , data from two observational clinical studies conducted in austrian hospitals were used . the first study : austrian tbi project focused on the introduction of evidence - based treatment of tbi in austria , ran from 2002 to 2005 , and collected data on 415 patients in five major austrian hospitals . all patients with a gcs of 8 or less and admitted to the hospital alive were included . the second study : early treatment of patients with severe and moderate tbi in austria , focused on evidence - based prehospital care and collected data on 778 patients with moderate or severe tbi ( gcs 12 or less ) in 14 austrian centers , in 20092012 . in both cases , compatible databases were used for data collection which allowed merging the two datasets in one , with a total of 1192 patient records . the study has been approved by the respective ethical committees of all participating centers where the data used in the paper were collected , in full accordance with the declaration of helsinki . the following data were collected for all patients : demographic characteristics ( age , sex ) ; data on injury type and severity ( including total gcs , its components , ais for the regions of head / neck , face , thorax , extremities , abdomen , and external ) ; data on ct scan ( allowing to categorize patients using the marshall and r - ct scores ) ; data on prehospital and intensive care unit ( icu ) treatment ; and data on outcome at icu discharge , hospital discharge , and 6 months postinjury . outcomes at icu discharge and hospital discharge were recorded as survival / death and the outcomes at 6 months were assessed using the glasgow outcome scale ( gos ) ; for this study , the gos at 6 months postinjury was categorized ( 1 ) as survival / death and ( 2 ) as favorable ( gos 45)/unfavorable ( gos 13 ) . the main line of analysis in this study is a side - by - side comparison of the prognostic value of the selected simple scores ( gcs , gcsm , ais - h , m - ct , and r - ct ) for the outcomes at icu discharge ( mortality ) , at hospital discharge ( mortality ) , and at 6 months postinjury ( mortality and favorable vs. unfavorable outcome ) . second , all scores are compared to the imp - e multivariable prognostic model selected as the gold standard . this model was selected as it showed good prognostic performance when externally validated for the prediction of outcomes in patients with tbi in austria . overview and characteristics of the scores that were analyzed for their prognostic value in predicting outcomes after traumatic brain injury binary logistic regression models were fit using the binary outcome ( either alive / dead or favorable / unfavorable ) as response variables , and the respective scores ( as single predictors ) or the set of variables defined in the imp - e model were used as predictors . to allow cross - comparisons of the size of the effect of the predicting variables on the outcome in each model , odds ratios with 95% confidence intervals are presented . to evaluate the prognostic value , the area under the receiver operating characteristics ( roc ) curve ( auc ) and nagelkerke 's r were used . the auc is a value ranging between 0.5 and 1 referring to the discrimination ability of the model ( discrimination between the two outcomes defined in the binary response variable ) : auc = 1 means perfect discrimination and auc = 0.5 means discrimination no better than by chance . nagelkerke 's r is a measure of the predictive ability ( ranging between 0 and 1 ) , indicating better performance when approaching 1 . model overfitting may eventually result in too optimistic expected performance on subjects other than those used for the development of the model . to correct for such optimism in the model predictions , the presented auc and nagelkerke 's r values are after such correction . to allow comparison of the performance of all models to the gold standard , the values of auc and nagelkerke 's r of each model were subtracted from the values calculated for the imp - e model . roc curves are presented to allow graphical assessment of the differences in aucs between the models . figure 1 presents the flow of participants throughout the study only patients for whom the outcomes at all three points of assessment were available were included in the analyses . table 2 presents the description of demographic characteristics of the sample , type and severity of their injury , their treatment , and outcome at different stages post injury . the mean age was 49.9 years and about three - fourth of patients were male . traffic injury and falls were with virtually equal share , the predominant causes ( together 85% of patients ) . in general , the mean iss ( 32.6 ) , mean ais - h ( 4.2 ) , low median of the first gcsm score ( 3 ) and total gcs score ( 4 ) , along with relatively high rates of hypotension and hypoxia indicate severe injuries . the most common predominant ct diagnoses were subdural hematoma ( 34% ) , diffuse edema ( 15% ) , and subarachnoid hemorrhage ( 14% ) . these ct findings were summarized using the marshall ct ( m - ct ) score ( 42% of patients fell into categories 5 or 6 ) and the r - ct score ( 28% had a score of 4 or higher ) . about half of the patients were transported by helicopter , most received prehospital fluid ( 95% of patients ; mean amount 864 ml ) , and almost three - fourth were intubated and underwent neurosurgical procedure . the mortality at icu discharge was 36% and increased to 41% at 6 months postinjury ; about half of the patients had unfavorable outcome at 6 months . gcsm : glasgow coma scale motor score ; gcst : total gcs ; ais - h : abbreviated injury scale for head and neck regions ; m - ct : marshall ct classification , r - ct : rotterdam ct score demographic characteristics , injury type and severity , treatment factors , and outcomes at different stages postinjury in the analyzed sample of patients with traumatic brain injury ( n=866 patients ) table 3 gives an overview of the effect size of the predictors ( simple scores and the imp - e model ) on the outcomes at different time points postinjury , presented as odds ratios . in general , the sizes of effects for the same predictor were similar throughout the outcomes with little variations . when comparing the gcs , gcsm , and ais - h with the r - ct and m - ct scores , the two latter had considerably higher effect , suggesting that the relation of the two ct scores to outcomes is higher . overview of odds ratios of the effects of tested predictors in the regression models with 95% confidence intervals table 4 presents the comparative analysis of the prognostic performance of the tested scores . when comparing the aucs and rs of the same model across the four outcomes , only little variation is observed in all cases which suggest that the performance of each score ( and model ) is similar for the prediction of outcome at icu discharge , hospital discharge , and 6 months ( both mortality and unfavorable outcome ) . a similar pattern is observed when comparing the performance of the models between each other ( for each outcome point separately ) : the variation in case of aucs is not larger than 0.09 points ; the rs vary somewhat more , by up to 0.17 points . both these comparisons suggest that all scores perform similarly when used for predicting outcomes at various points of assessment . in all cases , ais - h performed superior to all other tested scores , suggesting best prognostic value at each outcome point . prognostic value of the analyzed predictors for outcome in patients after traumatic brain injury at different stages post injury on the other hand , all analyzed scores perform significantly worse when compared to the imp - e model ( gold standard ) : in case of auc , the differences vary between 0.10 and 0.22 ( p < 0.05 in all comparisons of aucs between simple scores and gold standard ) , and in case of r , the differences are even more significant , ranging between 0.22 and 0.39 points . these findings are confirmed in figure 2 where the roc curves of all models are presented in a cross comparison : the rocs of all tested models are relatively close to each other , and a significantly lower performance is apparent in all cases when compared to the imp - e model . comparison of receiver operating characteristics curves of the analyzed prediction models to the gold standard ( receiver operating characteristics curve of the impact extended model ) . gcsm : glasgow coma scale motor score ; gcst : total gcs ; ais - h : abbreviated injury scale for head and neck ; m - ct : marshall ct classification , r - ct : rotterdam ct score ; imp - e : impact extended model . the thick curve represents the gold standard ( receiver operating characteristics of the impact extended model ) we present a study where we have performed a side - by - side comparison of the predictive value of simple injury severity scores between each other and against a gold standard , for which the prognostic performance of the imp - e multivariable prognostic model was taken . to the best of our knowledge , our main findings are as follows : the compared simple scores ( gcs , gcsm , m - ct , r - ct ) had similar prognostic performance in a side - by - side comparison for the prediction of outcomes at each time point ; the performance of ais - h was slightly superior to all other simple scores ; the performance of each single score ( gcs , gcsm , ais - h , m - ct , r - ct , imp - e ) was similar when used for predictions of outcome at various time points postinjury ; the prognostic performance of the simple scores ( gcs , gcsm , ais - h , m - ct , r - ct ) was significantly worse when compared to the performance of the imp - e model ( gold standard ) . although this is , to our knowledge , the first study presenting cross - comparison of prognostic performances of various injury severity scores , previous studies reported analyses of the performance of such scores separately . out of the scores reported in this study , the most studied in the context of outcome prediction in tbi patients is the gcs and its components . it must be noted that there are aspects of the gcs relevant to note in the context of this study . first , the gcs in tbi patients is routinely being assessed at various time points after injury ( typically in field and at admission ) and it has been shown that the predictive value may vary by time of assessment . these differences may be due to ability to validly assess the gcs in sedated and intubated patients . second , the values of the gcs may differ due to interrater disagreement when it is assessed at various time points by different persons . however , there is strong evidence that both the total gcs and the gcsm ( which was found to contain virtually all the prognostic value of the total gcs ) are strongly related to outcome in tbi patients . a study analyzing 21,657 patients from the uk reported an auc = 0.89 and nagelkerke 's r = 0.55 for the total gcs assessed in the field ; whereas for admission assessment , an auc = 0.9 and nagelkerke 's r = 0.59 were reported . an earlier study of the same predictor found an auc = 0.92 ( gcs defined as worse in 24 h and estimated if the patient received sedatives ) . a meta - analysis ( although including only 5 studies ) reported a pooled auc of 0.9 for the prediction of death . another study using a large dataset of 12,882 patients found aucs for field and admission assessments to be 0.84 . the performance of the gcsm was found to be similar to that of the total gcs ( auc = 0.91 for both field and admission assessment , nagelkerke 's r = 59 and 58 for field and admission assessment , respectively ) . the prognostic performance indices of the gcs and gcsm found in our study are somewhat lower which may be caused by various factors : ( 1 ) as opposed to using field or admission values of gcs , in our analyses we have used the first available gcs ( and gcsm ) as the predictor ( using the field assessment wherever available , and the admission value if the field assessment was not available ) , as suggested by the impact study ; ( 2 ) changes in prehospital treatment over time influence the values of gcs and its predictive value as shown in a study comparing patients over a 10-year lag period , and thus , different time of data collection in the compared studies could potentially affect the respective prognostic characteristics and partly explain the differences . although the ais - h as a predictor of outcome has been previously studied , no direct comparisons can be made as previous reports do not provide indices such as auc or nagelkerke 's r. a study compared the predictive ability of the head ais - h , injury severity scores , and gcs by correlating them with the 2 months outcome on the gos - extended scale in 270 tbi patients and concluded that ais - h , injury severity scores , and gcs in combination correlated with the outcome better than any of the three measures alone . ais - h had been previously proven to be significantly related to outcome of tbi patients in the population from which the patients for this study have been drawn . the predictive performance of the r - ct and m - ct scores was also previously studied . a side - by - side comparison of their predictive value reported an auc of 0.85 for both scores , somewhat higher than in our study . another study comparing the m - ct and r - ct in a side - by - side manner found aucs of 0.63 and 0.68 for 6 months unfavorable outcome and aucs of 0.64 and 0.7 for 6 months mortality ; the reported nagelkerke 's rs were 0.09 and 0.16 for the prediction of 6 months unfavorable outcome and 0.09 and 0.15 for the prediction of 6 months mortality . the performance indices reported in this study are similar to our findings and in favor of their comparability speaks the virtually the same line of statistical analysis that was followed in both studies ( including optimism correction ) . the imp - e study has been created along with other impact models in 2008 . it has been established as a prognostic model of choice by a number of external validation studies ( including a study on a population which was used for this study ) , which justifies its use as a gold standard in this paper . our question in this study was whether simple , readily available , and routinely used injury severity scores can be used for valid prognosis of outcome in tbi patients , when compared between each other and when compared to multivariable prognostic models . our findings suggest that the performance of multivariable prognostic models is far superior to simpler scores used in a univariable manner . this limits the potential use of these simple scores as primary , standalone prognostic tools in clinical or research settings . however , they still pose good prognostic characteristics and could be used for initial or preliminary prognosis . their relative simplicity provides a strong leverage over multivariable models , where usually a large set of predictors must be available to estimate the prognosis . the data used for the analyses come from two studies that were conducted a few years apart from each other . however , both studies used compatible databases , which ensured that all variables used in this paper were uniformly defined in both studies . furthermore , rigorous data quality control ( paper records were checked in detail against electronic records ) was in place to ensure the validity of the data . based on our analyses , all tested simple scores ( gcs , gcsm , ais - h , m - ct , and r - ct ) can provide reasonably valid prognosis . however , it is confirmed that well - developed multivariable prognostic models outperform these scores significantly and therefore should be used for prognosis in patients after tbi wherever possible . 15 , 2008 ) and by the austrian worker 's compensation board ( auva ; fk 11/2008 and fk 11/2010 ) . the work of marek majdan on this paper was partly funded by an institutional grant from the trnava university ( 2/tu/2015 ) . the project was funded jointly by the austrian ministry of health ( contract oct . 15 , 2008 ) and by the austrian worker 's compensation board ( auva ; fk 11/2008 and fk 11/2010 ) . the work of marek majdan on this paper was partly funded by an institutional grant from the trnava university ( 2/tu/2015 ) .

objectives : prognosis of outcome after traumatic brain injury ( tbi ) is important in the assessment of quality of care and can help improve treatment and outcome . the aim of this study was to compare the prognostic value of relatively simple injury severity scores between each other and against a gold standard model the impact - extended ( imp - e ) multivariable prognostic model.materials and methods : for this study , 866 patients with moderate / severe tbi from austria were analyzed . the prognostic performances of the glasgow coma scale ( gcs ) , gcs motor ( gcsm ) score , abbreviated injury scale for the head region , marshall computed tomographic ( ct ) classification , and rotterdam ct score were compared side - by - side and against the imp - e score . the area under the receiver operating characteristics curve ( auc ) and nagelkerke 's r2 were used to assess the prognostic performance . outcomes at the intensive care unit , at hospital discharge , and at 6 months ( mortality and unfavorable outcome ) were used as end-points.results:comparing aucs and r2s of the same model across four outcomes , only little variation was apparent . a similar pattern is observed when comparing the models between each other : variation of aucs < 0.09 and r2s by up to 0.17 points suggest that all scores perform similarly in predicting outcomes at various points ( aucs : 0.650.77 ; r2s : 0.090.27 ) . all scores performed significantly worse than the imp - e model ( with auc > 0.83 and r2 > 0.42 for all outcomes ) : aucs were worse by 0.100.22 ( p < 0.05 ) and r2s were worse by 0.220.39 points.conclusions:all tested simple scores can provide reasonably valid prognosis . however , it is confirmed that well - developed multivariable prognostic models outperform these scores significantly and should be used for prognosis in patients after tbi wherever possible .

I M Study design and participants Test methods and analysis R D C Financial support and sponsorship Conflicts of interest

prognosis of outcome after tbi is an important tool in the assessment of quality of care , policy - making , clinical research , and practice and thus can help improving treatment and outcome . one of the drawbacks of multivariable prognostic models is that they provide prognosis based on a large and strictly defined set of predictors which are not always available in clinical or research settings , thus making predictions impossible . alternatively to multivariable prognostic models , simpler and more readily available scores are often used to describe the severity and extent of the injury and prognosis after tbi ( for simplicity , we refer to them as simple scores throughout the paper ) . somewhat simpler when compared to multivariable models are scoring systems that are based on computed tomographic ( ct ) scans of the head and brain . the rotterdam ct score ( r - ct ) provides a score on a 16 scale that is based on the type and severity of lesions on the ct scan . both , the m - ct and the r - ct , have been proven as valid tools for prognosis of patients after tbi . the glasgow coma scale ( gcs ) , gcs motor score ( gcsm ) , and abbreviated injury scale ( ais ) provide yet even simpler and widely used measures of injury severity . the gcs , introduced by teasdale and jennett in 1974 , is used to assess the level of consciousness based on motoric , eye , and verbal responses of the patient . of the three components of the gcs , the motoric response was identified as containing virtually all the prognostic information carried by the summary score and had been used in multivariable prognostic models , such as the impact models . the ais has been developed by the ama committee on medical aspects of automotive safety to provide researchers with a comprehensive rating system to tissue damage in regions of the body including the region of head and neck ( ais for the head region [ ais - h ] ) . ais served as a basis for the development of the injury severity score a summary measure that describes the severity of the overall injury based on the assessment of the injury in each body part using the ais . the summary gcs , gcsm , and ais have all been previously used in prognostic models and have become standard measures of injury severity in patients after tbi . in this study , we sought to find out whether simple scores ( gcs , gcsm , ais - h , m - ct , or r - ct ) could be used for prognosis of outcome in patients after tbi alternatively to more complex multivariable models and how big is the difference between their predictive performances . the aim of this study was to perform a side - by - side comparison of the prognostic value of a set of simple injury severity measures ( gcs , gcsm , ais - h , m - ct , and r - ct ) between each other and against a gold standard presented by the impact - extended ( imp - e ) multivariable prognostic models . for this study , data from two observational clinical studies conducted in austrian hospitals were used . the second study : early treatment of patients with severe and moderate tbi in austria , focused on evidence - based prehospital care and collected data on 778 patients with moderate or severe tbi ( gcs 12 or less ) in 14 austrian centers , in 20092012 . the following data were collected for all patients : demographic characteristics ( age , sex ) ; data on injury type and severity ( including total gcs , its components , ais for the regions of head / neck , face , thorax , extremities , abdomen , and external ) ; data on ct scan ( allowing to categorize patients using the marshall and r - ct scores ) ; data on prehospital and intensive care unit ( icu ) treatment ; and data on outcome at icu discharge , hospital discharge , and 6 months postinjury . outcomes at icu discharge and hospital discharge were recorded as survival / death and the outcomes at 6 months were assessed using the glasgow outcome scale ( gos ) ; for this study , the gos at 6 months postinjury was categorized ( 1 ) as survival / death and ( 2 ) as favorable ( gos 45)/unfavorable ( gos 13 ) . the main line of analysis in this study is a side - by - side comparison of the prognostic value of the selected simple scores ( gcs , gcsm , ais - h , m - ct , and r - ct ) for the outcomes at icu discharge ( mortality ) , at hospital discharge ( mortality ) , and at 6 months postinjury ( mortality and favorable vs. unfavorable outcome ) . second , all scores are compared to the imp - e multivariable prognostic model selected as the gold standard . this model was selected as it showed good prognostic performance when externally validated for the prediction of outcomes in patients with tbi in austria . overview and characteristics of the scores that were analyzed for their prognostic value in predicting outcomes after traumatic brain injury binary logistic regression models were fit using the binary outcome ( either alive / dead or favorable / unfavorable ) as response variables , and the respective scores ( as single predictors ) or the set of variables defined in the imp - e model were used as predictors . to evaluate the prognostic value , the area under the receiver operating characteristics ( roc ) curve ( auc ) and nagelkerke 's r were used . the auc is a value ranging between 0.5 and 1 referring to the discrimination ability of the model ( discrimination between the two outcomes defined in the binary response variable ) : auc = 1 means perfect discrimination and auc = 0.5 means discrimination no better than by chance . nagelkerke 's r is a measure of the predictive ability ( ranging between 0 and 1 ) , indicating better performance when approaching 1 . model overfitting may eventually result in too optimistic expected performance on subjects other than those used for the development of the model . to correct for such optimism in the model predictions , the presented auc and nagelkerke 's r values are after such correction . to allow comparison of the performance of all models to the gold standard , the values of auc and nagelkerke 's r of each model were subtracted from the values calculated for the imp - e model . roc curves are presented to allow graphical assessment of the differences in aucs between the models . for this study , data from two observational clinical studies conducted in austrian hospitals were used . the second study : early treatment of patients with severe and moderate tbi in austria , focused on evidence - based prehospital care and collected data on 778 patients with moderate or severe tbi ( gcs 12 or less ) in 14 austrian centers , in 20092012 . the following data were collected for all patients : demographic characteristics ( age , sex ) ; data on injury type and severity ( including total gcs , its components , ais for the regions of head / neck , face , thorax , extremities , abdomen , and external ) ; data on ct scan ( allowing to categorize patients using the marshall and r - ct scores ) ; data on prehospital and intensive care unit ( icu ) treatment ; and data on outcome at icu discharge , hospital discharge , and 6 months postinjury . outcomes at icu discharge and hospital discharge were recorded as survival / death and the outcomes at 6 months were assessed using the glasgow outcome scale ( gos ) ; for this study , the gos at 6 months postinjury was categorized ( 1 ) as survival / death and ( 2 ) as favorable ( gos 45)/unfavorable ( gos 13 ) . the main line of analysis in this study is a side - by - side comparison of the prognostic value of the selected simple scores ( gcs , gcsm , ais - h , m - ct , and r - ct ) for the outcomes at icu discharge ( mortality ) , at hospital discharge ( mortality ) , and at 6 months postinjury ( mortality and favorable vs. unfavorable outcome ) . second , all scores are compared to the imp - e multivariable prognostic model selected as the gold standard . this model was selected as it showed good prognostic performance when externally validated for the prediction of outcomes in patients with tbi in austria . overview and characteristics of the scores that were analyzed for their prognostic value in predicting outcomes after traumatic brain injury binary logistic regression models were fit using the binary outcome ( either alive / dead or favorable / unfavorable ) as response variables , and the respective scores ( as single predictors ) or the set of variables defined in the imp - e model were used as predictors . to evaluate the prognostic value , the area under the receiver operating characteristics ( roc ) curve ( auc ) and nagelkerke 's r were used . the auc is a value ranging between 0.5 and 1 referring to the discrimination ability of the model ( discrimination between the two outcomes defined in the binary response variable ) : auc = 1 means perfect discrimination and auc = 0.5 means discrimination no better than by chance . nagelkerke 's r is a measure of the predictive ability ( ranging between 0 and 1 ) , indicating better performance when approaching 1 . model overfitting may eventually result in too optimistic expected performance on subjects other than those used for the development of the model . to correct for such optimism in the model predictions , the presented auc and nagelkerke 's r values are after such correction . to allow comparison of the performance of all models to the gold standard , the values of auc and nagelkerke 's r of each model were subtracted from the values calculated for the imp - e model . roc curves are presented to allow graphical assessment of the differences in aucs between the models . table 2 presents the description of demographic characteristics of the sample , type and severity of their injury , their treatment , and outcome at different stages post injury . in general , the mean iss ( 32.6 ) , mean ais - h ( 4.2 ) , low median of the first gcsm score ( 3 ) and total gcs score ( 4 ) , along with relatively high rates of hypotension and hypoxia indicate severe injuries . these ct findings were summarized using the marshall ct ( m - ct ) score ( 42% of patients fell into categories 5 or 6 ) and the r - ct score ( 28% had a score of 4 or higher ) . about half of the patients were transported by helicopter , most received prehospital fluid ( 95% of patients ; mean amount 864 ml ) , and almost three - fourth were intubated and underwent neurosurgical procedure . the mortality at icu discharge was 36% and increased to 41% at 6 months postinjury ; about half of the patients had unfavorable outcome at 6 months . gcsm : glasgow coma scale motor score ; gcst : total gcs ; ais - h : abbreviated injury scale for head and neck regions ; m - ct : marshall ct classification , r - ct : rotterdam ct score demographic characteristics , injury type and severity , treatment factors , and outcomes at different stages postinjury in the analyzed sample of patients with traumatic brain injury ( n=866 patients ) table 3 gives an overview of the effect size of the predictors ( simple scores and the imp - e model ) on the outcomes at different time points postinjury , presented as odds ratios . when comparing the gcs , gcsm , and ais - h with the r - ct and m - ct scores , the two latter had considerably higher effect , suggesting that the relation of the two ct scores to outcomes is higher . overview of odds ratios of the effects of tested predictors in the regression models with 95% confidence intervals table 4 presents the comparative analysis of the prognostic performance of the tested scores . when comparing the aucs and rs of the same model across the four outcomes , only little variation is observed in all cases which suggest that the performance of each score ( and model ) is similar for the prediction of outcome at icu discharge , hospital discharge , and 6 months ( both mortality and unfavorable outcome ) . a similar pattern is observed when comparing the performance of the models between each other ( for each outcome point separately ) : the variation in case of aucs is not larger than 0.09 points ; the rs vary somewhat more , by up to 0.17 points . both these comparisons suggest that all scores perform similarly when used for predicting outcomes at various points of assessment . prognostic value of the analyzed predictors for outcome in patients after traumatic brain injury at different stages post injury on the other hand , all analyzed scores perform significantly worse when compared to the imp - e model ( gold standard ) : in case of auc , the differences vary between 0.10 and 0.22 ( p < 0.05 in all comparisons of aucs between simple scores and gold standard ) , and in case of r , the differences are even more significant , ranging between 0.22 and 0.39 points . these findings are confirmed in figure 2 where the roc curves of all models are presented in a cross comparison : the rocs of all tested models are relatively close to each other , and a significantly lower performance is apparent in all cases when compared to the imp - e model . comparison of receiver operating characteristics curves of the analyzed prediction models to the gold standard ( receiver operating characteristics curve of the impact extended model ) . gcsm : glasgow coma scale motor score ; gcst : total gcs ; ais - h : abbreviated injury scale for head and neck ; m - ct : marshall ct classification , r - ct : rotterdam ct score ; imp - e : impact extended model . the thick curve represents the gold standard ( receiver operating characteristics of the impact extended model ) we present a study where we have performed a side - by - side comparison of the predictive value of simple injury severity scores between each other and against a gold standard , for which the prognostic performance of the imp - e multivariable prognostic model was taken . to the best of our knowledge , our main findings are as follows : the compared simple scores ( gcs , gcsm , m - ct , r - ct ) had similar prognostic performance in a side - by - side comparison for the prediction of outcomes at each time point ; the performance of ais - h was slightly superior to all other simple scores ; the performance of each single score ( gcs , gcsm , ais - h , m - ct , r - ct , imp - e ) was similar when used for predictions of outcome at various time points postinjury ; the prognostic performance of the simple scores ( gcs , gcsm , ais - h , m - ct , r - ct ) was significantly worse when compared to the performance of the imp - e model ( gold standard ) . although this is , to our knowledge , the first study presenting cross - comparison of prognostic performances of various injury severity scores , previous studies reported analyses of the performance of such scores separately . out of the scores reported in this study , the most studied in the context of outcome prediction in tbi patients is the gcs and its components . it must be noted that there are aspects of the gcs relevant to note in the context of this study . first , the gcs in tbi patients is routinely being assessed at various time points after injury ( typically in field and at admission ) and it has been shown that the predictive value may vary by time of assessment . second , the values of the gcs may differ due to interrater disagreement when it is assessed at various time points by different persons . however , there is strong evidence that both the total gcs and the gcsm ( which was found to contain virtually all the prognostic value of the total gcs ) are strongly related to outcome in tbi patients . a study analyzing 21,657 patients from the uk reported an auc = 0.89 and nagelkerke 's r = 0.55 for the total gcs assessed in the field ; whereas for admission assessment , an auc = 0.9 and nagelkerke 's r = 0.59 were reported . an earlier study of the same predictor found an auc = 0.92 ( gcs defined as worse in 24 h and estimated if the patient received sedatives ) . the prognostic performance indices of the gcs and gcsm found in our study are somewhat lower which may be caused by various factors : ( 1 ) as opposed to using field or admission values of gcs , in our analyses we have used the first available gcs ( and gcsm ) as the predictor ( using the field assessment wherever available , and the admission value if the field assessment was not available ) , as suggested by the impact study ; ( 2 ) changes in prehospital treatment over time influence the values of gcs and its predictive value as shown in a study comparing patients over a 10-year lag period , and thus , different time of data collection in the compared studies could potentially affect the respective prognostic characteristics and partly explain the differences . although the ais - h as a predictor of outcome has been previously studied , no direct comparisons can be made as previous reports do not provide indices such as auc or nagelkerke 's r. a study compared the predictive ability of the head ais - h , injury severity scores , and gcs by correlating them with the 2 months outcome on the gos - extended scale in 270 tbi patients and concluded that ais - h , injury severity scores , and gcs in combination correlated with the outcome better than any of the three measures alone . ais - h had been previously proven to be significantly related to outcome of tbi patients in the population from which the patients for this study have been drawn . a side - by - side comparison of their predictive value reported an auc of 0.85 for both scores , somewhat higher than in our study . another study comparing the m - ct and r - ct in a side - by - side manner found aucs of 0.63 and 0.68 for 6 months unfavorable outcome and aucs of 0.64 and 0.7 for 6 months mortality ; the reported nagelkerke 's rs were 0.09 and 0.16 for the prediction of 6 months unfavorable outcome and 0.09 and 0.15 for the prediction of 6 months mortality . the imp - e study has been created along with other impact models in 2008 . it has been established as a prognostic model of choice by a number of external validation studies ( including a study on a population which was used for this study ) , which justifies its use as a gold standard in this paper . our question in this study was whether simple , readily available , and routinely used injury severity scores can be used for valid prognosis of outcome in tbi patients , when compared between each other and when compared to multivariable prognostic models . our findings suggest that the performance of multivariable prognostic models is far superior to simpler scores used in a univariable manner . the data used for the analyses come from two studies that were conducted a few years apart from each other . based on our analyses , all tested simple scores ( gcs , gcsm , ais - h , m - ct , and r - ct ) can provide reasonably valid prognosis . however , it is confirmed that well - developed multivariable prognostic models outperform these scores significantly and therefore should be used for prognosis in patients after tbi wherever possible .

pulmonary fibrosis can occur as an idiopathic disease or as a consequence of a variety of connective tissue diseases with undefined aetiology , including scleroderma , dermatomyositis / polymyositis , systemic lupus erythematosus , and rheumatoid arthritis . pulmonary fibrosis is characterized by epithelial injury and activation , formation of distinctive subepithelial fibroblast / myofibroblast foci , and excessive extracellular matrix ( ecm ) accumulation . many lines of evidence have suggested that recurrent injuries to pulmonary epithelial cells and ineffective repair initiate aberrant fibroblastic responses . epithelial cells undergo phenotypic changes of epithelial to mesenchymal transition ( emt ) , in which the cells lose their epithelial characteristics and acquire a mesenchymal phenotype . it is estimated that up to one third of fibroblasts may be of epithelial origin according to lineage tracing in murine models of lung fibrosis in vivo [ 1 , 2 ] . although this view has been challenged by rock et al . , it needs further study not only in mice but also in tissues from patients with idiopathic pulmonary fibrosis . the key mesenchymal features of pathological fibrosis are increased numbers of transdifferentiated fibroblasts , named myofibroblasts . they overexpress -smooth muscle actin ( -sma ) and are probably responsible for the enhanced synthesis of abnormal matrix observed in pulmonary fibrosis . transforming growth factor ( tgf)-1 has been shown to play a key role in pulmonary fibrosis , not only through its functions to attract fibroblasts and to stimulate their proliferation , but also through induction of emt in alveolar epithelial cells by activating smad- or non - smad signaling pathways . interleukin-22 ( il-22 ) is a member of the il-10 cytokine family and plays a critical role in inflammation , immune surveillance , and homeostasis in tissues that serve a barrier function such as skin , respiratory ( trachea and lungs ) and gastrointestinal ( stomach , small intestines , and colon ) tracts as well as liver , pancreas , and kidney . il-22 binds to a membrane receptor complex composed of the il-22r1 ( il-22ra1 ) and il-10r2 ( il-10rb2 ) , and signals intracellularly mainly through transcription factor jak / stat . interestingly , it was also showed that tgf-/smad signaling contributes to blm - induced fibrosis by promoting emt , and recent studies demonstrated that tgf- downregulated the il-22 producing capacity of th17 cells in both human [ 9 , 10 ] and mouse systems and inhibited the development of th22 cells . similarly , il-17a could regulate the properties of il-22 in the airway damage and inflammation , whereas il-17a enhanced blm - induced fibrosis in a tgf- dependent manner . to date , however , the crosstalk between il-22 and tgf--driven emt in pulmonary fibrosis has remained unclear . in the present study , we investigated the role of il-22 in emt in blm - induced pulmonary fibrosis mouse model as well as in vitro . we found that il-22 inhibited blm - induced emt , suggesting a potential therapeutic role of il-22 in pulmonary fibrosis . c57bl/6 mice were purchased from the shanghai laboratory animal center ( chinese academy of sciences ) . the animal study was approved by the institutional animal care and use committee of huashan hospital , fudan university . all surgery was performed under chloral hydrate anesthesia , and all efforts were made to minimize suffering . six- to eight - week old female c57bl/6 mice were used for the studies of pulmonary fibrosis . for blm - induced pulmonary fibrosis , mice were anaesthetized with 2% chloral hydrate and administered blm ( nippon kayaku ) intratracheally at a dose ( ul ) of 3.5 units / kg dissolving in total 50 ul saline . mice were sacrificed at weeks 1 , 3 , 6 , and 8 after blm injection . the left lungs were fixed in 10% formalin , dehydrated , and embedded in paraffin . the right lungs were frozen in liquid nitrogen for the subsequent protein and mrna experiments . for the in vivo experiment , mice were divided into 4 groups at random : the first and second group were given blm as described above and injected intraperitoneally with 1.25 g anti - il-22 neutralizing monoclonal antibody ( ab ) or isotype ab ( both from perprotech ) suspended in saline for 2 consecutive weeks , respectively ; the third and fourth group were just given once blm or saline , respectively , through intratracheal route , serving as blm control and saline control . human type ii alveolar epithelial cells ( a549 ) were a gift from jiucun wang 's lab ( obtained from the american type culture collection , atcc ) . a549 cells were harvested in f-12 k medium containing 10% fetal bovine serum ( fbs ) with 100 u / ml penicillin and 100 ug / ml streptomycin at 37c in a humidified 5% co2 atmosphere . confluent cultures of a549 were serum - starved for 12 hours ( h ) and then cultured with or without 100 mu / ml blm , subsequently stimulated with recombinant human il-22 ( perprotech ) of different concentrations for 48 h. cell viability was measured by cell counting kit-8 ( cck-8 , dojindo ) . after sterile phosphate buffered saline ( pbs ) was infused through the pulmonary vasculature by right heart puncture to remove any contaminating peripheral blood mononuclear cells , the whole lung was digested with collagenase iv ( gibco ) and dnase i ( sigma ) at 37c for 60 minutes ( min ) on the shaker . after filtering , erythrocyte lysing , and two washes with pbs , mononuclear cells from lung homogenates were incubated in 24-well plates with rpmi 1640 medium containing 10% fbs . for intracellular cytokine staining , total lung cells were cultured at 10 cells / ml in complete rpmi 1640 medium containing cell stimulation cocktail ( plus protein transport inhibitors ) ( ebioscience , including phorbol 12-myristate 13-acetate ( pma ) , ionomycin , and protein transport inhibitors brefeldin a and monensin ) at 37c for 5 h. the cells were washed and stained with monoclonal antibodies directed against cd3 ( apc , ebioscience ) , cd4 ( fitc , ebioscience ) , tcr ( fitc , ebioscience ) , or nkp46 ( fitc , ebioscience ) . cells were fixed and permeabilized with flow cytometry staining buffer ( ebioscience ) and permeabilization buffer ( ebioscience ) per manufacturer 's instructions , followed by staining with il-22 ( pe , ebioscience ) , or il-17a ( pe , ebioscience ) , or isotype controls for 30 min at room temperature . the lymphocyte population was identified using forward and 90 light scatter patterns , and fluorescence intensity was analyzed using a facs canto cytometer ( bd ) . total rna was isolated from frozen lung specimens using trizol reagent ( invitrogen ) in accordance with the manufacturer 's protocols . primescript rt reagent kit ( takara ) was used to reverse - transcribe 1 ug rna to complementary dna . real - time rt - pcr ( 40 cycles of denaturation at 95c for 15 seconds and annealing at 60c for 60 seconds ) was performed on an abi prism 7500 sequence detector ( applied biosystems ) with sybr premix ex taq ( takara ) . the relative expressions of pcr products were determined according to the ct method which compares target gene and gapdh messenger rna ( mrna ) expression . total protein concentration was measured using the bca protein assay kit ( biyuntian ) with bovine serum albumin ( bsa , sigma - aldrich ) as the standard protein . thirty g of protein were loaded for each lane of 10% sds page gels , followed by electrophoresis , and protein transfers to pvdf membranes ( millipore ) . immunoblots were probed with primary antibody against stat3 ( cell signaling technology ) , pstat3 ( cell signaling technology ) , -sma ( epitomics ) , e - cadherin ( bio - world ) , il-22 ( millipore ) , smad2 ( cell signaling technology ) , psmad2 ( cell signaling technology ) , or gadph ( epitomics ) at 4c overnight followed by goat anti - rabbit secondary antibodies ( jackson immunoresearch , 1 : 10,000 dilution ) for 30 min at room temperature . after extensive washing , the immunoblots were visualized by ecl ( pierce ) and the band densities for each phenotype marker were quantified using image reader las-3000 ( fijifilm ) after scanning with a las-3000 imaging densitometer ( fujifilm ) . paraffin - embedded sections ( 4 m in thickness ) were stained with hematoxylin and eosin ( h&e ) and masson 's trichrome ( sigma - aldrich ) according to the manufacturer 's instructions . for immunohistochemistry analysis , sections were treated with 3% hydrogen peroxide for 10 minutes at room temperature to block endogenous peroxidase . subsequently , the sections were incubated with antibody against il-22 ( millipore ) , -sma ( epitomics ) , tgf- ( santa cruz ) , collagen i ( abcam ) , or collagen iii ( abcam ) overnight at 37c , and then incubated at 37c for 1 hour with horseradish peroxidase - conjugated goat anti - rabbit igg secondary antibody . sections were washed 3 times with pbs between each incubation . after development with 3,3-diaminobenzidine tetrahydrochloride and hydrogen peroxide , sections were counterstained with hematoxylin . values were expressed as the mean sd . an independent two - group t - test or one - way analysis of variance ( anova ) test with lsd 's multiple comparison test was used to evaluate the significance of differences between groups . to determine if the emt response was involved in pulmonary fibrosis after blm administration , we examined pathological changes and emt markers ( e - cadherin for epithelial cells and -sma for myofibroblasts ) in the lungs over an 8-week period . in this study , pulmonary fibrosis in c57bl/6 mice was examined by h&e , masson 's trichrome , immunohistochemistry for collagen ( col ) i and col iii staining at the 1st , 3rd , 6th , and 8th week after blm or saline treatment , showing that the treatment with blm enhanced the collagen deposition in the lung tissues ( figure 1(a ) ) . apart from smooth muscle cells of vascular and bronchiolar walls , -sma was mainly expressed in myofibroblast ( figure 1(a ) ) . it was shown that -sma was upregulated , whereas e - cadherin was downregulated in blm - treated mice ( figure 1(c ) ) . the increased transcripts of col1a2 and col3a1 ( figure 1(b ) ) and protein level of -sma ( figure 1(c ) ) showed that the lung fibrosis aggravated after blm treatment and peaked around the 3rd week . these data indicated that blm - induced lung fibrosis is characterized by an emt response . tgf- expression in the lung tissues of blm - treated mice was higher than that of saline - treated mice ( figure 1(a ) ) . meanwhile , both phosphorylated ( psmad2 ) and total smad2 in the lung tissues of blm - treated mice were found to be elevated ( figure 1(c ) ) , and the ratio of psmad2/smad2 was significantly increased at the 1st , 3rd , and 6th week ( figure 1(d ) ) . these results suggested that the tgf-/smad2 related emt process participated in the initiation and development of the pulmonary fibrosis . to determine whether il-22 was involved in blm - induced pulmonary fibrosis , the expression of il-22 was evaluated by western blotting ( figure 2(a ) ) , or immunohistochemistry ( figure 2(b ) ) . as shown by immunoblots , total il-22 production in the lung tissue was significantly decreased in the blm - treated mice during 8-week period ( figure 2(a ) ) , which was in agreement with the histological findings ( figure 2(b ) ) . in addition , most il-22-positive cells were showed to distribute mainly subepithelially , within the alveoli and vessels . decrement of il-22 level , either secreted or in situ , implicated a potential role of il-22 in pulmonary fibrosis . to better understand the origin of il-22 and il-17 in blm - induced pulmonary fibrosis , the percentages of il-22 and il-17 produced cells were examined in the lung and spleen tissues of c57bl/6 mice after blm treatment by flow cytometry . in the lung tissues , as compared with saline - treated mice , the percentages of cd4il-22 , tcril-22 , and cd4il-17 cells were significantly reduced in blm - treated mice , especially at the 3rd week after the treatment ( figure 3(a ) ) . in contrast , blm - treated mice showed significantly increased percentage of tcril-17a t cells at the 1st week in the lung tissues of blm - treated mice . in the spleen tissues , the percentages of tcril-22 , nkp46il-22 , and cd4il-17 cells were reduced , but cd4il-22 and tcril-17a were increased at the 1st week ( figure 3(b ) ) . in addition , very few il-17ankp46 cells were found in the lung and spleen tissues within the same period ( data not shown ) . these data indicated that cd4 and tcr t cells differentially expressed il-17a and il-22 in response to blm treatment , suggesting that the subsets of cd4il-22 , tcril-22 , cd4il-17a , and tcril-17a t cells may have distinct functions in blm - induced pulmonary fibrosis . to further explore the underlying mechanism of il-22 in blm - induced pulmonary fibrosis , we examined il-22 expression in the lung . studies have shown that il-22r1 , a specific receptor , was mainly expressed in primary alveolar epithelial cell ( aec ) of lung tissue . our study showed that il-22r1 mrna was expressed in the whole murine and human lung tissues , as well as in aec line a549 . however , il-22 was not detected in fibroblast cell line hfl1 ( figure 4(a ) ) . the phosphorylation of stat3 was detected rapidly after ril-22 stimulation and reached the peak around 30 min ( figure 4(b ) ) , corroborating that the a549 cell line is responsive to ril-22 . to test whether il-22 could influence blm - induced pulmonary damage , blm was added to epithelial cell cultures in either the presence or absence of ril-22 . the a549 epithelial cells after blm ( 100 nu / ml ) treatment for over 48 h was shown a significant increase in the expression of mesenchymal markers--sma , which is suggestive of the process of emt . furthermore , we demonstrated that the addition of exogenous ril-22 to the culture medium significantly downregulated blm - induced -sma in epithelial cells in a dose - dependent manner ( figure 4(c ) ) . furthermore , analysis of cell viability by cck-8 revealed that treatment of il-22 at indicated concentrations could partially reverse the decreased cell viability induced by blm ( figure 4(d ) ) . collectively , these results suggest that il-22 may protect aecs from development towards emt and impaired viability induced by blm . since substitute rhil-22 could ameliorate blm - induced emt , we suspect that blockage of il-22 may deteriorate blm - induced lung fibrosis . to further address our hypothesis , we administrated anti - il-22 neutralizing antibody ( ab ) intraperitoneally daily for 2 consecutive weeks starting from the day of blm treatment . the levels of il-22 both in the serum and balf were decreased significantly confirmed by elisa ( data not shown ) . treatment of anti - il-22 ab led to even higher lymphocytes in balf than isotype antibody treated control , suggesting il-22 may have potential to inhibit the assembly of lymphocytes in balf induced by blm ( figure 5(a ) ) . strikingly , the blm - induced pulmonary fibrosis was much worse after the il-22 neutralizing ab treatment as compared with the isotype ab - control , shown by h&e and masson 's trichrome - stained lung sections ( figure 5(b ) ) . immunohistochemical stains of the lung tissues showed an increased expression of col i and col iii , which was in line with the elevated relative transcript levels of col1a2 and col3a1 measured by real - time rt - pcr ( figures 5(b ) and 5(c ) ) . -sma - expressing myofibroblasts were shown to be increased and mainly distributed peritracheally and perivascularly . of note , -sma was also expressed in some epithelial cells , especially in anti - il-22 neutralizing ab treated mice . expression of tgf- examined by immunohistochemistry was higher than that in the isotype ab - treated lungs ( figure 5(b ) ) . conversely , neutralizing il-22 antibodies enhanced blm - induced transcription levels of -sma and mmp2 ( figure 5(c ) ) . the increased expression of transcript for tgf-by real - time rt - pcr was shown in the anti - il-22 neutralizing ab - treated lung tissues as compared with isotype ab - treated mice , but this did not reach statistical significance ( figure 5(d ) ) . the ratio of psmad2/total smad2 was significantly elevated by 147.9% in the anti - il-22 neutralizing ab - treated lungs relative to that of isotype ab - treated control ( figure 5(d ) ) . taken together , these data provide the evidence that il-22 regulates the process of blm - induced emt and pulmonary fibrosis , likely via tgf-/smad2 signaling pathway . having emerged as an important cytokine in innate immunity , regeneration , and protection from damage , il-22 plays either a protective or a pathogenic role in different conditions . in the present study , we investigated a blm - induced pulmonary fibrosis model for 8 weeks and found a progressive process of emt , aberrant reepithelization , ultimate deposition of ecm , and destruction of lung architectures , accompanied by significantly decreased production of il-22 . though il-22 has been reported to have both pathogenic and protective properties depending on the nature of the affected tissue and the local cytokine milieu , here we showed that anti - il-22 antibody treatment exacerbated the lung fibrosis in vivo , indicating a potential protective role of il-22 in the development of lung fibrosis . also il-22 inhibited the overexpression of -sma and partially reversed the cell viability of epithelial cells induced by blm in vitro , which further confirmed the in vivo results . in addition , in order to identify which il-22 expressed cell subsets play a role in this case , we examined the cd4il-22 , tcril-22 , nkp46il-22 cell both in the lung and spleen at the indicated time points by flow cytometry . and we found that il-22 mainly produced t cells were decreased significantly both in the lung and spleen at the 3rd week , indicating tcril-22 cell may participate in the regulation of pulmonary fibrosis . taken together , our studies identified il-22 as a critical regulator of pulmonary fibrosis after blm administration , implicating the potential utility of il-22 in the treatment of pulmonary fibrosis . simonian et al . showed that il-22-secreting t cells could protect lung fibrosis by inhibiting recruitment of t cells in a mouse model of bacillus subtilis - induced hypersensitivity pneumonitis fibrosis . on the other hand , sonnenberg 's group reported a pathogenic role of il-22 in a model of high - dose - blm - induced acute lung damage and inflammation . il-22 has been shown to act synergistically with il-17a to promote acute pathological airway inflammation . conversely , anti - il-22 ab was found to exacerbate blm - induced airway inflammation in il17a mice , indicating that il-22 is tissue protective in the absence of il-17a . braun et al . reported colv - pretreated animals led to a significant reduction in lung inflammation , which was associated with a significant decrease in the relative expression of interleukin ( il)-6 , il-17 , and il-22 in cells present in bal fluid at 7 and 14 days after blm instillation . however , the evidence of lung fibrosis needs to be supplied , and more functional details of il-22 in the development of blm induced lung fibrosis need further study . evidence has shown that activated t cells , nk cells , nkt cells , lymphoid tissue inducer ( lti ) cells , and lti - like cells express il-22 , whereas resting or activated b cells , monocytes , monocyte - derived macrophages and immature and mature dcs were not able to produce this cytokine [ 1825 ] . interestingly , we found that the percentages of il-22 expressing cd3cd4cd8 oligoclonal t cells were significantly decreased both in the lung and spleen at the 3rd week , consistent with the decreased levels of il-22 as confirmed by western blotting and immunohistochemistry . in the previously reported mouse model of hypersensitivity pneumonitis , the authors found that il-22-secreting t cells could protect from lung fibrosis by diminishing recruitment of cd4 t cells to the lung . v6/v1 t cells are the predominant cell type in the lung producing il-22 in response to chronic b. subtilis exposure . hence , t cells may play the critical role as an inhibitor of pulmonary fibrosis via il-22 . cd3nkp46 cells have been reported to be present in the bone marrow , spleen , thymus , liver , intestines , and lymph nodes and play a role in epithelial cell homeostasis as a distinct subset of nk cell , so - called nk22 cells which express il-22 and rort but lack the capacity to perform classical nk cell functions such as cytotoxicity and ifn- production . our data show that il-22 expressed cd3nkp46 cells also existed in lung , with no significant difference between blm - induced lung fibrosis and the saline - treated control . thus , our findings are unique in that nk22 cells are also present in the lung , but the function of nk22 cell needs further research . il-22 has been shown to bind to the il-22r1/il-10r2 receptor complex to mediate its biological effects . importantly , only the expression of il-22r1 determines cellular sensitivity towards il-22 due to the ubiquitous expression of il-10r2 . we demonstrated that il-22r1 mrna was expressed in both human and murine lung tissue , and il-22r1 was expressed only in alveolar epithelial cell line a549 but not in fibroblast cell line hfl1 . this result was in agreement with the previous study that il-22 was found only in primary epithelial cells , but not in alveolar macrophages , monocytes , or neutrophils . taken together , these data suggest that alveolar epithelial cells may act as the exclusive target cell of il-22 in the lung . in the next step , we will test the expression of il-22r1 in the primary cells from the lung tissues of human and mouse . our data that il-22 induced the phosphorylation of stat3 after treating a549 immediately with rhil-22 , reaching the peak around 30 minutes further corroborates this notion . interestingly , evidence has shown that tgf- could inhibit the il-22 producing capacity of th17 cells both in the human [ 9 , 10 ] and the mouse . so whether il-22 could perform the feedback regulation on tgf- signaling deserved our intense investigation . stimulation of tgf- on the receptor complex led to the activation of smad2 and smad3 through direct c - terminal phosphorylation by tri . studies have indicated that lung epithelial cell - specific loss of 3 integrin expression reduced emt and protected from lung fibrosis , apparently by inhibiting tyrosine phosphorylation of -catenin and formation of -catenin / smad2 complex , and it was confirmed in ipf patients . these findings demonstrated smad2 was required for the epithelial integrin - dependent profibrotic crosstalk between -catenin and smad signaling during process of emt . additional line of evidence has shown that tgf- could induce a549 cells with an alveolar epithelial type ii cell phenotype to undergo emt , dependent of phosphorylation of smad2 . recently rock et al . proposed that type ii alveolar cells ( aec2 ) are not a major source of myofibroblasts through emt , which is an innovative idea about the origin of myofibroblast . but a lot of humans , mice , and cell data still support emt as an important event in the development of lung fibrosis [ 2833 ] . the phosphorylation of smad2 was found elevated in the lungs of blm - treated mice . administration of anti - il-22 neutralizing antibody resulted in further increase of phosphorylated smad2 , as well as the increment level of tgf-. we think that il-22 may play a protective role in pulmonary fibrosis through inactivating tgf-/smad signaling . however , we could not exclude other pathway altered by il-22 in downregulation of emt . non - smad signaling , such as erk map kinases , rho gtpases , and the pi3 kinase / akt pathway , could also mediate tgf- induced emt , and more detailed functional characterization are warranted . pulmonary fibrosis in human can be a fatal disorder characterized by progressive disease that leads to respiratory failure . furthermore , we demonstrated that neutralizing il-22 could lead to the exacerbation of emt process and an excessive deposition of ultimate collagen . in turn thus , the ability of il-22 to regulate blm - induced pulmonary fibrosis both in vivo and in vitro raises the possibility that il-22 may act as a potential target to treat diseases characterized by chronic lung inflammation and fibrosis .

pulmonary fibrosis is a progressive and fatal fibrotic disease of the lungs with unclear etiology . recent insight has suggested that early injury / inflammation of alveolar epithelial cells could lead to dysregulation of tissue repair driven by multiple cytokines . although dysregulation of interleukin- ( il- ) 22 is involved in various pulmonary pathophysiological processes , the role of il-22 in fibrotic lung diseases is still unclear and needs to be further addressed . here we investigated the effect of il-22 on alveolar epithelial cells in the bleomycin- ( blm- ) induced pulmonary fibrosis . blm - treated mice showed significantly decreased level of il-22 in the lung . il-22 produced t cells were also decreased significantly both in the tissues of lungs and spleens . administration of recombinant human il-22 to alveolar epithelial cell line a549 cells ameliorated epithelial to mesenchymal transition ( emt ) and partially reversed the impaired cell viability induced by blm . furthermore , blockage of il-22 deteriorated pulmonary fibrosis , with elevated emt marker ( -smooth muscle actin ( -sma ) ) and overactivated smad2 . our results indicate that il-22 may play a protective role in the development of blm - induced pulmonary fibrosis and may suggest il-22 as a novel immunotherapy tool in treating pulmonary fibrosis .

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

pulmonary fibrosis can occur as an idiopathic disease or as a consequence of a variety of connective tissue diseases with undefined aetiology , including scleroderma , dermatomyositis / polymyositis , systemic lupus erythematosus , and rheumatoid arthritis . pulmonary fibrosis is characterized by epithelial injury and activation , formation of distinctive subepithelial fibroblast / myofibroblast foci , and excessive extracellular matrix ( ecm ) accumulation . epithelial cells undergo phenotypic changes of epithelial to mesenchymal transition ( emt ) , in which the cells lose their epithelial characteristics and acquire a mesenchymal phenotype . they overexpress -smooth muscle actin ( -sma ) and are probably responsible for the enhanced synthesis of abnormal matrix observed in pulmonary fibrosis . transforming growth factor ( tgf)-1 has been shown to play a key role in pulmonary fibrosis , not only through its functions to attract fibroblasts and to stimulate their proliferation , but also through induction of emt in alveolar epithelial cells by activating smad- or non - smad signaling pathways . interleukin-22 ( il-22 ) is a member of the il-10 cytokine family and plays a critical role in inflammation , immune surveillance , and homeostasis in tissues that serve a barrier function such as skin , respiratory ( trachea and lungs ) and gastrointestinal ( stomach , small intestines , and colon ) tracts as well as liver , pancreas , and kidney . il-22 binds to a membrane receptor complex composed of the il-22r1 ( il-22ra1 ) and il-10r2 ( il-10rb2 ) , and signals intracellularly mainly through transcription factor jak / stat . interestingly , it was also showed that tgf-/smad signaling contributes to blm - induced fibrosis by promoting emt , and recent studies demonstrated that tgf- downregulated the il-22 producing capacity of th17 cells in both human [ 9 , 10 ] and mouse systems and inhibited the development of th22 cells . similarly , il-17a could regulate the properties of il-22 in the airway damage and inflammation , whereas il-17a enhanced blm - induced fibrosis in a tgf- dependent manner . to date , however , the crosstalk between il-22 and tgf--driven emt in pulmonary fibrosis has remained unclear . in the present study , we investigated the role of il-22 in emt in blm - induced pulmonary fibrosis mouse model as well as in vitro . we found that il-22 inhibited blm - induced emt , suggesting a potential therapeutic role of il-22 in pulmonary fibrosis . for blm - induced pulmonary fibrosis , mice were anaesthetized with 2% chloral hydrate and administered blm ( nippon kayaku ) intratracheally at a dose ( ul ) of 3.5 units / kg dissolving in total 50 ul saline . a549 cells were harvested in f-12 k medium containing 10% fetal bovine serum ( fbs ) with 100 u / ml penicillin and 100 ug / ml streptomycin at 37c in a humidified 5% co2 atmosphere . confluent cultures of a549 were serum - starved for 12 hours ( h ) and then cultured with or without 100 mu / ml blm , subsequently stimulated with recombinant human il-22 ( perprotech ) of different concentrations for 48 h. cell viability was measured by cell counting kit-8 ( cck-8 , dojindo ) . after sterile phosphate buffered saline ( pbs ) was infused through the pulmonary vasculature by right heart puncture to remove any contaminating peripheral blood mononuclear cells , the whole lung was digested with collagenase iv ( gibco ) and dnase i ( sigma ) at 37c for 60 minutes ( min ) on the shaker . cells were fixed and permeabilized with flow cytometry staining buffer ( ebioscience ) and permeabilization buffer ( ebioscience ) per manufacturer 's instructions , followed by staining with il-22 ( pe , ebioscience ) , or il-17a ( pe , ebioscience ) , or isotype controls for 30 min at room temperature . after extensive washing , the immunoblots were visualized by ecl ( pierce ) and the band densities for each phenotype marker were quantified using image reader las-3000 ( fijifilm ) after scanning with a las-3000 imaging densitometer ( fujifilm ) . to determine if the emt response was involved in pulmonary fibrosis after blm administration , we examined pathological changes and emt markers ( e - cadherin for epithelial cells and -sma for myofibroblasts ) in the lungs over an 8-week period . in this study , pulmonary fibrosis in c57bl/6 mice was examined by h&e , masson 's trichrome , immunohistochemistry for collagen ( col ) i and col iii staining at the 1st , 3rd , 6th , and 8th week after blm or saline treatment , showing that the treatment with blm enhanced the collagen deposition in the lung tissues ( figure 1(a ) ) . it was shown that -sma was upregulated , whereas e - cadherin was downregulated in blm - treated mice ( figure 1(c ) ) . the increased transcripts of col1a2 and col3a1 ( figure 1(b ) ) and protein level of -sma ( figure 1(c ) ) showed that the lung fibrosis aggravated after blm treatment and peaked around the 3rd week . these data indicated that blm - induced lung fibrosis is characterized by an emt response . tgf- expression in the lung tissues of blm - treated mice was higher than that of saline - treated mice ( figure 1(a ) ) . meanwhile , both phosphorylated ( psmad2 ) and total smad2 in the lung tissues of blm - treated mice were found to be elevated ( figure 1(c ) ) , and the ratio of psmad2/smad2 was significantly increased at the 1st , 3rd , and 6th week ( figure 1(d ) ) . these results suggested that the tgf-/smad2 related emt process participated in the initiation and development of the pulmonary fibrosis . to determine whether il-22 was involved in blm - induced pulmonary fibrosis , the expression of il-22 was evaluated by western blotting ( figure 2(a ) ) , or immunohistochemistry ( figure 2(b ) ) . as shown by immunoblots , total il-22 production in the lung tissue was significantly decreased in the blm - treated mice during 8-week period ( figure 2(a ) ) , which was in agreement with the histological findings ( figure 2(b ) ) . decrement of il-22 level , either secreted or in situ , implicated a potential role of il-22 in pulmonary fibrosis . to better understand the origin of il-22 and il-17 in blm - induced pulmonary fibrosis , the percentages of il-22 and il-17 produced cells were examined in the lung and spleen tissues of c57bl/6 mice after blm treatment by flow cytometry . in the lung tissues , as compared with saline - treated mice , the percentages of cd4il-22 , tcril-22 , and cd4il-17 cells were significantly reduced in blm - treated mice , especially at the 3rd week after the treatment ( figure 3(a ) ) . in contrast , blm - treated mice showed significantly increased percentage of tcril-17a t cells at the 1st week in the lung tissues of blm - treated mice . in the spleen tissues , the percentages of tcril-22 , nkp46il-22 , and cd4il-17 cells were reduced , but cd4il-22 and tcril-17a were increased at the 1st week ( figure 3(b ) ) . in addition , very few il-17ankp46 cells were found in the lung and spleen tissues within the same period ( data not shown ) . these data indicated that cd4 and tcr t cells differentially expressed il-17a and il-22 in response to blm treatment , suggesting that the subsets of cd4il-22 , tcril-22 , cd4il-17a , and tcril-17a t cells may have distinct functions in blm - induced pulmonary fibrosis . to further explore the underlying mechanism of il-22 in blm - induced pulmonary fibrosis , we examined il-22 expression in the lung . studies have shown that il-22r1 , a specific receptor , was mainly expressed in primary alveolar epithelial cell ( aec ) of lung tissue . our study showed that il-22r1 mrna was expressed in the whole murine and human lung tissues , as well as in aec line a549 . however , il-22 was not detected in fibroblast cell line hfl1 ( figure 4(a ) ) . the phosphorylation of stat3 was detected rapidly after ril-22 stimulation and reached the peak around 30 min ( figure 4(b ) ) , corroborating that the a549 cell line is responsive to ril-22 . to test whether il-22 could influence blm - induced pulmonary damage , blm was added to epithelial cell cultures in either the presence or absence of ril-22 . the a549 epithelial cells after blm ( 100 nu / ml ) treatment for over 48 h was shown a significant increase in the expression of mesenchymal markers--sma , which is suggestive of the process of emt . furthermore , we demonstrated that the addition of exogenous ril-22 to the culture medium significantly downregulated blm - induced -sma in epithelial cells in a dose - dependent manner ( figure 4(c ) ) . furthermore , analysis of cell viability by cck-8 revealed that treatment of il-22 at indicated concentrations could partially reverse the decreased cell viability induced by blm ( figure 4(d ) ) . collectively , these results suggest that il-22 may protect aecs from development towards emt and impaired viability induced by blm . since substitute rhil-22 could ameliorate blm - induced emt , we suspect that blockage of il-22 may deteriorate blm - induced lung fibrosis . the levels of il-22 both in the serum and balf were decreased significantly confirmed by elisa ( data not shown ) . treatment of anti - il-22 ab led to even higher lymphocytes in balf than isotype antibody treated control , suggesting il-22 may have potential to inhibit the assembly of lymphocytes in balf induced by blm ( figure 5(a ) ) . strikingly , the blm - induced pulmonary fibrosis was much worse after the il-22 neutralizing ab treatment as compared with the isotype ab - control , shown by h&e and masson 's trichrome - stained lung sections ( figure 5(b ) ) . immunohistochemical stains of the lung tissues showed an increased expression of col i and col iii , which was in line with the elevated relative transcript levels of col1a2 and col3a1 measured by real - time rt - pcr ( figures 5(b ) and 5(c ) ) . of note , -sma was also expressed in some epithelial cells , especially in anti - il-22 neutralizing ab treated mice . expression of tgf- examined by immunohistochemistry was higher than that in the isotype ab - treated lungs ( figure 5(b ) ) . conversely , neutralizing il-22 antibodies enhanced blm - induced transcription levels of -sma and mmp2 ( figure 5(c ) ) . the increased expression of transcript for tgf-by real - time rt - pcr was shown in the anti - il-22 neutralizing ab - treated lung tissues as compared with isotype ab - treated mice , but this did not reach statistical significance ( figure 5(d ) ) . the ratio of psmad2/total smad2 was significantly elevated by 147.9% in the anti - il-22 neutralizing ab - treated lungs relative to that of isotype ab - treated control ( figure 5(d ) ) . taken together , these data provide the evidence that il-22 regulates the process of blm - induced emt and pulmonary fibrosis , likely via tgf-/smad2 signaling pathway . having emerged as an important cytokine in innate immunity , regeneration , and protection from damage , il-22 plays either a protective or a pathogenic role in different conditions . in the present study , we investigated a blm - induced pulmonary fibrosis model for 8 weeks and found a progressive process of emt , aberrant reepithelization , ultimate deposition of ecm , and destruction of lung architectures , accompanied by significantly decreased production of il-22 . though il-22 has been reported to have both pathogenic and protective properties depending on the nature of the affected tissue and the local cytokine milieu , here we showed that anti - il-22 antibody treatment exacerbated the lung fibrosis in vivo , indicating a potential protective role of il-22 in the development of lung fibrosis . also il-22 inhibited the overexpression of -sma and partially reversed the cell viability of epithelial cells induced by blm in vitro , which further confirmed the in vivo results . in addition , in order to identify which il-22 expressed cell subsets play a role in this case , we examined the cd4il-22 , tcril-22 , nkp46il-22 cell both in the lung and spleen at the indicated time points by flow cytometry . and we found that il-22 mainly produced t cells were decreased significantly both in the lung and spleen at the 3rd week , indicating tcril-22 cell may participate in the regulation of pulmonary fibrosis . taken together , our studies identified il-22 as a critical regulator of pulmonary fibrosis after blm administration , implicating the potential utility of il-22 in the treatment of pulmonary fibrosis . showed that il-22-secreting t cells could protect lung fibrosis by inhibiting recruitment of t cells in a mouse model of bacillus subtilis - induced hypersensitivity pneumonitis fibrosis . on the other hand , sonnenberg 's group reported a pathogenic role of il-22 in a model of high - dose - blm - induced acute lung damage and inflammation . conversely , anti - il-22 ab was found to exacerbate blm - induced airway inflammation in il17a mice , indicating that il-22 is tissue protective in the absence of il-17a . reported colv - pretreated animals led to a significant reduction in lung inflammation , which was associated with a significant decrease in the relative expression of interleukin ( il)-6 , il-17 , and il-22 in cells present in bal fluid at 7 and 14 days after blm instillation . however , the evidence of lung fibrosis needs to be supplied , and more functional details of il-22 in the development of blm induced lung fibrosis need further study . interestingly , we found that the percentages of il-22 expressing cd3cd4cd8 oligoclonal t cells were significantly decreased both in the lung and spleen at the 3rd week , consistent with the decreased levels of il-22 as confirmed by western blotting and immunohistochemistry . in the previously reported mouse model of hypersensitivity pneumonitis , the authors found that il-22-secreting t cells could protect from lung fibrosis by diminishing recruitment of cd4 t cells to the lung . v6/v1 t cells are the predominant cell type in the lung producing il-22 in response to chronic b. subtilis exposure . hence , t cells may play the critical role as an inhibitor of pulmonary fibrosis via il-22 . cd3nkp46 cells have been reported to be present in the bone marrow , spleen , thymus , liver , intestines , and lymph nodes and play a role in epithelial cell homeostasis as a distinct subset of nk cell , so - called nk22 cells which express il-22 and rort but lack the capacity to perform classical nk cell functions such as cytotoxicity and ifn- production . our data show that il-22 expressed cd3nkp46 cells also existed in lung , with no significant difference between blm - induced lung fibrosis and the saline - treated control . thus , our findings are unique in that nk22 cells are also present in the lung , but the function of nk22 cell needs further research . we demonstrated that il-22r1 mrna was expressed in both human and murine lung tissue , and il-22r1 was expressed only in alveolar epithelial cell line a549 but not in fibroblast cell line hfl1 . this result was in agreement with the previous study that il-22 was found only in primary epithelial cells , but not in alveolar macrophages , monocytes , or neutrophils . taken together , these data suggest that alveolar epithelial cells may act as the exclusive target cell of il-22 in the lung . in the next step , we will test the expression of il-22r1 in the primary cells from the lung tissues of human and mouse . interestingly , evidence has shown that tgf- could inhibit the il-22 producing capacity of th17 cells both in the human [ 9 , 10 ] and the mouse . studies have indicated that lung epithelial cell - specific loss of 3 integrin expression reduced emt and protected from lung fibrosis , apparently by inhibiting tyrosine phosphorylation of -catenin and formation of -catenin / smad2 complex , and it was confirmed in ipf patients . additional line of evidence has shown that tgf- could induce a549 cells with an alveolar epithelial type ii cell phenotype to undergo emt , dependent of phosphorylation of smad2 . but a lot of humans , mice , and cell data still support emt as an important event in the development of lung fibrosis [ 2833 ] . the phosphorylation of smad2 was found elevated in the lungs of blm - treated mice . administration of anti - il-22 neutralizing antibody resulted in further increase of phosphorylated smad2 , as well as the increment level of tgf-. we think that il-22 may play a protective role in pulmonary fibrosis through inactivating tgf-/smad signaling . furthermore , we demonstrated that neutralizing il-22 could lead to the exacerbation of emt process and an excessive deposition of ultimate collagen . in turn thus , the ability of il-22 to regulate blm - induced pulmonary fibrosis both in vivo and in vitro raises the possibility that il-22 may act as a potential target to treat diseases characterized by chronic lung inflammation and fibrosis .

the most common disorders are depression , substance abuse , disturbance of attention , and eating disorders . it is defined by who as a state of well - being in which every individual realizes his or her own potential , can cope with the normal stresses of life , can work productively and fruitfully , and is able to make a contribution to her or his community . positive mental health is seen as a resource which is essential to general well - being . the promotion of mental health is a more extensive concept than merely preventing mental health problems . mental health promotion strives to find and enhance factors and processes that protect mental health and reduce factors harmful to it . mental health promotion can improve people 's survival skills and ability to feel empathy and , consequently , protect their mental health and improve their ability to support other members of their community with mental health problems . the promotion of mental health not only consists of the support of the mental health of individuals and provision of mental health services but also includes activities at the community and society levels [ 1014 ] . actions to promote mental health include national mental health programmes , laws and policies , development of mentally healthy communities and physical environments and providing of opportunities for leisure activities . as mental health is an integral part of health , mental health promotion is an integral part of overall health promotion . an environment that promotes or hinders schoolchildren 's mental health involves the person 's entire mental , physical , and social environment , especially the school , home , and friends . there is evidence of how mental health promotion in schools has positive effects on the age group . taking into account the whole of the school community especially , environment and the families of the pupils as well as focusing on positive mental health and adequate lengths of the treatments are factors which contribute to the success of mental health promotion . preventing mental health problems and doing mental health promotion have an effect in one 's success in school and well - being in life in general . the mental health and behavioural problems of children and adolescents can often be seen as difficulties in later phases of their lives [ 2125 ] . barry and jenkins made an overview of the evidence from systematic reviews and several mental health promoting programmes . these are high - quality implementation , evaluation and sustainability and such things as adopting a whole - school and a social competence approach , performing interventions over multiple years and having a strong theoretical base . used theoretical foundations are mostly psychology theories such like child development theory or cognitive behavioural therapy but in many programs there is no theory basis at all [ 10 , 26 ] . cooperation and the participation of all community members in mental health promotion have been found to be significant factors for the success of planned mental health programs [ 4 , 10 , 27 , 28 ] . self - expression and self - ruling are key factors for well - being ; therefore , just being heard affects mental health and well - being [ 2729 ] . who european ministerial conference also focuses in its mental health declaration fostering awareness of importance of mental well - being ( priority 1 ) and recognizing knowledge and experiences of service users and carers ( family member , friend , or another informal caregiver ) as an important basis on planning and developing mental health services ( priority 5 ) . schoolchildren are the main target of mental health promotion , which makes them key elements in the discussion on promoting mental health at school . it is the primary , most important , and most influential system to which the child belongs . schoolchildren and their families represent service users in this study material . despite the large number of projects and recommendations on the promotion of schoolchildren 's mental health , the literature does not offer a comprehensive theoretical description of what mental health work with schoolchildren is as a whole [ 10 , 26 ] . having a theoretical description of mental health promotion work helps in the development of facilities [ 10 , 26 , 28 ] . the employees , the schoolchildren , and their families as well as previous knowledge of the subject . this study is a part of a more extensive research to produce a theory of mental health promotion in the upper level of comprehensive school . in these schools , this study aims at describing the concepts referring to the promotion of mental health from the schoolchildren 's and their families ' perspective and , consequently , facilitating the development of the practices of mental health promotion . in this study , families ' perspective was studied by interviewing mothers of schoolchildren . in this study , it was intended to especially highlight the perspective of the schoolchildren and their families . therefore , the analysis was left on the level of axial coding to produce and describe the concepts . after that , it is easier to develop practices and support their self - ruling ability . schoolchildren are the main targets of mental health promotion , which makes them key elements in the discussion on promoting mental health at school . self - expression and self - rule are key factors for well - being ; therefore , just being heard affects mental health and well - being [ 2729 ] . the purpose of this study was to find the key aspects and requirements for promoting mental health in the way the pupils and their families see them . this study was conducted in a finnish upper - level comprehensive school with 446 pupils in 20052007 . this is the only upper - level comprehensive school in the quite small city where the school is located . in finland , the last three grades of comprehensive school attended from age of 12 to age of 16are usually , and also in this case , located in a bigger school further away from the homes . the school offers an excellent environment to reach the whole age group because of the overarching school system . in finland , almost every child ( 99.7% ) completes the nine - year - long basic education . the first type consisted of material gathered from the interviews with school pupils and their parents . the parents who agreed to be interviewed were all mothers , but they were asked to speak from the viewpoint of the whole family . the second type consisted of the pupils ' written replies to the well - being survey conducted by their school . the school well - being profile is a tool that is used to measure well - being in school communities . what is the best feature in your school ? and what things in your school should especially be improved ? . because positive mental health is an intregral part of general well - being , it was assumed that statements regarding mental health could be found in this study 's survey material . in this study the responses to these two questions were incorporated into the other data sets and the statements drawn from them were analysed together with the rest of the data . a sufficient amount of data was considered having been collected when the analysis no longer resulted in statements that did not fit into the categories . the interviews with the schoolchildren and mothers produced 54 pages of text and the verbal answers to the school well - being profile produced 436 statements that were also added to the research material . a total of 1523 statements were written up as a result of the interviews and the survey questions . the themes of the interviews were as follows : the conceptions of the interviewees of mental health work in general and in this school;school as a working environment;particular concerns , such as bullying and absences , and actions to prevent them;getting help in mental health problem situations ; cooperation between the home of the pupil and the school;cooperation between primary health care , social services , and special health care;the most crucial development needs in mental health promotion of schoolchildren . the conceptions of the interviewees of mental health work in general and in this school ; school as a working environment ; particular concerns , such as bullying and absences , and actions to prevent them ; getting help in mental health problem situations ; cooperation between the home of the pupil and the school ; cooperation between primary health care , social services , and special health care ; the most crucial development needs in mental health promotion of schoolchildren . the interviews were conducted by the researcher and they were carried out in the school during the school day . the principal was aware of the aims and methods of the study and of the fact that there was a need for interviewees who have things to say through their own experience and who represent the schoolchildren comprehensively . she approached a group of parents and these four were chosen to be the respondents due to their willingness to participate . the criteria for selecting the pupils to be interviewed were that the pupils represented different grades and were of both sexes . the relatively open interview themes helped to see the world from the perspective of participants and to develop deeper understanding of their viewpoint . in the interview material of the parents , for the schoolchildren , it seemed to be easier to produce statements anonymously in the survey than it was in the interview . with the survey material , it was possible to reach many schoolchildren and get their opinions on their school . the open questions in the survey also provided a way to find aspects the schoolchildren themselves found important . in an interview situation , the school well - being profile survey was completed by 423 out of 426 pupils present in school on the day of the survey . it aims at creating a substantive theory , including concepts and assumptions on the relationships among them [ 32 , 37 , 38 ] . grounded theory was selected because it is analysing method that takes account of people and their experiences . in grounded theory , the researcher aims to come close to the actor 's perspective and tries to capture his or her viewpoint . this perspective of schoolchildren and families is needed to understand the mental health promotion in school . to understand experience , it must be located within the larger events in a social , political , cultural , ethnic and gender related , informational , and technological framework . developing knowledge helps to develop practice [ 32 , 40 ] . the theoretical background of the grounded theory method lies in symbolic interactionism and pragmatism . symbolic interactionism brings attention to human behaviour and interaction by focusing on the significance of events and things in people 's everyday lives . it is useful for conceptualising complex situations , understanding unsolved social problems , and understanding the impact of new ideologies . the objective of grounded theory is to generate basic models as explanations of social life in general . one , the purely inductive method represented by glaser , develops theory purely on the basis of the material . the other , favoured by strauss and applied in this study , inspects the existing theoretical knowledge and combines it with the data gained through research [ 32 , 37 , 39 ] . because of the relatively large number of articles about some aspects of mental health promotion at school , it is reasonable to examine and use previous articles about the promotion of schoolchildren 's mental health while producing a comprehensive theoretical description this is why the strauss method was selected for this study [ 32 , 37 , 39 ] . the schoolchildren 's and families ' perspective that comes from the material of this study can be compared to the existing knowledge and viewpoints [ 37 , 39 ] . some points should be taken into account when under - age respondents are used in a study . , respondents aged 12 to 16 are considered old enough to participate in some cases even without permission from their parents . such cases are situations where sensitive questions are not asked and it is seen that the questions do not convulse the youngsters . however , in this study , it was still ensured that the pupils ' parents were aware of the interviews and gave their consent . letters were sent to the homes of the interviewed pupils to inform their parents of the interviews . consent forms were sent with these letter and the parents were asked to give their child their consent to participate in the interview by signing the form . before the interview , all respondents gave their written consent to the use of the interview as research material . the participating pupils and parents received a brief introduction to the study and its objectives . the questions were designed in such a way that the objectives were approached with as familiar situations and concepts as possible . the pupils were also informed of the use of the well - being profile responses in the promotion of mental health and well - being at school . the study was endorsed by permissions from the joint municipal authority for primary healthcare , the social welfare board , the board of education , and also the ethics committee of the local hospital district . grounded theory focuses on concept formation . they express time , place , people , and people 's patterns of action [ 32 , 42 ] . , the researcher also spent a lot of time discussing with the school personnel to do so . getting familiar with the school context and with the literature also enhanced the researcher 's preunderstanding about the subject [ 32 , 38 ] . both materials , the transcribed interview material and the written answers of the survey , were analysed together . statements the informants used to illustrate mental health promotion were picked from the text [ 32 , 38 ] . then the researcher analysed the collected material with the constant comparative method to identify similarities and differences between the statements . the questions that were used to support the categorisation of the material included the following : the process of grouping concepts which seem to pertain to the same phenomena is called categorizing . as a result of the regrouping and comparison , all of the statements were placed in a category of statements pertaining to the same theme and a shared concept illustrating the phenomenon was identified . table 1 presents an example of how the statements were placed into categories and subcategories and shows the concepts used to name the categories . data analysis occurred simultaneously with data collection . during the data collection categories developed in terms of their properties and dimensions . the codes produced by the analysis determined the direction of the data collection . following the principle of saturation , the amount of data was considered sufficient when no new utterances emerged from the data collected but the same themes began to recur . sufficient sampling was determined to occur when categories offered considerable depth and breadth of understanding about the phenomenon and relationships to other categories were clear . in the next phase , the axial coding , the data was examined to identify structural factors , contextual factors , and factors related to the phenomenon , circumstances , and action . this way , the five key concepts which the parents and the schoolchildren used in describing the promotion of mental health in the upper - level comprehensive school emerged [ 32 , 40 ] . at this stage of the research therefore , the analysis was left on the level of axial coding . on this level it is possible to form causal conditions , context , phenomena , and factors related to action and interaction from the material . finally , the generated concepts were compared with the existing research information on the topic . with regard to mental health promotion , five key concepts were discovered in the material : school environment , school friends and teachers , cooperation , actions to promote well - being and mental health , and getting help with problems . table 2 shows the key concepts related to promoting mental health and properties and describes them from the schoolchildren 's and their parents ' perspective . school environment comprises the physical conditions and educational equipment in the school including resources , teaching , rules , and services for pupils , such as school lunch and their impacts on the pupils . this concept also comprises the requirements of the teaching , the flexibility and individuality of the teaching , and the characteristics of the pupils themselves . they were pleased with the high - quality teaching tools and spacious classrooms but thought that the recesses should offer more activity opportunities ; just standing around is not enough . on the other hand , the rules governing recess - time activities and school practices aroused divergent opinions : some were against these rules and some wanted them to be adhered to and controlled more actively . also , the parents considered that the school environment influences children 's well - being and they had made efforts to improve the school premises , for example , via the parents ' association.the schoolyard could use some improving , there has been some opinions about that . about that something should be done there there ( sic).the atmosphere is good.needs to be improved : rules the schoolyard could use some improving , there has been some opinions about that . needs to be improved : rules learning was considered to be a positive thing . the pupils were happy with the idea of optional subjects , but , on the other hand , they wanted to have more nontheoretical subjects such as physical exercise , music , and home economics . also the parents wished the curricula to be more favourable for pupils who are more inclined towards artistic performance than theoretical learning . the pupils and parents wanted to add individual aspects and flexibility to the teaching and teaching methods with an increased degree of student participation . individualism was considered to be a mental health - promoting factor in the form of promoting the self - esteem and feelings of success.it's that supporting of growth and finding those positive capabilities.the best thing in this school are ( sic ) the electives . learning difficulties , vandalism , and absences were further issues associated with the school environment although to a lesser extent . in general , factors related to the pupils ' age and personal properties were essential in this respect . the available human and financial resources were brought up as potential obstacles to detecting and resolving problems.well , the curator - situation has been inadequate.the school nurse is nevertheless always available every day . well , the curator - situation has been inadequate . the school nurse is nevertheless always available every day . school friends and teachers include social relationships in school and the whole school community . friends were also considered to be important and to make the school feel like a comfortable place . breaking up groups of friends when transferring from primary school to the upper - level comprehensive school is an issue that aroused much thought , especially in the transfer phase . safety in the school community was also considered important.i think it 's that the teachers treat the pupils equally.nice school friends the class teacher was expected to notice pupils ' needs for support and respond to them . the pupils often complimented their teachers as being nice , skilled , and good teachers . however , there were also comments about too high demands and dictator - like and unfair behaviour.teachers have the expertise to see in my opinion.i wished remedial education for my son in some of the subjects and the teacher did carry my wish forward then.and the teachers are quite nice . i wished remedial education for my son in some of the subjects and the teacher did carry my wish forward then . and the teachers are quite nice . co - operation and communication between parents and employees were things that the parents particularly expected . this concept also includes cooperation between employees and between children and adults and sufficient amount of information . the parents considered that their task is to take an interest in the child 's life , monitor the child 's health and motivation , and ensure sufficient rest and nutrition . child upbringing was seen as a joint project in which parents and teachers function as partners , both relying on their own expertise ; parents know the child 's personality and background , whereas teachers are competent at issues related to learning.i ( a mother ) myself try to look after resting , bedtimes . i ( a mother ) myself try to look after resting , bedtimes . parents are not professionals of education . according to the pupils , contacts between their homes and the school were scarce , but they believed that information will be relayed whenever necessary . the parents said that they get information on the school work from bulletins and parents ' meetings . one specific channel for information is the parents ' association which gathers information on the school and tries to contribute to the school environment 's development by , for example , arranging fund - raising events.there's not much contacting from home , but the school , however , contacts even for smaller matters.when it 's needed , everything is told . like , when something comes up that needs to be told , the school is informed of it . there 's not much contacting from home , but the school , however , contacts even for smaller matters . like , when something comes up that needs to be told , the school is informed of it . actions to promote well - being and mental health are related to having an effect on enhancing the school 's conditions and social environment . when talking about the factors that influence mental health , both the parents and the pupils mentioned friendship and stress . furthermore , the parents believed that learning experiences influence adolescent 's self - confidence and , consequently , mental health . the results indicated that the school had paid attention to the development of friendships and community spirit , as well as preparing for other changes related to the transition from lower - level to upper - level comprehensive school.in spring , they ( the new students getting to know their future school ) already visited this place more than once . and then they had their future class together and they , like , already got to know each other . in spring , they ( the new students getting to know their future school ) already visited this place more than once . and then they had their future class together and they , like , already got to know each other . with regard to well - being and feeling well in general , there was a lot of discussion related to the above - mentioned school environment factors , resources , and social relationships . ensuring a sensible eating routine and a sufficient amount of sleep , improving the safety and cosiness of the school environment , fair treatment and possibilities to influence school matters , these things can be influenced by the parents and the pupils , for example , via the pupils ' council.well this is pretty good , you know , because there is a well arranged pupils ' council in here . it has improved the cosiness . well this is pretty good , you know , because there is a well arranged pupils ' council in here . the pupils said they had received some information about mental health - related issues from their teachers . health education lessons were mentioned as one channel in learning to take care of your well - being . the parents stated that they got information about the school 's practices and services in parents ' meetings.in parents ' meetings , it 's told how they do act in these situations which , for example , concern bullying . in parents ' meetings , it 's told how they do act in these situations which , for example , concern bullying . getting help with problems includes skills to notice mental health problems and possibilities to get proper and timely help . the respondents had a little knowledge of instances that offer actual mental health care services . the school health care was seen as the primary instance to contact such matters and it was considered to be easy to access . some respondents were aware of the existence of the pupil welfare group but not of its activities or members . the respondents believed that the main responsibility for seeking help was with the individual concerned . they did not believe that the chances of detecting problems were great , especially with quiet , socially withdrawn pupils . the respondents ' views on mental health work were mainly based on experienced problem situations , such as problems and negative feelings related to learning difficulties and experiences of offering help in stressful situations . cooperation and communication between the home and the school were considered important and preferable factors with regard to mental health problems.well , we have a school nurse who 's spezialised ( sic ) in mental health . like , i was immediately asked whether i would like to go there to talk to him / her because i worry a lot about the exams.well , in my opinion , it works well now that people contact each other immediately . like , the form teacher contacts the parents when there 's something that 's puzzling him or her , and the parents do the same if they are puzzled . well , we have a school nurse who 's spezialised ( sic ) in mental health . like , i was immediately asked whether i would like to go there to talk to him / her because i worry a lot about the exams . well , in my opinion , it works well now that people contact each other immediately . like , the form teacher contacts the parents when there 's something that 's puzzling him or her , and the parents do the same if they are puzzled . in grounded theory , previous literature is used to support and to validate the findings . the founded literature did not provide a comprehensive description of mental health promotion in school , but there was a good amount of studies and articles related to the individual categories . previous studies were searched with such terms as mental health promotion and school , children , or adolescents in several databases . several studies argued about the same kind of aspects being important as these study results did . this supports the conclusion that concepts discovered from study material are actually things that are essential in promoting mental health . school environment and school friends and teachers especially were mentioned in many studies . konu and lintonen , and goodman et al . wrote about the importance of developing an environment that supports the well - being of the pupils . in their studies on mental health promotion projects and reviews thereof , they found strong evidence of the social competence approach , which brings focus on the promotion of resourcefulness and generic coping and competence skills . also family members should help the nursing staff to gain a clearer picture of the depth and the diversity of the family health and support the resources that promote family health . cooperation in mental health projects is also a key element in poole 's project on preventive mental health work , in which families , schools , municipalities , and authorities worked in extensive cooperation to develop a model of partnership between the school and the families . adelman and taylor [ 46 , 47 ] named the collaborative work of school staff , health professionals , and the community resources as one very important part of successful mental health promotion work in their reviews of school mental health promotion . according to deschesnes et al . cooperation between the family , school , and community was a significant factor in the promotion of schoolchildren 's mental health . in this study this is why it was important to include the adolescents ' and families ' viewpoint . the influence of the entire social school environment , including the teachers and their major role , is stated in the results and enhancing these elements is seen important in many studies [ 10 , 29 , 35 , 43 ] . barry and jenkins wrote about the significance of social and problem - solving skills for well - being . in the light of barry 's results , it is easy to understand how important role the schoolchildren and their parents give to human relations and co - operation . the results of getting help with problems especially reflect the viewpoints of the pupils and their families . in previous studies anyway , previous results of co - operation [ 31 , 44 , 45 , 48 ] , mental health and social skills training and being heard [ 49 , 50 ] are things that also support this finding . the importance of being heard and possibilities to have influence in one 's own school matters can be seen in the results of this study . . wrote in their study on children 's viewpoints on child psychiatric care that relationships with other children seem to be of extreme importance . according to the unicef 's convention on the rights of the child , children have a right to express their opinions and to have their views taken seriously and given due weight . children also have a responsibility to respect the rights of others , especially those of their parents . the convention refers to the family as the fundamental group of society and the natural environment for the growth and well - being of children . in qualitative research , validity and reliability data generated using the grounded theory method can be assessed in terms of credibility , conformability , and truth value [ 38 , 40 ] . the views and opinions of the parents and youngsters , on which matters are important , can be considered credible . this study features interviews with four youngsters and a survey among a large group of schoolchildren on their opinions of their school . the material from the interviews with parents was rich and versatile and described the parents ' thoughts on their children 's schooling from various angles . combining several types of data proved to be a helpful way to find more information and a richer description about the studied phenomenon . for example , using the open questions from the survey supplemented the interview material remarkably . as the material collection proceeded , certain statements became more frequent , which means that the material itself can be considered in order to confirm its own credibility . the impression of the promotion of schoolchildren 's mental health gained from the material provides a versatile insight into the youngsters ' and parents ' viewpoints from various angles . a particular objective of this study was to bring out human experiences , which was well achieved with the interview material . the schoolchildren 's views on mental health promotion were clearly visible [ 32 , 40 ] . a substantive theory produced by grounded theory one concern is whether the mothers ' viewpoints can be extrapolated to the general family , even though they were told to represent the whole family . no fathers agreed to be interviewed , but mothers were asked to speak from the viewpoint of the whole family . at least their viewpoint can be seen to be a remarkable part of family viewpoints . however , a quality theory will inevitably identify a basic process that is also relevant more generally . details related to the school 's practices and human resources vary between schools and countries , which is why detailed practices , such as group - formation on transition to the upper - level school , can not be generalised into a description of mental health promotion practices in schools as a whole . instead , the process of collecting and analysing the pupils ' viewpoints used in this study , as well as the founded key aspects of mental health promotion in school , can be considered to apply to a wider context and internationally . grounded theory proved to be an effective mean of eliciting people 's personal viewpoints . when theory is developed from the personal descriptions of the actors and the objects of action , it is easily translatable into a development tool . pupils and their families are the key targets of mental health promotion work in schools and bringing up their viewpoints is important and forms a significant research result . the founded viewpoints help the school personnel develop their work and select the focus areas of development , even in schools with different human resources and practices [ 10 , 26 , 51 ] . schools are one of the most important settings for promoting mental health of young people . according to previous literature , the term mental health is often misunderstood and misused . pupils and parents , as well , need knowledge of mental health promotion and problem solving in school . this study , by giving names and contents to different fields of mental health promotion , helps the whole school community to find key development areas . this should lead to services being better tailored to people 's needs and better used . the results indicate that the pupils find teaching and teachers to be very important factors at school . with these factors in mind , it is important to pay attention to the opportunities to increase flexibility and individuality . finding competent teachers would also make the school work seem more worthwhile for the pupils . the school staff needs guidance in mental health promotion and theory - based means for the practical implementation thereof . it is often the case that the operators are not aware of the underlying theory behind development programmes , which makes the practical application more difficult . through the perspectives of different actors , it is easier to find an understandable and practical description of the action . with the help of the produced concepts , the mental health promotion of schoolchildren can thus be observed and promoted , which is the central benefit of this study . however , co - operation between the families and the school staff , as well as increasing the pupils ' possibilities to influence , could help in this respect as well . the whole school community must be taken into consideration . combining viewpoints of children , families , and professionals and then discussing and developing mental health promotion activities in cooperation would not only increase awareness but also enable development that takes all viewpoints into account . the next phase of the research is to combine these different viewpoints into a substantive theory on mental health promotion in school . the theory which will be built will help the whole school community to develop mental health promoting work by giving a framework and helping to find key development areas .

while developing mental health work in schools , it is very important to consider the viewpoint of pupils . parents can also give remarkable information on their children 's viewpoint . the purpose of this study was to produce a description of the concepts used by schoolchildren aged 1216 years and their families associated with promoting mental health in schools . the research material comprised interviews with schoolchildren and mothers , and verbal answers from the school well - being profile survey ( n = 426 ) . the analysis was conducted by applying the grounded theory method as introduced by strauss . the study was conducted in a finnish comprehensive school .

1. Introduction 2. Materials and Methods 3. Results: Concepts of Mental Health Promotion as Described by the Schoolchildren and Their Families 4. Discussion 5. Conclusions 6. Recommendations for Practice and Research

it is defined by who as a state of well - being in which every individual realizes his or her own potential , can cope with the normal stresses of life , can work productively and fruitfully , and is able to make a contribution to her or his community . positive mental health is seen as a resource which is essential to general well - being . the promotion of mental health is a more extensive concept than merely preventing mental health problems . the promotion of mental health not only consists of the support of the mental health of individuals and provision of mental health services but also includes activities at the community and society levels [ 1014 ] . actions to promote mental health include national mental health programmes , laws and policies , development of mentally healthy communities and physical environments and providing of opportunities for leisure activities . an environment that promotes or hinders schoolchildren 's mental health involves the person 's entire mental , physical , and social environment , especially the school , home , and friends . there is evidence of how mental health promotion in schools has positive effects on the age group . taking into account the whole of the school community especially , environment and the families of the pupils as well as focusing on positive mental health and adequate lengths of the treatments are factors which contribute to the success of mental health promotion . preventing mental health problems and doing mental health promotion have an effect in one 's success in school and well - being in life in general . barry and jenkins made an overview of the evidence from systematic reviews and several mental health promoting programmes . self - expression and self - ruling are key factors for well - being ; therefore , just being heard affects mental health and well - being [ 2729 ] . who european ministerial conference also focuses in its mental health declaration fostering awareness of importance of mental well - being ( priority 1 ) and recognizing knowledge and experiences of service users and carers ( family member , friend , or another informal caregiver ) as an important basis on planning and developing mental health services ( priority 5 ) . schoolchildren are the main target of mental health promotion , which makes them key elements in the discussion on promoting mental health at school . it is the primary , most important , and most influential system to which the child belongs . schoolchildren and their families represent service users in this study material . despite the large number of projects and recommendations on the promotion of schoolchildren 's mental health , the literature does not offer a comprehensive theoretical description of what mental health work with schoolchildren is as a whole [ 10 , 26 ] . having a theoretical description of mental health promotion work helps in the development of facilities [ 10 , 26 , 28 ] . the employees , the schoolchildren , and their families as well as previous knowledge of the subject . this study is a part of a more extensive research to produce a theory of mental health promotion in the upper level of comprehensive school . in these schools , this study aims at describing the concepts referring to the promotion of mental health from the schoolchildren 's and their families ' perspective and , consequently , facilitating the development of the practices of mental health promotion . in this study , it was intended to especially highlight the perspective of the schoolchildren and their families . therefore , the analysis was left on the level of axial coding to produce and describe the concepts . after that , it is easier to develop practices and support their self - ruling ability . schoolchildren are the main targets of mental health promotion , which makes them key elements in the discussion on promoting mental health at school . self - expression and self - rule are key factors for well - being ; therefore , just being heard affects mental health and well - being [ 2729 ] . the purpose of this study was to find the key aspects and requirements for promoting mental health in the way the pupils and their families see them . this study was conducted in a finnish upper - level comprehensive school with 446 pupils in 20052007 . this is the only upper - level comprehensive school in the quite small city where the school is located . in finland , the last three grades of comprehensive school attended from age of 12 to age of 16are usually , and also in this case , located in a bigger school further away from the homes . the school offers an excellent environment to reach the whole age group because of the overarching school system . the first type consisted of material gathered from the interviews with school pupils and their parents . the parents who agreed to be interviewed were all mothers , but they were asked to speak from the viewpoint of the whole family . the second type consisted of the pupils ' written replies to the well - being survey conducted by their school . the school well - being profile is a tool that is used to measure well - being in school communities . because positive mental health is an intregral part of general well - being , it was assumed that statements regarding mental health could be found in this study 's survey material . in this study the responses to these two questions were incorporated into the other data sets and the statements drawn from them were analysed together with the rest of the data . the interviews with the schoolchildren and mothers produced 54 pages of text and the verbal answers to the school well - being profile produced 436 statements that were also added to the research material . a total of 1523 statements were written up as a result of the interviews and the survey questions . the themes of the interviews were as follows : the conceptions of the interviewees of mental health work in general and in this school;school as a working environment;particular concerns , such as bullying and absences , and actions to prevent them;getting help in mental health problem situations ; cooperation between the home of the pupil and the school;cooperation between primary health care , social services , and special health care;the most crucial development needs in mental health promotion of schoolchildren . the conceptions of the interviewees of mental health work in general and in this school ; school as a working environment ; particular concerns , such as bullying and absences , and actions to prevent them ; getting help in mental health problem situations ; cooperation between the home of the pupil and the school ; cooperation between primary health care , social services , and special health care ; the most crucial development needs in mental health promotion of schoolchildren . the interviews were conducted by the researcher and they were carried out in the school during the school day . the principal was aware of the aims and methods of the study and of the fact that there was a need for interviewees who have things to say through their own experience and who represent the schoolchildren comprehensively . the relatively open interview themes helped to see the world from the perspective of participants and to develop deeper understanding of their viewpoint . in the interview material of the parents , for the schoolchildren , it seemed to be easier to produce statements anonymously in the survey than it was in the interview . with the survey material , it was possible to reach many schoolchildren and get their opinions on their school . in an interview situation , the school well - being profile survey was completed by 423 out of 426 pupils present in school on the day of the survey . grounded theory was selected because it is analysing method that takes account of people and their experiences . in grounded theory , the researcher aims to come close to the actor 's perspective and tries to capture his or her viewpoint . this perspective of schoolchildren and families is needed to understand the mental health promotion in school . to understand experience , it must be located within the larger events in a social , political , cultural , ethnic and gender related , informational , and technological framework . the theoretical background of the grounded theory method lies in symbolic interactionism and pragmatism . it is useful for conceptualising complex situations , understanding unsolved social problems , and understanding the impact of new ideologies . the other , favoured by strauss and applied in this study , inspects the existing theoretical knowledge and combines it with the data gained through research [ 32 , 37 , 39 ] . because of the relatively large number of articles about some aspects of mental health promotion at school , it is reasonable to examine and use previous articles about the promotion of schoolchildren 's mental health while producing a comprehensive theoretical description this is why the strauss method was selected for this study [ 32 , 37 , 39 ] . the schoolchildren 's and families ' perspective that comes from the material of this study can be compared to the existing knowledge and viewpoints [ 37 , 39 ] . however , in this study , it was still ensured that the pupils ' parents were aware of the interviews and gave their consent . before the interview , all respondents gave their written consent to the use of the interview as research material . the pupils were also informed of the use of the well - being profile responses in the promotion of mental health and well - being at school . the study was endorsed by permissions from the joint municipal authority for primary healthcare , the social welfare board , the board of education , and also the ethics committee of the local hospital district . getting familiar with the school context and with the literature also enhanced the researcher 's preunderstanding about the subject [ 32 , 38 ] . statements the informants used to illustrate mental health promotion were picked from the text [ 32 , 38 ] . as a result of the regrouping and comparison , all of the statements were placed in a category of statements pertaining to the same theme and a shared concept illustrating the phenomenon was identified . table 1 presents an example of how the statements were placed into categories and subcategories and shows the concepts used to name the categories . the codes produced by the analysis determined the direction of the data collection . following the principle of saturation , the amount of data was considered sufficient when no new utterances emerged from the data collected but the same themes began to recur . this way , the five key concepts which the parents and the schoolchildren used in describing the promotion of mental health in the upper - level comprehensive school emerged [ 32 , 40 ] . at this stage of the research therefore , the analysis was left on the level of axial coding . on this level it is possible to form causal conditions , context , phenomena , and factors related to action and interaction from the material . with regard to mental health promotion , five key concepts were discovered in the material : school environment , school friends and teachers , cooperation , actions to promote well - being and mental health , and getting help with problems . table 2 shows the key concepts related to promoting mental health and properties and describes them from the schoolchildren 's and their parents ' perspective . school environment comprises the physical conditions and educational equipment in the school including resources , teaching , rules , and services for pupils , such as school lunch and their impacts on the pupils . this concept also comprises the requirements of the teaching , the flexibility and individuality of the teaching , and the characteristics of the pupils themselves . also , the parents considered that the school environment influences children 's well - being and they had made efforts to improve the school premises , for example , via the parents ' association.the schoolyard could use some improving , there has been some opinions about that . individualism was considered to be a mental health - promoting factor in the form of promoting the self - esteem and feelings of success.it's that supporting of growth and finding those positive capabilities.the best thing in this school are ( sic ) the electives . learning difficulties , vandalism , and absences were further issues associated with the school environment although to a lesser extent . the school nurse is nevertheless always available every day . i wished remedial education for my son in some of the subjects and the teacher did carry my wish forward then . the parents considered that their task is to take an interest in the child 's life , monitor the child 's health and motivation , and ensure sufficient rest and nutrition . child upbringing was seen as a joint project in which parents and teachers function as partners , both relying on their own expertise ; parents know the child 's personality and background , whereas teachers are competent at issues related to learning.i ( a mother ) myself try to look after resting , bedtimes . the parents said that they get information on the school work from bulletins and parents ' meetings . one specific channel for information is the parents ' association which gathers information on the school and tries to contribute to the school environment 's development by , for example , arranging fund - raising events.there's not much contacting from home , but the school , however , contacts even for smaller matters.when it 's needed , everything is told . actions to promote well - being and mental health are related to having an effect on enhancing the school 's conditions and social environment . the results indicated that the school had paid attention to the development of friendships and community spirit , as well as preparing for other changes related to the transition from lower - level to upper - level comprehensive school.in spring , they ( the new students getting to know their future school ) already visited this place more than once . with regard to well - being and feeling well in general , there was a lot of discussion related to the above - mentioned school environment factors , resources , and social relationships . ensuring a sensible eating routine and a sufficient amount of sleep , improving the safety and cosiness of the school environment , fair treatment and possibilities to influence school matters , these things can be influenced by the parents and the pupils , for example , via the pupils ' council.well this is pretty good , you know , because there is a well arranged pupils ' council in here . the pupils said they had received some information about mental health - related issues from their teachers . health education lessons were mentioned as one channel in learning to take care of your well - being . the parents stated that they got information about the school 's practices and services in parents ' meetings.in parents ' meetings , it 's told how they do act in these situations which , for example , concern bullying . the school health care was seen as the primary instance to contact such matters and it was considered to be easy to access . some respondents were aware of the existence of the pupil welfare group but not of its activities or members . the respondents ' views on mental health work were mainly based on experienced problem situations , such as problems and negative feelings related to learning difficulties and experiences of offering help in stressful situations . cooperation and communication between the home and the school were considered important and preferable factors with regard to mental health problems.well , we have a school nurse who 's spezialised ( sic ) in mental health . like , the form teacher contacts the parents when there 's something that 's puzzling him or her , and the parents do the same if they are puzzled . the founded literature did not provide a comprehensive description of mental health promotion in school , but there was a good amount of studies and articles related to the individual categories . previous studies were searched with such terms as mental health promotion and school , children , or adolescents in several databases . this supports the conclusion that concepts discovered from study material are actually things that are essential in promoting mental health . wrote about the importance of developing an environment that supports the well - being of the pupils . in their studies on mental health promotion projects and reviews thereof , they found strong evidence of the social competence approach , which brings focus on the promotion of resourcefulness and generic coping and competence skills . cooperation in mental health projects is also a key element in poole 's project on preventive mental health work , in which families , schools , municipalities , and authorities worked in extensive cooperation to develop a model of partnership between the school and the families . adelman and taylor [ 46 , 47 ] named the collaborative work of school staff , health professionals , and the community resources as one very important part of successful mental health promotion work in their reviews of school mental health promotion . cooperation between the family , school , and community was a significant factor in the promotion of schoolchildren 's mental health . in this study this is why it was important to include the adolescents ' and families ' viewpoint . the influence of the entire social school environment , including the teachers and their major role , is stated in the results and enhancing these elements is seen important in many studies [ 10 , 29 , 35 , 43 ] . barry and jenkins wrote about the significance of social and problem - solving skills for well - being . in the light of barry 's results , it is easy to understand how important role the schoolchildren and their parents give to human relations and co - operation . the results of getting help with problems especially reflect the viewpoints of the pupils and their families . in previous studies anyway , previous results of co - operation [ 31 , 44 , 45 , 48 ] , mental health and social skills training and being heard [ 49 , 50 ] are things that also support this finding . the importance of being heard and possibilities to have influence in one 's own school matters can be seen in the results of this study . wrote in their study on children 's viewpoints on child psychiatric care that relationships with other children seem to be of extreme importance . the convention refers to the family as the fundamental group of society and the natural environment for the growth and well - being of children . in qualitative research , validity and reliability data generated using the grounded theory method can be assessed in terms of credibility , conformability , and truth value [ 38 , 40 ] . this study features interviews with four youngsters and a survey among a large group of schoolchildren on their opinions of their school . the material from the interviews with parents was rich and versatile and described the parents ' thoughts on their children 's schooling from various angles . the impression of the promotion of schoolchildren 's mental health gained from the material provides a versatile insight into the youngsters ' and parents ' viewpoints from various angles . a particular objective of this study was to bring out human experiences , which was well achieved with the interview material . the schoolchildren 's views on mental health promotion were clearly visible [ 32 , 40 ] . no fathers agreed to be interviewed , but mothers were asked to speak from the viewpoint of the whole family . details related to the school 's practices and human resources vary between schools and countries , which is why detailed practices , such as group - formation on transition to the upper - level school , can not be generalised into a description of mental health promotion practices in schools as a whole . instead , the process of collecting and analysing the pupils ' viewpoints used in this study , as well as the founded key aspects of mental health promotion in school , can be considered to apply to a wider context and internationally . when theory is developed from the personal descriptions of the actors and the objects of action , it is easily translatable into a development tool . pupils and their families are the key targets of mental health promotion work in schools and bringing up their viewpoints is important and forms a significant research result . the founded viewpoints help the school personnel develop their work and select the focus areas of development , even in schools with different human resources and practices [ 10 , 26 , 51 ] . schools are one of the most important settings for promoting mental health of young people . this study , by giving names and contents to different fields of mental health promotion , helps the whole school community to find key development areas . with these factors in mind , it is important to pay attention to the opportunities to increase flexibility and individuality . the school staff needs guidance in mental health promotion and theory - based means for the practical implementation thereof . it is often the case that the operators are not aware of the underlying theory behind development programmes , which makes the practical application more difficult . through the perspectives of different actors , it is easier to find an understandable and practical description of the action . with the help of the produced concepts , the mental health promotion of schoolchildren can thus be observed and promoted , which is the central benefit of this study . combining viewpoints of children , families , and professionals and then discussing and developing mental health promotion activities in cooperation would not only increase awareness but also enable development that takes all viewpoints into account . the next phase of the research is to combine these different viewpoints into a substantive theory on mental health promotion in school .

micrornas ( mirnas ) are a group of small rnas , with around 19 to 25 nucleotides , resulting from cleavage of larger non - coding rnas . they act as post - transcriptional regulators of gene expression , both in plants and in animals ( bartel , 2004 ) . in 1993 , the first mirna , named lin-4 ( lineage - deficient-4 ) , was discovered in caenorhabditis elegans , and was found to be associated with regulation of larval development . the second mirna , let-7 , was discovered in 2000 , also in caenorhabditis elegans . the fundamental discovery of the regulatory processes governed by mirnas was a major stimulus for the scientific community and led to a large number of important research on mirnas ( ambros , 2003 ) . over the last seven years , more than 4500 of mirnas have been identified in the genomes of nematodes , flies , plants , viruses and humans . estimates suggest that mirna regulatory processes may modulate the expression of 1 to 4% of human genes , thus making mirnas one of the largest classes of genomic regulators ( calin et al . , 2004 ) . in mammals , mirnas have been associated with diverse molecular pathways including regulation of proliferation , apoptosis , differentiation , cell cycle regulation hematopoiesis , and many more cellular processes . recent studies have emphasized the importance of understanding the mechanism of mrna regulation by studying changes in mirna expression in a variety of human pathological conditions , including cancers ( vanderboom et al . , 2008 ) . given their importance in development , it was to be expected that mirnas would also have a significant role in tumorigenesis . since their discovery close to 3000 publications , including over 700 reviews , documented associations between mirnas and cancer , ( garofalo and croce , 2010 ; medina and slack , 2008 ) . the mirnas of different organisms have very similar overall patterns of three - dimensional structure , even though they frequently differ significantly in their precise cellular functions ( miyoshi et al . , 2010 ) . the biogenesis of mirnas begins with their transcription by rna polymerase ii , generating a long primary transcript ( pri - mirna ) with a cap5 start and a polya tail . this transcript has a secondary hairpin - shaped structure and while still in the nucleus it is cleaved by rnaseiii and drosha and their cofactor dgcr8 ( di george syndrome critical region gene 8) , thus generating a precursor molecule of approximately 70 nucleotides . the pre - mirna is then quickly transported to the cytoplasm via exportina-5 ( exp5 ) , a nuclear exportation protein that makes use of ran - gtp as a cofactor . once in the cytoplasm , the pre - mirna is processed by rnase iii and dicer , thereby generating a double strand of mirna , of approximately 22 nucleotides in length . this product binds to the risc complex ( an induction complex for rna silencing ) and directs sequence - specific cleavage of target mrnas altyernatively , the mirna may repress translation by remaining bound to the mrna , thereby impeding its translation ( figure 1 ) . such transcriptional repression has been shown to play an important role in regulating growth and differentiation ( ambros , 2003 ; medina and slack , , 2008 ) , and abnormal expression of components of the mirna regulatory complex has been correlated with different human tumors ( discussed below ) . post - transcriptional regulation by mirnas at the untranslated 3 region depends on the extent of sequence homology with the target mrna ( templates ) , and this may influence either inhibition of translation of the template , or facilitate degradation of the mrna target transcript . imperfect matching with mrna leads to variable inhibition of translation of the target , and this mechanism is the main mode of action of mirnas in mammals . the observation that mirnas are typically short sequences that can act without the need for complete matching means that a single mirna can regulate many target mrnas . the converse also holds , so that individual mirnas may cooperate and collectively control one single mrna target ( calin et al . , 2004 ) . the molecular biology of mirnas and how they de facto act in organisms is still only beginning to be understood . there is a growing number of studies that reveal the importance of these small rnas in diverse biological processes . moreover , through the overall regulation of cellular gene expression and associations with different functional pathways it has become clear that mirnas may be involved in various human diseases . the current challenge is to identify target transcripts and pathways that are regulated by mirnas . studies have shown that a single mirisc complex may bind to more than 200 target genes , which may have a variety of functions , such as transcription factors , receptors and transporters . thus , mirnas can control the expression of almost one third of the human mrna population , and deletions or modifications in this expression may contribute towards a variety of diseases , as well as disrupt pathways of fundamental importance in neoplasia ( miyoshi et al . , 2010 ) . cancer is a complex genetic disease involving changes in structures and gene expression . for almost three decades , carcinogenesis has been primarily attributed to abnormalities in oncogenes and tumor - suppressing genes . it is now recognized that mirna also have a primary role in cancer onset and progression . such mirnas were initially linked to tumorigenesis due to their proximity to chromosomal breakpoints ( calin et al . , 2004b ) and their dysregulated expression levels in many malignancies ( calin et al . , 2004a ) . abnormal gene expression by mirnas has been correlated with several types of tumors , and such genes may function as oncogenes or tumor - suppressing genes ( figure 2 ) . in humans , 50% of the mirna genes are located at genomic sites associated with cancer - specific chromosomal rearrangements . a prime example are the genes mir-15 and mir-16 , which are located on chromosome 13q14 , a region that is deleted in more than half of the cases of chronic lymphocytic leukemia and b - cell leukemia ( calin et al . , 2002 ) . it has also been reported that mir-15a and mir-16 - 1 negatively regulate the expression of bcl2 , an anti - apoptotic oncogene that is generally overexpressed in a variety of tumors , including leukemias and lymphomas ( calin et al . , 2008 ) . these findings suggest that mir-15a and mir-16 may act as tumor - suppressing genes in human cancer . the mirnas that code for the let-7 family were the first group of oncomirs identified . mutations of the ras oncogene are present in around 25%30% of all human tumors , and overexpression of the ras oncogene is very common in lung cancer cases . ras proteins are membrane proteins that regulate cell growth and differentiation through map kinase signaling . in vitro experiments on a pulmonary adenoma cell lineage showed that let-7 was able to inhibit cell proliferation though ras , inferring that let-7 may function as a tumor suppressor in this context ( johnson et al . , 2005 ) . thus , the let-7 mirna that regulates the expression of the ras protein is also able to indirectly alter the cell proliferation rate through its downstream map signaling cascade . the strongest evidence for an association between mirnas and cancer was demonstrated by a sequence of three concurrent studies published in nature in june 2005 ( he et al . the myc oncogene , which codes for a transcription factor and functions as a cell growth regulator to induce proliferation and apoptosis , frequently appears mutated or amplified in human tumors . these authors reported that the mirna group mir-17 - 92 ( composed of seven mirnas : mir-17 - 5p , mir-17 - 3p , mir-18 , mir-19a , mir-20 , mir-19b-1 and mir-92 - 1 ) was deregulated leading to increased expression of myc , culminating with development of b - cell neoplasia . recent studies have demonstrated that the expression levels of mir-143 and mir-145 are significantly lower in colorectal tumors , thus suggesting that these mirnas act as potential tumor suppressors ( arndt et al . , 2009 ) . several classes of deregulated mirnas have also been shown to be differentially expressed in breast cancer , compared with healthy breast tissue ( volinia et al . , 2006 ) . moreover , the expression signatures of informative mirna subsets have enabled better molecular classification than mrna expression profiles in several types of human cancer ( lu et al . , 2005 ) . among the mirnas differentially expressed in breast cancer , mir-10b , mir-125 , mir-145 , mir-21 and mir-155 have consistently presented the highest degree of deregulation . downregulation of mir-10b , mir-125b and mir-145 and upregulation of mir-21 and mir-155 suggest that these mirnas may play an important role as tumor - suppressors or oncogenes ( blenkiron et al . , 2007 ) . in particular , the mir-145 mirna is progressively downregulated when passing from healthy breast tissue to breast cancer with high cell proliferation rates . similarly , but in the opposite direction , the expression of mir-21 is progressively upregulated when comparing normal breast tissue with breast cancer at advanced stages . thus , the deregulation of these mirnas may affect molecular events that are critical for tumor progression ( yang et al . , 2008 ) . some specific mirnas have also been associated with tumor invasion and metastasis in breast cancer . for example , the level of mir-10b expression in primary breast carcinomas has been correlated with clinical progression of the disease ( ma et al . , 2007 ) . recently it was also observed that the expression of mir-7 , mir-128a , mir-210 and mir-516 - 3p was associated with aggressiveness in cases that were positive for estrogen - receptor tumors and negative for lymph node tumors ( foekens et al . , 2008 ) . in recent years , studies on mirnas , especially on a large scale using microarrays , have provided a more comprehensive picture on the role of abnormal mirna expression in neoplasia . upon using molecular profiling methods such as bead - based flow cytometry , real - time pcr or mirage ( sage analysis ) in line with this , novel molecular classifications of tumors based on their mirna expression , have provided a wealth of new resources for predictive and prognostic biomarkers for clinical applications in cancer . when a sequence polymorphism is present within a mirna transcript it is called a mir - snp . these mir - snps are single - base polymorphisms ( snps ) in the mirna sequences and are considered to be an important new class of functional polymorphisms in the human genome . since the mode of action of mirna is highly sequence - dependent , changing a single base in a mirna sequence that alters binding specificities may affect multiple genes , thus impacting on one or several biological pathways . the function of mirnas may be altered through variations in their own sequence ( mir - snps ) or in their target sequences ( called mir - ts - snps ) ( figure 3 ) . since a single mirna can have multiple mrna target sites , sequence polymorphisms in general have deeper and more extensive effects from a biological point of view than would sequence alterations to mrna ( sun et al . , 2009 ) . duan and pak ( 2007 ) identified a snp within an essential region of mir-125 that significantly changed the mir-125a sequence and abolished recognition of its target site . functional experiments in vivo confirmed that the presence of this polymorphism blocked the maturation of mir-125a . another example of this process is a snp in the precursor of mir - k5 , that is encoded by the human herpes virus in association with kaposi s sarcoma . this polymorphism correlated with abnormalities in the mirna cleavage processing by the drosha enzyme . similarly , yang et al ( 2008 ) analyzed 41 potentially functional polymorphisms in mirnas , pre - micrornas and pri - micrornas that predisposed to bladder cancer ( yang et al . , 2008 ) and found a snp in gemin3 gene and a common haplotype in gemin4 that showed significant associations with higher risk of bladder cancer . moreover , it could be demonstrated that combinations of certain genotypes were strongly associated with predisposition towards bladder cancer , and that the presence of these specific mirna genotypes could be used as a tool for predicting the risk of developing such tumors . these examples illustrate that variations in the different biogenesis routes for mirnas affect their own function and consequently the expression of the target messenger rna . one of the first epidemiological studies showing a relationship between polymorphisms in the binding region of mirnas and cancer was published by landi et al ( 2008 ) , wherein a relationship between the allele variants of cd86 ( polymorphism rs17281995 , c > g ) and the micrornas mir-337 , mir-582 , mir-200a , mir-184 and mir-212 was found to lead to a higher risk of colorectal cancer . it was also shown that there is a relationship between the presence of polymorphism rs1051690 in the insulin receptor ( insr ) and a higher risk of colorectal cancer due to modification of the binding affinity of the mirnas 618 and 612 ( landi et al . , 2008 ) . subsequently , a snp was identified at the target site of let-7 in the 3utr region of the kras gene ( lcs6-kras ) ( trang et al . , 2008 ) . the presence of this polymorphism was associated with increased risk of lung cancer among smokers . this allele variant was detected in 20% of the patients with lung carcinoma ( nsclc non - small cell lung carcinoma ) . in asymptomatic individuals , lcs6-kras was found in 6% of the sample analyzed ( n = 2433 ) . in a case - control study on lung cancer , the presence of this allele was associated with an increased risk of lung cancer ( relative risk of 2.3 ) among individuals with a history of smoking ( mean of 820 cigarettes / year ) . functional studies demonstrated that the presence of this polymorphism diminished the binding affinity of let-7 to its target site in kras , and consequently , increased expression of kras . nonetheless , christensen et al ( 2009 ) reported that in head and neck cancer , this same polymorphism ( lcs6-kras ) was not associated with any general increase in cancer risk , but significantly so with reduced survival ( christensen et al . , 2009 ) . amongst the polymorphisms affecting mirna target sites , tchatchou et al ( 2009 ) analyzed a group of 11 snps and found a strong correlation between the variant rs2747648 ( c / t ) in the estrogen receptor 1 ( esr1 ) gene and an increased risk of breast cancer . this risk was shown to be higher for premenopausal women with a positive family history of cancer ( tchatchou et al . , 2009 ) . the mode of action was inferred to be due to a lower binding affinity of mir-453 in the presence of the t allele , and concomitant reduced repression of the esr1 gene . since loss of binding of mir-453 , led to overexpression of both the esr1 mrna and its receptor protein , the breast cancer cancer risk is expected to be increased . taken together the regulation and role of mirnas and the large number of target sites in functionally important genes and associated pathways provides new insights into cancer risk . it is clear that both a single mir - snp or a specific combination of mirna gene variants may act together on their target mrna sites to constitute a new previously unappreciated mechanism for predisposition to cancer . recent studies have indicated that mirna expression can be regulated by different epigenetic mechanisms , including changes in dna methylation in promoter regions and histone modification ( scott et al . , 2006 ; esteller , 2008 ) ( figure 4 ) . when expression of such a mirna is affected , any methylation of this area will simultaneously alter the expression of any target mrna and proteins modulated by the epigenetically modified mirna ( scott et al . , 2006 ) . recent studies involving epigenetic factors and changes to mirna expression have mostly been restricted to assays on tumor cells . one of the first studies to be published involved mir-127 , which acts to repress the tumor - specific expression of the proto - oncogene bcl-6 . after treating tumor cells with chromatin modifying agents , mir-127 showed activity in different types of human cancer cell lines , inferring that its inactivation in these cells could involve chromatin - induced epigenetic alterations ( saito et al . , 2006 ) . suppression of hsa - mir-9 - 1 , hsa - mir-129 - 2 and hsa - mir-137 in colorectal cancer is , at least partly , mediated by epigenetic mechanisms such as dna hypermethylation and histone deacetylation , as demonstrated in a recent study by bandres et al ( 2009 ) . this study also emphasized that frequent hypermethylation of these mirna loci in colorectal cancer was correlated with clinicopathological abnormalities . expression of hsa - mir-9 - 1 was associated with positive lymph node biopsies in patients with advanced stages of colorectal cancer . dna methylation was considered to be the most likely mechanism for diminishing or inhibiting the expression of these specific mirnas . considering that these mirnas are not usually expressed in normal mucosal tissue , this epigenetic alteration would diminish the tracking of disease evolution ( bandres et al . , 2009 ) . in breast cancer cell cultures , the homozygous variant of the mirna hsa - mir-196a-2 ( rs11614913 , ct ) was shown to be significantly associated with diminished risk of breast cancer , and hypermethylation of a cpg island located 700 base pairs above the precursor region of mir196a-2 led to a reduction in the risk of breast cancer ( hoffman et al . , 2009 ) . based on a series of molecular analyses , these authors suggested that mir-196a-2 might have oncogenic potential in breast cell tumorigenesis , and that functional genetic variations in this mirna could serve as biomarkers for susceptibility to breast cancer . 2008 ) reported that mir-34a expression was consistently silenced in different types of cancer by aberrant methylation of cpg in the promoter region ( lodygin et al . , 2008 ) . it was shown that 79.1% of primary prostate carcinomas had cpg methylation and concomitant loss of mir-34a expression . similar observations with differing proportions were made in carcinoma cells in breast ( 25% ) , lung ( 29.1% ) , colon ( 13% ) , kidney ( 21.4% ) and pancreas tissue ( 15.7% ) , and in melanomas ( 43.2% ) and primary melanomas ( 62.5% ) . several methods for evaluating mirna expression profiles have been implemented , including rt - pcr , microarrays and serial analysis of gene expression ( mi - rage ) . independent of the approach , success in applying these techniques is essentially limited by the availability of fresh or frozen clinical tissue samples , which are considered to be the most reliable sources of integral rna ( zhang et al . , 2008 ) . nevertheless , mirnas turned out to be less affected by fixation in formalin and embedding in paraffin than mrnas because of their slower degradation , smaller size and lack of a poly - a tail . good correlations were denoited formirna profiles of rna extracted from frozen samples and those embedded in paraffin ( zhang et al . , 2008 ) . samples preserved in paraffin are also useful in evaluating the action of a mirna on a target gene and determining whether it may result in a change in the expression of the corresponding protein . changes in the expression of proteins regulating the biogenesis of mirna can also be evaluated at the cellular level . for such analyses , immunohistochemical techniques are useful as these make it possible to detect the location and/or site of subcellular action of the target protein . as an example , the proteins that bind rna lin28 and lin28b prevent precursors of mirna let-7 from being processed by mature mirna ( newman et al . , 2008 ) . using immunohistochemistry and tissue microarrays , lin28 and lin28b increased expression was associated with physiological repression of let-7 levels and tumor progression , implying a tumor - suppressing role for this mirna ( viswanathan et al . , 2009 ) . in addition , the role of mirna in tumor tissue samples can be evaluated by means of in situ hybridization ( ish ) tests to measure expression levels of specific mirnas in target cells . recently , a highly sensitive technique was described for detecting single mirna molecules in individual cells ( lu and tsourkas , 2009 ) . the method known as lna - elf - fish employs oligonucleotides from locked nucleic acids with fluorescence for signal amplification , thus allowing mirnas to be spatially located and quantified inside cells . in a study on gliomas , and especially multiform glioblastomas , the mirnas regulated by dicer , mir-222 and mir-339 were identified using ish , while the endonuclease and the intercellular adhesion molecule icam-1 were evaluated by means of immunohistochemistry . these mirnas were shown to be expressed by the tumors and negatively regulated icam-1 , given that the expression of these molecules presented inverse associations in the tissue samples ( ueda et al . , 2009 ) . in expression microarrays , there was no difference in dicer expression between normal prostatic tissue and organ - confined prostate cancer . however , immunohistochemical analysis demonstrated that in normal tissues , dicer immunoreactivity was detected only in basal cells , proliferative neoplastic cells , and in invasive cancer . the redistribution of dicer among the cell types seemed to be biologically significant with cancer progression and metastasis , and the level of this endonuclease continued to increase in the abnormal cells ( chiosea et al . , 2009 ) . the mir-21 is one of the most - studied mirnas in cancer cases and it is highly expressed in breast cancer . in a cohort analysis using ish , a progressive increase in the percentage of patient tumors positive for mir-21 was observed , from normal breast tissue ( 13% ) , flat epithelial atypia ( 47% ) , ductal carcinoma in situ ( 75% ) to invasive ductal carcinoma ( 88% ) . in addition , the expression of mir-21 target genes such as pten , pdcd4 and tm1 was evaluated in the same tumor samples from the cohort at cellular level , and the cell transformation suppressor tm1 was confirmed as a target of mir-21 in breast tumors , presenting reduced tissue immunoreactivity with progressive lesions , i.e. an inverse relationship with the marker pattern of mir-21 ( qi et al . , 2009 ) . despite the fast advances in comprehending the biogenesis and action mechanism of mirnas , many questions regarding their function and influence on central signaling pathways and cell cycle control remain . the complex stochastic nature of gene expression in mammalian cells has wide - ranging impact on phenotypic diversity . it is therefore likely that evaluating mean mirna expression levels in mixtures of cell populations may result in loss of crucial information for linking mirna expression to cellular functions . thus , the physiological role of mirna in single cells should be more informative in distinguishing the impact of mirna on signaling networks and cellular pathways relevant to disease ( lu and tsourkas , 2009 ) . recently , a new technology of directed artificial - site mirnas ( templates ) , for increasing or inhibiting endogenous mirna regulation was describes ( brown et al . , 2009 ) . this strategy has been used to detect site - specific target genes in cells , in relation to stem cell therapies and studies on transgenic animals . through this highly specific new approach a promising strategy has emerged , combining gene therapy with mirna templates ( viruses carrying the target sequence ) , in an attempt to fully or partially inhibit the expression of these mirnas . as described earlier , mirnas can exert their effects either by acting as tumor suppressors or through favoring cancer development ( oncomirs ) . when hyperexpression of mirnas contributes towards oncogenesis , the rational strategy is to reduce their expression . in this regard , inhibition of specific endogenous mirnas has been used through administration of antisense synthetic oligonucleotides , which are complementary to endogenous mature mirnas . antagomirs , currently constitute the majority of mirna inhibition tools ( soifer et al . , 2007 ) . basically three different types of omas are used for inhibiting mirnas , these being oligonucleotides with modifications to the 2-oh group of the ribose residues that involve replacement with 2-o - methyl ( 2-ome ) , 2-o - methoxy - ethyl ( 2-moe ) or locked nucleic acid ( lna ) . these modifications have been incorporated through knowledge gained from interference rna techniques ( rnai ) , and were essential for offering resistance to enzymatic degradation , thereby improving oma stability when exposed to the large quantities of nucleases present in blood and the cell environment . another important structural change incorporated into these oligonucleotides , with a view to improving their pharmacokinetic properties ( such as plasma half - life ) and increasing the uptake of the molecule by cells , was the introduction of a cholesterol molecule in the 3 terminal region of the nucleic acid ( bijsterbosch et al . , 2000 ) . applications of oligonucleotides that specifically inhibit oncomirs , such as mir-21 , have been demonstrated in cultures on glioblastoma cells and breast cancer cells , thereby promoting increased caspase activation and mediating apoptosis in these cells ( chan et al . , 2005 ; si et al . , 2007 ) . furthermore , suppression of mir-21 gave rise to significant reductions in invasions and lung metastases in cultures on mda - mb231 breast cancer cells ( zhu et al . , 2008 ) . the efficacy and significance of several omas have been examined and validated in several pioneering studies using a model that eliminates mir-122 , which is hyper - expressed in rat livers after administration of specifically developed omas ( krutzfeldt et al . , 2005 ) . these authors were the first to demonstrate nontoxic long - duration silencing generated through intravenous injection of ( 2-ome ) that were complementary to mir-122 , in mice . notwithstanding , one of the major obstacles to applying antagomirs in clinical screening is achieving effective release of rnai in the target tissue ( soifer et al . , 2007 ) . strategies for overcoming these problems have been developed , for example complexation or covalent bonding of lipids and/or proteins released in small rna molecules ( dykxhoorn et al . , 2006 ) . other alternatives , such as the use of cationic liposomes and cholesterol , conjugation with rna - packaging phages and rna aptamers that bind to receptors have been developed to release small rnas in target cells ( soutschek et al . , 2004 ) . research on local release of biomolecules should substantially improve the therapeutic opportunities for using mirnas as targets . recently , a new form of mirnas inhibitors called mirna sponges were developed which may be transitorily expressed in mammal cell cultures ( ebert et al . , 2007 ) . mirna sponges are transcripts that are under the control of strong gene promoters ( rna polymerase ii ) containing tandemly arranged multiple sites for binding to mirnas of interest and are capable of inhibiting mirnas as strongly as omas ( ebert et al . , 2007 ) . for mirnas that present reduced expression in cancer cases , restoration of mirna levels in the diseased tissue should provide a therapeutic benefit through replacement of the target gene regulation . introduction of double - stranded mirnas , which are equivalent to the products from endogenous dicer and analogous in structure to a sirna ( small interference rna ) , may provide transient restoration for underexpressed mirnas . however , in vivo application of double - strand mimetic mirna , resembling sirna , still needs to be evaluated . to achieve greater persistence of mirna replacement , a transgenic approach is needed so that expression of specific mirnas may be induced , starting from a plasmid or viral vector containing the promoters for both the polymerases ( ii or iii ) that control the expression of a short hairpin rna , which is processed subsequent to the mature mirna . at present only few studies on the use of mirnas for in vivo cancer therapy have been published . ( short hairpin rna ) with an anti - cancer focus employed liposomes , polymers and nanoparticles . similar strategies and technologies used for sirna release in cells may also be used with mirnas . mirnas have emerged over recent years as new regulatory components of the complex mechanisms of gene expression , with implications for many diseases , including cancer . emerging evidence increasingly demonstrates that mirnas may also affect or be affected by genetic and epigenetic mechanisms . in addition , mirnas and mir - snps are powerful tools for studying disease prognoses and , in the near future , hold tremendous therapeutic promise for clinical medicine and for improvements in cancer control and in curing rates .

micrornas are key regulators of various fundamental biological processes and , although representing only a small portion of the genome , they regulate a much larger population of target genes . mature micrornas ( mirnas ) are single - stranded rna molecules of 2023 nucleotide ( nt ) length that control gene expression in many cellular processes . these molecules typically reduce the stability of mrnas , including those of genes that mediate processes in tumorigenesis , such as inflammation , cell cycle regulation , stress response , differentiation , apoptosis and invasion . microrna targeting is mostly achieved through specific base - pairing interactions between the 5 end ( seed region ) of the mirna and sites within coding and untranslated regions ( utrs ) of mrnas ; target sites in the 3 utr diminish mrna stability . since mirnas frequently target hundreds of mrnas , mirna regulatory pathways are complex . calin and croce were the first to demonstrate a connection between micrornas and increased risk of developing cancer , and meanwhile the role of micrornas in carcinogenesis has definitively been evidenced . it needs to be considered that the complex mechanism of gene regulation by micrornas is profoundly influenced by variation in gene sequence ( polymorphisms ) of the target sites . thus , individual variability could cause patients to present differential risks regarding several diseases . aiming to provide a critical overview of mirna dysregulation in cancer , this article reviews the growing number of studies that have shown the importance of these small molecules and how these micrornas can affect or be affected by genetic and epigenetic mechanisms .

MicroRNAs: Characterization and Biogenesis Regulation of Gene Expression Through MicroRNAs and Implications for Cancer Association Between SNPS at MicroRNA Binding Sites and the Risk of Cancer Association Between Epigenetic Alterations and MicroRNAs in Cancer Preservation, Expression and Localization of MicroRNAs in Paraffinized Tissue Samples Gene Therapy and MicroRNAs Conclusion

micrornas ( mirnas ) are a group of small rnas , with around 19 to 25 nucleotides , resulting from cleavage of larger non - coding rnas . they act as post - transcriptional regulators of gene expression , both in plants and in animals ( bartel , 2004 ) . in 1993 , the first mirna , named lin-4 ( lineage - deficient-4 ) , was discovered in caenorhabditis elegans , and was found to be associated with regulation of larval development . the fundamental discovery of the regulatory processes governed by mirnas was a major stimulus for the scientific community and led to a large number of important research on mirnas ( ambros , 2003 ) . over the last seven years , more than 4500 of mirnas have been identified in the genomes of nematodes , flies , plants , viruses and humans . estimates suggest that mirna regulatory processes may modulate the expression of 1 to 4% of human genes , thus making mirnas one of the largest classes of genomic regulators ( calin et al . in mammals , mirnas have been associated with diverse molecular pathways including regulation of proliferation , apoptosis , differentiation , cell cycle regulation hematopoiesis , and many more cellular processes . recent studies have emphasized the importance of understanding the mechanism of mrna regulation by studying changes in mirna expression in a variety of human pathological conditions , including cancers ( vanderboom et al . given their importance in development , it was to be expected that mirnas would also have a significant role in tumorigenesis . since their discovery close to 3000 publications , including over 700 reviews , documented associations between mirnas and cancer , ( garofalo and croce , 2010 ; medina and slack , 2008 ) . this transcript has a secondary hairpin - shaped structure and while still in the nucleus it is cleaved by rnaseiii and drosha and their cofactor dgcr8 ( di george syndrome critical region gene 8) , thus generating a precursor molecule of approximately 70 nucleotides . once in the cytoplasm , the pre - mirna is processed by rnase iii and dicer , thereby generating a double strand of mirna , of approximately 22 nucleotides in length . this product binds to the risc complex ( an induction complex for rna silencing ) and directs sequence - specific cleavage of target mrnas altyernatively , the mirna may repress translation by remaining bound to the mrna , thereby impeding its translation ( figure 1 ) . such transcriptional repression has been shown to play an important role in regulating growth and differentiation ( ambros , 2003 ; medina and slack , , 2008 ) , and abnormal expression of components of the mirna regulatory complex has been correlated with different human tumors ( discussed below ) . post - transcriptional regulation by mirnas at the untranslated 3 region depends on the extent of sequence homology with the target mrna ( templates ) , and this may influence either inhibition of translation of the template , or facilitate degradation of the mrna target transcript . imperfect matching with mrna leads to variable inhibition of translation of the target , and this mechanism is the main mode of action of mirnas in mammals . the molecular biology of mirnas and how they de facto act in organisms is still only beginning to be understood . there is a growing number of studies that reveal the importance of these small rnas in diverse biological processes . moreover , through the overall regulation of cellular gene expression and associations with different functional pathways it has become clear that mirnas may be involved in various human diseases . studies have shown that a single mirisc complex may bind to more than 200 target genes , which may have a variety of functions , such as transcription factors , receptors and transporters . thus , mirnas can control the expression of almost one third of the human mrna population , and deletions or modifications in this expression may contribute towards a variety of diseases , as well as disrupt pathways of fundamental importance in neoplasia ( miyoshi et al . abnormal gene expression by mirnas has been correlated with several types of tumors , and such genes may function as oncogenes or tumor - suppressing genes ( figure 2 ) . in humans , 50% of the mirna genes are located at genomic sites associated with cancer - specific chromosomal rearrangements . the mirnas that code for the let-7 family were the first group of oncomirs identified . mutations of the ras oncogene are present in around 25%30% of all human tumors , and overexpression of the ras oncogene is very common in lung cancer cases . thus , the let-7 mirna that regulates the expression of the ras protein is also able to indirectly alter the cell proliferation rate through its downstream map signaling cascade . these authors reported that the mirna group mir-17 - 92 ( composed of seven mirnas : mir-17 - 5p , mir-17 - 3p , mir-18 , mir-19a , mir-20 , mir-19b-1 and mir-92 - 1 ) was deregulated leading to increased expression of myc , culminating with development of b - cell neoplasia . recent studies have demonstrated that the expression levels of mir-143 and mir-145 are significantly lower in colorectal tumors , thus suggesting that these mirnas act as potential tumor suppressors ( arndt et al . several classes of deregulated mirnas have also been shown to be differentially expressed in breast cancer , compared with healthy breast tissue ( volinia et al . among the mirnas differentially expressed in breast cancer , mir-10b , mir-125 , mir-145 , mir-21 and mir-155 have consistently presented the highest degree of deregulation . similarly , but in the opposite direction , the expression of mir-21 is progressively upregulated when comparing normal breast tissue with breast cancer at advanced stages . thus , the deregulation of these mirnas may affect molecular events that are critical for tumor progression ( yang et al . for example , the level of mir-10b expression in primary breast carcinomas has been correlated with clinical progression of the disease ( ma et al . recently it was also observed that the expression of mir-7 , mir-128a , mir-210 and mir-516 - 3p was associated with aggressiveness in cases that were positive for estrogen - receptor tumors and negative for lymph node tumors ( foekens et al . in recent years , studies on mirnas , especially on a large scale using microarrays , have provided a more comprehensive picture on the role of abnormal mirna expression in neoplasia . upon using molecular profiling methods such as bead - based flow cytometry , real - time pcr or mirage ( sage analysis ) in line with this , novel molecular classifications of tumors based on their mirna expression , have provided a wealth of new resources for predictive and prognostic biomarkers for clinical applications in cancer . these mir - snps are single - base polymorphisms ( snps ) in the mirna sequences and are considered to be an important new class of functional polymorphisms in the human genome . since the mode of action of mirna is highly sequence - dependent , changing a single base in a mirna sequence that alters binding specificities may affect multiple genes , thus impacting on one or several biological pathways . functional experiments in vivo confirmed that the presence of this polymorphism blocked the maturation of mir-125a . another example of this process is a snp in the precursor of mir - k5 , that is encoded by the human herpes virus in association with kaposi s sarcoma . this polymorphism correlated with abnormalities in the mirna cleavage processing by the drosha enzyme . , 2008 ) and found a snp in gemin3 gene and a common haplotype in gemin4 that showed significant associations with higher risk of bladder cancer . moreover , it could be demonstrated that combinations of certain genotypes were strongly associated with predisposition towards bladder cancer , and that the presence of these specific mirna genotypes could be used as a tool for predicting the risk of developing such tumors . these examples illustrate that variations in the different biogenesis routes for mirnas affect their own function and consequently the expression of the target messenger rna . one of the first epidemiological studies showing a relationship between polymorphisms in the binding region of mirnas and cancer was published by landi et al ( 2008 ) , wherein a relationship between the allele variants of cd86 ( polymorphism rs17281995 , c > g ) and the micrornas mir-337 , mir-582 , mir-200a , mir-184 and mir-212 was found to lead to a higher risk of colorectal cancer . it was also shown that there is a relationship between the presence of polymorphism rs1051690 in the insulin receptor ( insr ) and a higher risk of colorectal cancer due to modification of the binding affinity of the mirnas 618 and 612 ( landi et al . subsequently , a snp was identified at the target site of let-7 in the 3utr region of the kras gene ( lcs6-kras ) ( trang et al . the presence of this polymorphism was associated with increased risk of lung cancer among smokers . this allele variant was detected in 20% of the patients with lung carcinoma ( nsclc non - small cell lung carcinoma ) . in asymptomatic individuals , lcs6-kras was found in 6% of the sample analyzed ( n = 2433 ) . in a case - control study on lung cancer , the presence of this allele was associated with an increased risk of lung cancer ( relative risk of 2.3 ) among individuals with a history of smoking ( mean of 820 cigarettes / year ) . functional studies demonstrated that the presence of this polymorphism diminished the binding affinity of let-7 to its target site in kras , and consequently , increased expression of kras . nonetheless , christensen et al ( 2009 ) reported that in head and neck cancer , this same polymorphism ( lcs6-kras ) was not associated with any general increase in cancer risk , but significantly so with reduced survival ( christensen et al . amongst the polymorphisms affecting mirna target sites , tchatchou et al ( 2009 ) analyzed a group of 11 snps and found a strong correlation between the variant rs2747648 ( c / t ) in the estrogen receptor 1 ( esr1 ) gene and an increased risk of breast cancer . this risk was shown to be higher for premenopausal women with a positive family history of cancer ( tchatchou et al . the mode of action was inferred to be due to a lower binding affinity of mir-453 in the presence of the t allele , and concomitant reduced repression of the esr1 gene . taken together the regulation and role of mirnas and the large number of target sites in functionally important genes and associated pathways provides new insights into cancer risk . it is clear that both a single mir - snp or a specific combination of mirna gene variants may act together on their target mrna sites to constitute a new previously unappreciated mechanism for predisposition to cancer . recent studies have indicated that mirna expression can be regulated by different epigenetic mechanisms , including changes in dna methylation in promoter regions and histone modification ( scott et al . one of the first studies to be published involved mir-127 , which acts to repress the tumor - specific expression of the proto - oncogene bcl-6 . suppression of hsa - mir-9 - 1 , hsa - mir-129 - 2 and hsa - mir-137 in colorectal cancer is , at least partly , mediated by epigenetic mechanisms such as dna hypermethylation and histone deacetylation , as demonstrated in a recent study by bandres et al ( 2009 ) . dna methylation was considered to be the most likely mechanism for diminishing or inhibiting the expression of these specific mirnas . considering that these mirnas are not usually expressed in normal mucosal tissue , this epigenetic alteration would diminish the tracking of disease evolution ( bandres et al . in breast cancer cell cultures , the homozygous variant of the mirna hsa - mir-196a-2 ( rs11614913 , ct ) was shown to be significantly associated with diminished risk of breast cancer , and hypermethylation of a cpg island located 700 base pairs above the precursor region of mir196a-2 led to a reduction in the risk of breast cancer ( hoffman et al . based on a series of molecular analyses , these authors suggested that mir-196a-2 might have oncogenic potential in breast cell tumorigenesis , and that functional genetic variations in this mirna could serve as biomarkers for susceptibility to breast cancer . similar observations with differing proportions were made in carcinoma cells in breast ( 25% ) , lung ( 29.1% ) , colon ( 13% ) , kidney ( 21.4% ) and pancreas tissue ( 15.7% ) , and in melanomas ( 43.2% ) and primary melanomas ( 62.5% ) . several methods for evaluating mirna expression profiles have been implemented , including rt - pcr , microarrays and serial analysis of gene expression ( mi - rage ) . independent of the approach , success in applying these techniques is essentially limited by the availability of fresh or frozen clinical tissue samples , which are considered to be the most reliable sources of integral rna ( zhang et al . nevertheless , mirnas turned out to be less affected by fixation in formalin and embedding in paraffin than mrnas because of their slower degradation , smaller size and lack of a poly - a tail . samples preserved in paraffin are also useful in evaluating the action of a mirna on a target gene and determining whether it may result in a change in the expression of the corresponding protein . changes in the expression of proteins regulating the biogenesis of mirna can also be evaluated at the cellular level . for such analyses , immunohistochemical techniques are useful as these make it possible to detect the location and/or site of subcellular action of the target protein . as an example , the proteins that bind rna lin28 and lin28b prevent precursors of mirna let-7 from being processed by mature mirna ( newman et al . in addition , the role of mirna in tumor tissue samples can be evaluated by means of in situ hybridization ( ish ) tests to measure expression levels of specific mirnas in target cells . the method known as lna - elf - fish employs oligonucleotides from locked nucleic acids with fluorescence for signal amplification , thus allowing mirnas to be spatially located and quantified inside cells . these mirnas were shown to be expressed by the tumors and negatively regulated icam-1 , given that the expression of these molecules presented inverse associations in the tissue samples ( ueda et al . however , immunohistochemical analysis demonstrated that in normal tissues , dicer immunoreactivity was detected only in basal cells , proliferative neoplastic cells , and in invasive cancer . the redistribution of dicer among the cell types seemed to be biologically significant with cancer progression and metastasis , and the level of this endonuclease continued to increase in the abnormal cells ( chiosea et al . the mir-21 is one of the most - studied mirnas in cancer cases and it is highly expressed in breast cancer . in addition , the expression of mir-21 target genes such as pten , pdcd4 and tm1 was evaluated in the same tumor samples from the cohort at cellular level , and the cell transformation suppressor tm1 was confirmed as a target of mir-21 in breast tumors , presenting reduced tissue immunoreactivity with progressive lesions , i.e. despite the fast advances in comprehending the biogenesis and action mechanism of mirnas , many questions regarding their function and influence on central signaling pathways and cell cycle control remain . the complex stochastic nature of gene expression in mammalian cells has wide - ranging impact on phenotypic diversity . thus , the physiological role of mirna in single cells should be more informative in distinguishing the impact of mirna on signaling networks and cellular pathways relevant to disease ( lu and tsourkas , 2009 ) . this strategy has been used to detect site - specific target genes in cells , in relation to stem cell therapies and studies on transgenic animals . through this highly specific new approach a promising strategy has emerged , combining gene therapy with mirna templates ( viruses carrying the target sequence ) , in an attempt to fully or partially inhibit the expression of these mirnas . basically three different types of omas are used for inhibiting mirnas , these being oligonucleotides with modifications to the 2-oh group of the ribose residues that involve replacement with 2-o - methyl ( 2-ome ) , 2-o - methoxy - ethyl ( 2-moe ) or locked nucleic acid ( lna ) . these modifications have been incorporated through knowledge gained from interference rna techniques ( rnai ) , and were essential for offering resistance to enzymatic degradation , thereby improving oma stability when exposed to the large quantities of nucleases present in blood and the cell environment . another important structural change incorporated into these oligonucleotides , with a view to improving their pharmacokinetic properties ( such as plasma half - life ) and increasing the uptake of the molecule by cells , was the introduction of a cholesterol molecule in the 3 terminal region of the nucleic acid ( bijsterbosch et al . applications of oligonucleotides that specifically inhibit oncomirs , such as mir-21 , have been demonstrated in cultures on glioblastoma cells and breast cancer cells , thereby promoting increased caspase activation and mediating apoptosis in these cells ( chan et al . these authors were the first to demonstrate nontoxic long - duration silencing generated through intravenous injection of ( 2-ome ) that were complementary to mir-122 , in mice . notwithstanding , one of the major obstacles to applying antagomirs in clinical screening is achieving effective release of rnai in the target tissue ( soifer et al . strategies for overcoming these problems have been developed , for example complexation or covalent bonding of lipids and/or proteins released in small rna molecules ( dykxhoorn et al . other alternatives , such as the use of cationic liposomes and cholesterol , conjugation with rna - packaging phages and rna aptamers that bind to receptors have been developed to release small rnas in target cells ( soutschek et al . for mirnas that present reduced expression in cancer cases , restoration of mirna levels in the diseased tissue should provide a therapeutic benefit through replacement of the target gene regulation . introduction of double - stranded mirnas , which are equivalent to the products from endogenous dicer and analogous in structure to a sirna ( small interference rna ) , may provide transient restoration for underexpressed mirnas . however , in vivo application of double - strand mimetic mirna , resembling sirna , still needs to be evaluated . to achieve greater persistence of mirna replacement , a transgenic approach is needed so that expression of specific mirnas may be induced , starting from a plasmid or viral vector containing the promoters for both the polymerases ( ii or iii ) that control the expression of a short hairpin rna , which is processed subsequent to the mature mirna . mirnas have emerged over recent years as new regulatory components of the complex mechanisms of gene expression , with implications for many diseases , including cancer . emerging evidence increasingly demonstrates that mirnas may also affect or be affected by genetic and epigenetic mechanisms . in addition , mirnas and mir - snps are powerful tools for studying disease prognoses and , in the near future , hold tremendous therapeutic promise for clinical medicine and for improvements in cancer control and in curing rates .

together with the actin microfilaments and the microtubules , intermediate filaments ( ifs ) are the components of the cytoskeleton of eukaryotic cells , which is involved in the maintenance of cell shape , locomotion , intracellular organization , and transport ( bershadsky and vasiliev 1988 ; ku et al . if proteins comprise a large family , which includes ~70 different genes ( omary et al . they are further divided into six subtypes ( table 1 ) , which are , at least in part , expressed in a cell type ( and differentiation)-dependent manner ( omary et al . 2004 ; kim and coulombe 2007 ; goldman et al . accordingly , ifs serve as cell type markers and antibodies to if proteins are widely used today in diagnostic pathology ( wick 2000 ; barak et al . individual if proteins consist of a conserved central coiled - coil -helical rod domain ( interrupted by linkers ) which is flanked by n - terminal ( head ) and c - terminal ( tail ) domains ( omary et al . the n- and c - terminal domains contribute to the structural heterogeneity and are major sites of posttranslational modifications with phosphorylation being the best characterized one ( omary et al . this makes them important regulatory domains , since dynamic changes in phosphorylation status are responsible for alterations in if dynamics , solubility , and organization ( omary et al . 2006).table 1intermediate filament proteinstypename / localizationdisease locationremarksi ( n = 28)k9 - 28 ( epithelia)k10,14,16,17-skinacidic keratinsk31 - 40 ( hair / nail)k12-cornea(pi < 5.7)k13-stratified mucosatype i / ii obligate 1:1 polymersk16,17-nailk18-liverii ( n = 26)k1 - 8 , k71 - 80 ( epithelia)k1,2e,5,9-skinbasic keratinsk81 - 86 ( hair / nail)k3-cornea(pi 6.0)k4-stratified mucosatype i / ii obligate 1:1k6a,6b - nailpolymersk8-liverk75,81,83,85,86-hairiiidesmin ( muscle)muscle , heartdesmin , vimentin and gfap are found in stellate cellsvimentin ( mesenchymal)peripherin ( neurons)brain , spinal cordgfap ( astrocytes / glia)brainivnf - l ( neurons)brain , spinal cord-internexin forms polymers with nfsnf - m ( neurons)brain , spinal cordnf - h ( neurons)brain , spinal cordsynemin is also called desmuslin.-internexin ( cns neurons)nestin - stem cell markersynemins ( muscle)syncoilin ( muscle)nestinvlamin a / c ( ubiquitous)heart , muscle , fat , premature aging , complex defectsthe only nuclear ifs , longer rod domainlamin b1/2 ( ubiquitous)orphanphakinin ( lens)lensbeaded filaments in lens epitheliafilensin ( lens)lens not a causative association , variants represent a risk factor . for an overview about the new keratin nomenclature , ( 2006 ) in addition to the posttranslational modification , if function is modified and complemented through interaction with a variety of if - associated proteins ( ifap ; table 2 ; green et al . 2005 ; omary et al . 2006 ; kim and coulombe 2007 ) . these proteins can be subdivided into several subgroups , which reflect multiple if functions ( green et al . 2005 ) . for example , ifs interact with a variety of anchoring proteins thereby forming transcellular networks which contribute to proper tissue architecture . ifaps also include several cytolinker proteins , which provide the structural framework for coordinated cytoskeletal function ( table 2 ) . by doing that , they are important mechanical stabilizers and accordingly , if disruption results in increased mechanical fragility ( omary et al . 2004 ; ku et al . 2007 ; herrmann et al . the scaffolding function of ifs is best seen in if - deficient animals , who exhibit disrupted cellular architecture , protein mistargeting as well as alterations in organelle localization and function ( toivola et al . 2005 ) . in the case of lamin deficiencies , the impairment of nuclear composition has profound impact on many aspects of normal nuclear functions such as epigenetic changes , chromatin organization or dna transcription , and repair ( dechat et al . 2008).table 2examples of if - associated proteinstype of interactionexamplesfunctionanchoringdesmoplakin , bpag1 , -dystobrevintissue architecturecytolinkerplectin , filaggrin , cellular architecturechaperoneshsp27 , -b - crystallin , hsp70protein foldingkinasespkc , cdk5if regulation , phosphate sink , cell cycleadaptor proteins14 - 3 - 3 protein , ap-3multiple effectsmembrane proteinspolycystin-1unknownapoptotic proteinstradd , tnfr2 , c - flip , caspase3/9apoptosis regulationmotor proteinsdynein , kinesinmovement of if components examples of if - associated proteins ifs are not just simple cellular scaffolds , they rather build complex signaling platforms ( pallari and eriksson 2006 ; kim and coulombe 2007 ) . ifs interact with a variety of enzymatic and adaptor proteins , thereby affecting a multitude of cellular functions . for example , keratins associate with 14 - 3 - 3 proteins in a phosphorylation - dependent manner and this interaction regulates cell growth and cell cycle progression ( ku et al . 2007 ; kim and coulombe 2007 ) . if phosphorylation through associated kinases does not only regulate if properties , but ifs also serve as phosphate sponge thereby preventing activation of other , potentially pro - apoptotic , substrates ( omary et al . in addition to that , ifs also directly participate in apoptosis regulation through binding of several apoptosis - related molecules ( marceau et al . 2007 ) . in contrast to the actin and tubulin system , ifs emerged later in the evolution and are important supportive elements of the cell rather than their essential components . therefore , if variants are observed in various human diseases , which reflect their tissue specific distribution , whereas only few actin and tubulin variants have been described so far , likely due to their embryolethality ( ku et al . . currently , more than 30 diseases are caused by / associated with if mutations ( omary et al . 2004 ) . among them , keratin - related- and lamin - related disorders are the best studied ones . for example , mutations in keratins 5/14 cause a blistering skin disease termed epidermolysis bullosa simplex , whereas mutations in lamins a and c result , among others , in different diseases including premature aging , cardiomyopathy , and lipodystrophy ( omary et al . the disease relevance of ifs is also highlighted by a variety of if - containing inclusion bodies , which represent the pathological hallmarks of several neurodegenerative , muscular , and other disorders ( goebel 1998 ; ross and poirier 2004 ; omary et al . these aggregates share a variety of features such as presence of misfolded , ubiquitinated structural proteins together with variable amounts of chaperones and p62 ( kuusisto et al . 2001 ; zatloukal et al . 2002 ; ross and poirier 2004 ) . among the if - related inclusions , mallory - denk bodies ( mdbs ) are the most common and also the best studied ones due to the availability of animal mdb models ( denk et al . 1975 ; yokoo et al . 1982 ; jensen and gluud 1994 ; denk et al . therefore , one focus of our review will be to describe mdb as a prototype of if - related inclusion body , which should offer useful insights into the formation of if - related aggregates in multiple human diseases . keratins represent the largest subfamily of ifs consisting of > 50 unique gene product members ( schweizer et al . 2006 ; kim and coulombe 2007 ; godsel et al . 2008 ) which include 37 epithelial and 17 hair keratin members in humans ( table 1 ; schweizer et al . 2006 ) . based on their pi , epithelial keratins can be subdivided in types i ( acidic ) and ii ( basic ) corresponding to keratins 920 ( k9k28 ) and keratins 18 plus keratins 7180 ( k1k8 ; k71k80 ) , respectively ( table 1 ; coulombe and omary 2002 ; schweizer et al . keratins are found as obligatory type i and type ii heteropolymers ( i.e. , consisting of at least one type i and one type ii keratin ) and a homozygous disruption of a keratin results in degradation of its keratin partner at the protein level ( ku and omary 2000 ; omary et al . 2004 ) . similarly to ifs , keratins are expressed in a tissue - specific manner , with different pairs being the major cellular ifs in different cell populations ( moll et al . ( i.e. , single layered ) epithelia , as found in digestive organs , express k8 together with variable levels of k7 , k18 , k19 , and k20 depending on the tissue ( moll et al . 1982 ; ku et al . 1999 ; coulombe and omary 2002 ; ku et al . in contrast , stratified epithelia , like epidermis , express k5/k14 in the basal and k1/k10 in the suprabasal keratinocytes , respectively ( moll et al . 1982 ; lane and mclean 2004 ; coulombe and omary 2002 ) . despite their similar molecular composition , simple and stratified keratins are not interchangeable , as shown in k14-null mice , whose phenotype was only partially restored by addition of k18 ( hutton et al . 1998 ) . furthermore , keratins have their preferential binding partners in vivo and the lack of such partner leads to their rapid degradation ( magin 1998 ) . this contrasts with the in vitro situation , where if assembly is more promiscuous ( hatzfeld and franke 1985 ) . k8/k18/k19 are promising serological markers based on their high abundance ( approximately 0.3% of total liver protein ) and intracellular localization under basal conditions with release into blood upon liver injury ( omary et al . however , one important caveat is the fact , that k8/k18/k19 are expressed in most simple epithelial cells and are therefore not liver - specific ( moll et al . 1982 ; ku et al . the k8/k18/k19 epitopes used in serologic diagnosis can be divided into two classes , that is , non - specific and apoptosis - generated epitopes . the former class constitutes established tumor markers such as tissue polypeptide antigen ( tpa , represents total k8/k18/k19 ) , tissue polypeptide specific antigen ( tps , derived from k18 ) , and cytokeratin fragment 21 - 1 ( cyfra 21 - 1 , derived from k19 ) . their original clinical use was to monitor treatment response and to detect recurring tumors ( barak et al . however , later studies showed that these epitope serum levels are also elevated in non - malignant diseases and might be a general marker of tissue injury ( gonzalez - quintela et al . the apoptosis - specific keratin antibodies are based on the finding , that type i keratins are cleaved at a conserved vemd / vevd residue during apoptosis . in addition to that , k18 posseses a second , k18 specific caspase - cleavage site at asp396 , which is an early event during apoptosis preceding the cleavage at the vemd / vevd motif ( oshima 2002 ; marceau et al . the cleavage of human k18 at asp396 can be monitored using the m30 antibody ( leers et al . 1999 ) and m30-ab elisa has become a useful serologic test for determining liver disease severity . for example , elevated serum m30 titers can distinguish simple steatosis from non - alcoholic steatohepatitis ( wieckowska et al . 2006 ) and predict several important prognostic parameters in patients with chronic hepatitis c infection ( bantel et al . 2004 2006 ) . as another tool for detecting apoptotic keratin fragments , an antibody specific to the conserved k18/k19 cleavage site at asp237 it detects both mouse and human k18/k19 fragments and appears to be more sensitive than the established asp396-ab ( tao et al . 2008 ) . measuring the serum levels of apoptosis - specific keratin fragments should also improve our understanding of chronic liver disease , where apoptotic cell death is an important pathogenic feature ( malhi et al . in addition to that , monitoring the phosphorylation status of the circulating keratin fragments might be useful . keratins undergo dynamic phosphorylation during mitosis , apoptosis and a variety of stress situations ( omary et al . 1998 ; omary et al . 2006 ) and their in situ phosphorylation status is a marker of human liver disease progression ( toivola et al . . however , it is currently unknown whether the phosphorylation status of the circulating keratin fragments correlates with the situation in situ . the liver consists of different cell types with characteristic if composition ( table 3 ; omary et al . adult hepatocytes are unique among simple epithelial cells in that they express exclusively k8 and k18 , whereas other glandular epithelia exhibit a more complex keratin expression pattern ( omary et al . the hepatocytic keratin if network is dense , particularly around bile canaliculi and at the cell periphery , and acts as cytoskeletal backbone to the functionally more dynamic and contractile actin microfilament system ( zatloukal et al . biliary epithelial cells differ from hepatocytes by additional expression of keratin 7 and 19 ( omary et al . keratins in cholangiocytes , but not hepatocytes , exhibit polarized and compartment - specific expression pattern ( omary et al . the biological significance of such an expression is enigmatic , but it may be related to polarity and secretory processes . among non - epithelial cells , stellate cells express variable amounts of gfap , desmin , vimentin , and nestin dependent on their activation status , localization , and other parameters ( table 3 ; geerts 2001).table 3ifs of liver cell populationscell typeif compositionhepatocytek8/k18oval cellsk7/k8/k18/k19cholangiocytek7/k8/k18/k19kupffer cellvimentinstellate cellgfap , vimentin , desmin , nestinendothelial cellvimentinmodified from omary et al . ( 2002 ) during embryogenesis , hepatocytes also express variable levels of k19 ( vassy et al . 1997 ) stellate cells represent a highly heterogeneous population with variable if expression dependent on species , activation status of the cell , location within the hepatic lobe and many other parameters ( geerts 2001 ) ifs of liver cell populations modified from omary et al . ( 2002 ) during embryogenesis , hepatocytes also express variable levels of k19 ( vassy et al . 1997 ) stellate cells represent a highly heterogeneous population with variable if expression dependent on species , activation status of the cell , location within the hepatic lobe and many other parameters ( geerts 2001 ) studies in keratin knock - out mice revealed that the regular liver development does not require the presence of k8 , k18 , or k19 ( ku et al . 2007 ) . however , k8/k18 knockout mice exhibited mild chronic hepatitis , hepatocyte fragility and were markedly more sensitive to a variety of stress conditions ( omary et al . , k8/k18 transgenic mice were developed , which over - express different single - amino - acid variants ( ku et al . 2007 ) . among them , the k18 r89c variant resulted in disruption of hepatocyte if network and exhibited a phenotype reminiscent of the situation in k8/k18-knockout mice ( ku et al . 1995 , 1996 ) . k18 r89c mice also predisposed to development of thioacetamide - induced liver fibrosis ( strnad et al . 2008 ) . ablations of different k8/k18 phosphorylation sites usually led to a somewhat milder phenotype which became apparent in stress situations , but not under basal conditions ( ku et al . for example , k18 s52a variant resulted in increased sensitivity to microcystin - lr - induced liver injury , whereas k8 s73a mice were predisposed to fas - induced liver apoptosis ( ku et al . 1998 ; ku and omary 2006 ) . the ablation of k18 s33 phosphorylation site , which regulates the binding to 14 - 3 - 3 proteins , caused limited mitotic arrest and accumulation of abnormal mitotic figures after partial hepatectomy ( ku et al . in contrast , mice lacking k19 did not have an obvious liver phenotype ( tao et al . gfap / vimentin - knockouts displayed compromised astrocytic function with attenuated reactive gliosis , but no obvious alteration in the in vitro activation of hepatic stellate cells despite the lack of if network ( geerts et al . however , in vivo studies are needed to conclusively address the fibrogenic potency of these transgenic mice . the large body of evidence from animal studies showing the importance of k8/k18 for liver homeostasis led to a search for keratin mutations in patients with liver diseases . several k8/k18 variants were found to associate with the development of cryptogenic liver disease ( ku et al . , k8/k18 were shown to represent susceptibility genes for development of end - stage liver disease of multiple etiologies ( ku et al . 2003a , 2005 ) . in particular , biologically significant k8/k18 variants were found in 44 of 467 liver explants ( 12.4% ) , but only in 11 out of 349 analyzed blood bank donors , which were used as a control group ( p < 0.0001 , prevalence or3.8 ; 95% ci = 2.17 ) . furthermore , k8/k18 variants associate with liver fibrosis progression in patients with chronic hepatitis c ( strnad et al . k8 r340h represents the most common amino acid altering k8/k18 variant and it is the only one , which was significantly associated with development of end - stage liver disease ( ku et al . larger studies are needed to analyze the pathological significance of the other less common k8/k18 variants ( ku et al . in contrast to human association studies , experiments in k8/k18-deficient mice were shown to predispose mainly to acute liver injury ( ku et al . , we recently analyzed a large cohort of patients with acute liver failure ( alf ) . k8/k18 variants were significantly more frequent in total alf patient cohort ( 46/345 ; 13.3% ) and in patients with acetaminophen - induced alf ( 21/169 ; 12.4% ) when compared to blood bank donors ( 11/349 ; 3.7% ; p < 0.002 for both comparisons ) . among the single polymorphisms , the k8 r340h variant was found at significantly higher frequencies in the whole alf cohort as well as in the acetaminophen - induced alf subgroup ( frequency 6.6 and 7.1% , respectively vs. 3.1% in the control group ; p < 0.03 for both comparisons ) . in addition , transgenic mice over - expressing k8 r340 variants displayed augmented acetaminophen - induced liver toxicity . in conclusion , k8/k18 are also susceptibility genes for development of alf and k8/k18 variants may predispose to drug - induced liver injury ( strnad et al . , unpublished data ) . up to date , only one published study analyzed the polymorphisms in k19 gene and found no association between k19 variants and inflammatory bowel disease ( tao et al . 2007 ) . interestingly , we recently observed k19 g17s variant in three out of 190 patients with primary biliary cirrhosis , but none was found in control blood bank donors ( 200 samples ; zhong et al . , unpublished data ) . however , larger studies are needed to address the importance of k19 in biliary diseases . the human k8/k18 variants described above do not cause a particular liver disease per se , they just pose a risk factor for its development . this is different from the situation in stratified epithelia , where keratin mutations result in several monogenic keratin diseases ( lane and mclean 2004 ; omary et al . this discrepancy may be caused either by the intrinsic difference between keratins of simple and stratified epithelia ( hutton et al . 1998 ) or by the different localization of the variants within the protein backbone . to that end , disease - causing keratin mutations in stratified epithelia are clustered in the highly conserved helix initiation and termination motif , whereas k8/k18 disease - predisposing variants are observed in more variable domains ( owens and lane 2004 ; omary et al . the disturbance in cytoprotective function of keratins is the likely mechanisms by which k8 and k18 variants predispose to liver disorders . for example , k8/k18 are anti - apoptotic proteins and this ability is hampered in keratin - deficient animals ( oshima 2002 ; ku and omary 2006 ; marceau et al . k8/k18 bind to several apoptotic proteins and type i keratins are established caspase substrates ( oshima 2002 ; green et al . 2005 ; marceau et al . 2007 ) . in addition , k8/k18 serve as physiologic kinase substrates in vitro and in transgenic mice and an ablation of the k8 s73 phosphorylation site or introduction of the naturally occurring k8 g61c variant leads to increased apoptosis through increased phosphorylation of pro - apoptotic proteins ( ku et al . . the keratin - mediated anti - apoptotic function may be highly relevant given the importance of apoptosis in liver disease and the pro - fibrogenic properties of elevated rate of apoptotic cell death ( friedman 2004 ; malhi et al . keratins exhibit anti - oxidative properties , as they sequester oxidatively damaged proteins , and similarly , the k18 r89c variant primes the liver towards oxidative injury ( tao et al . this keratin property might be helpful in attenuating both liver fibrosis and acetaminophen - induced liver injury ( parola and robino 2001 ; jaeschke et al . keratins are also established stress - inducible proteins , which are upregulated both in humans and mice under several , mainly cholestatic conditions ( fickert et al . 2002 , 2003 keratin variants may interfere with keratin upregulation or simply result in decreased keratin levels due to protein instability ( as seen for k18 r89c variant in the liver ; ku et al . the naturally occurring k8/k18 variants interfere with basic if properties such as k8/k18 filament assembly and keratin solubility ( ku et al . 2007 ) . due to altered protein conformation , some of them impair potentially cytoprotective keratin phosphorylation at adjacent residues ( as seen in k8 g61c and k8 g434s variant ; ku et al . 2005 ; ku and omary 2006 ) . k8/k18 variants may also affect organelle function , as seen in k8 knockout mice , which exhibit altered mitochondrial shape , localization , and alterations in several mitochondrial proteins ( toivola et al . 2005 ) . despite the various cytoprotective effects of keratins , their impact seems to be limited to certain conditions . for example , k18 r89c mice are predisposed to fas but not tnf - induced apoptosis and the same mice develop more pronounced liver fibrosis after thioacetamide , but not after carbon tetrachloride injection ( ku et al . it is also unknown which one of the keratin - mediated effects is important in particular disease settings . the recently established transgenic mouse lines overexpressing the naturally occurring k8 g61c and r340h variants will likely offer valuable insights in this respect ( ku and omary 2006 ; zhou et al . , unpublished data ) . steatohepatitis is characterized by hepatocyte ballooning , that is , swelling and rounding with clearing of the cytoplasm , which prevails in the perivenular zone and is often associated with pericellular fibrosis , predominantly granulocytic inflammation ( satellitosis ) , steatosis ( usually of macrovesicular type ) , and cholestasis ( brunt 2004 ; lefkowitch 2005 ) . ballooned cells are often associated with granular , rope- or clump - like cytoplasmic inclusions , called mallory - denk bodies ( mdbs ) ( originally also designated alcoholic hyalin , which is , however , a misnomer since they are not specific for alcoholic etiology ) together with derangement or even disappearance of the keratin if cytoskeleton ( mallory 1911 ; denk et al . the disappearance of the keratin immunostaining in ballooned hepatocytes is reasonably specific for ash and nash , since it is not seen in ballooned cells of viral hepatitis or toxic damage and can therefore be used as an objective morphologic parameter in grading of steatohepatitis ( lackner et al . however , ballooning ( with concomitant cytoskeletal alterations ) and mdb formation are not entirely interchangeable since not all ballooned hepatocytes contain mdbs . mdbs are usually detected as eosinophilic aggregates in standard hematoxylin and eosin stained sections ( a ) . after chromotrope aniline blue staining , mdbs appear as blue structures , often with red center ( b ) . immunofluorescence or immunohistochemical staining represents a more sensitive method for mdb detection than conventional histological stainings , but is strongly dependent on the antibody used as well as the staining protocol . mdbs can be reliably detected with antibodies against k8/k18 [ green and redchannel in ( c ) and ( d ) , respectively ] or p62 [ red channel in ( c ) ] , whereas only some mdbs stain with antibodies to phosphorylated keratins such as k8 ps431 antibody [ green channel in ( d ) ] . in both immunofluorescence pictures , mdbs are seen as yellow structure due to co - localization of both visualized epitopes mdbs are readily detected by different methods . in clinical routine , mdbs are usually detected as eosinophilic aggregates in standard hematoxylin and eosin stained sections ( a ) . after chromotrope aniline blue staining , mdbs appear as blue structures , often with red center ( b ) . immunofluorescence or immunohistochemical staining represents a more sensitive method for mdb detection than conventional histological stainings , but is strongly dependent on the antibody used as well as the staining protocol . mdbs can be reliably detected with antibodies against k8/k18 [ green and redchannel in ( c ) and ( d ) , respectively ] or p62 [ red channel in ( c ) ] , whereas only some mdbs stain with antibodies to phosphorylated keratins such as k8 ps431 antibody [ green channel in ( d ) ] . in both immunofluorescence pictures , mdbs are seen as yellow structure due to co - localization of both visualized epitopes mdbs are typical morphological features of ash and nash , although nash usually exhibits slightly less prominent mdbs than the ones seen in ash ( brunt 2004 ; zatloukal et al . 2007 ) . mdbs can also be detected after intestinal bypass surgery for morbid obesity , in chronic cholestasis , particularly in late stages of primary biliary cirrhosis , wilson disease and other types of copper toxicosis , various metabolic disturbances , and hepatocellular neoplasms ( mller et al . in contrast , mdbs have not been observed in the context of acute cholestasis , acute viral hepatitis and a variety of acute toxic or drug - induced liver diseases ( jensen and gluud 1994 , zatloukal et al . however , even in potentially mdb - forming liver diseases , mdbs are found only in a subset of patients , partially ( but not completely ) depending on the sensitivity of the detection method used . for example , when using immunohistochemistry for keratin or ubiquitin , mdbs were found in about 70% of ash cases in contrast to 40% seen in hematoxilin - eosin - stained sections ( ray 1987).fig . immunohistochemical staining with p62 antibody visualizes the presence of multiple irregularly shaped aggregates in patients with alcoholic steatohepatitis ( a ) , non - alcoholic steatohepatitis ( b ) , indian childhood cirrhosis and ( c ) , idiopathic copper toxicosis ( d ) mdbs are seen in various human liver diseases . immunohistochemical staining with p62 antibody visualizes the presence of multiple irregularly shaped aggregates in patients with alcoholic steatohepatitis ( a ) , non - alcoholic steatohepatitis ( b ) , indian childhood cirrhosis and ( c ) , idiopathic copper toxicosis ( d ) this suggests that mdbs require either a specific pathogenetic constellation or genetic predisposition for its formation , which is present only in a subset of patients . for example , a low percentage of hepatocytes express keratin 7 and to a lesser extent keratin 19 which indicate that these cells acquire features of precursor cells which normally express keratin 8 , 18 , 7 , and 19 during regeneration ( van eyken et al . 1988 ; zatloukal et al . mdbs may coincide with another type of cytoplasmic inclusions , termed intracellular hyaline bodies ( ihbs ) , which share several components with mdbs , but do not contain keratins ( stumptner et al . however , correlation between the clinical disease manifestation / progression on one side and hepatocyte ballooning with mdb formation on the other is imperfect . for example , patients with severe clinical symptoms of ash sometimes show only moderate histopathological alterations with few or no mdbs , whereas patients with pronounced histological alterations do not necessarily exhibit significant clinical and laboratory abnormalities ( zatloukal et al . controlled clinical - pathologic studies comparing nash patients with ambulatory and hospitalized alcoholics revealed that hepatocellular damage , presence of mdbs , inflammation , and fibrosis collectively correlated with disease severity ( cortez - pinto et al . also in other studies , hepatocellular ballooning and mdb formation was positively correlated with disease progression , development of fibrosis , and cirrhosis and liver - related mortality ( orrego et al . mdbs are irregularly shaped , usually dense cytoplasmic inclusions of different sizes ( mallory 1911 ) . small mdbs arise in association with if bundles throughout the cytoplasm , whereas larger mdbs are often seen in the perinuclear region ( denk et al . ultrastructurally , they usually consist of haphazardly arranged filamentous rods , approximately 1015 nm in diameter , covered by a fuzzy coat ( designated as type ii by yokoo et al . 1972 ) . in addition , mdbs with an electron dense granular to amorphous center ( designated type 3 ) are seen predominantly in older inclusions , and filaments in parallel arrangement were also described ( designated as type i ) but seem to be exceedingly rare ( yokoo et al . keratins 8 and 18 , sequestosome 1/p62 ( p62 ) and ubiquitin are major , and low and high molecular weight heat shock proteins ( hsp 70 , hsp 25 ) , but also proteins of the protein degradation machinery , are minor constituents ( denk et al . 2000 ; riley et al . keratins 7 , 19 , and 20 have also occasionally been detected ( cadrin et al . 1990 ; dinges et al . 1992 ) . 1993 ) , are hyperphosphorylated , partially degraded and cross - linked ( hazan et al . the list of mdb components is likely to grow since , in addition to intrinsic components , proteins non - specifically incorporated in the aggregate have to be expected . studies on mdb formation and composition are greatly enhanced by the availability of animal models . mdbs can be induced under standardized conditions and their fate followed in mouse liver by chronic griseofulvin or 3,5-diethoxycarbonyl-1,4-dihydrocollidine ( ddc ) administration ( denk et al . liver sections were double labeled with antibodies to k8/k18 ( green ) and p62 ( red ) . samples from mouse fed ddc for 12 weeks ( d ) and from a patient with alcoholic steatohepatitis ( b ) exhibit multiple irregularly shaped inclusion bodies , which appear yellow due to presence of both epitopes . in contrast , control human ( a ) and mouse liver ( c ) display an unaffected keratin network with no apparent p62 staining . note that ddc feeding leads to deposition of protoporphyrin , which can be seen as occasional blue pigment in ( d ) mdbs formed in ddc - fed animals resemble inclusion bodies observed in human diseases . liver sections were double labeled with antibodies to k8/k18 ( green ) and p62 ( red ) . samples from mouse fed ddc for 12 weeks ( d ) and from a patient with alcoholic steatohepatitis ( b ) exhibit multiple irregularly shaped inclusion bodies , which appear yellow due to presence of both epitopes . in contrast , control human ( a ) and mouse liver ( c ) display an unaffected keratin network with no apparent p62 staining . note that ddc feeding leads to deposition of protoporphyrin , which can be seen as occasional blue pigment in ( d ) in patients , mdb formation is usually a chronic process requiring several years of alcohol intoxication or metabolic imbalance , that is , in association with the metabolic syndrome , wilson disease , other metabolic disorders , and chronic cholestasis . an exception is idiopathic copper toxicosis of children in which end stage liver disease associated with mdb formation occurs at very early age ( mller et al . surprisingly , in alcoholics recovered from ash , an alcohol excess may almost instantaneously lead to mdb recurrence , a situation which has been compared to an immunologic response and termed toxic memory does not only reproduce the structural and morphological features seen in humans , but also the phenomenon of toxic memory mdb formation in mice is reversible , since mdbs disappear after recovery on standard diet for 1 month . however , after rechallenge of the recovered ( primed ) animals , mdbs reappear within a few days ( denk et al . rapid mdbs formation in primed mice is rather non - specific , since it can be triggered by a variety of stress conditions including colchicine ( but not by lumicolchicine ) , bile acids , bile duct ligation , several other toxins , and proteasome inhibitors which were unable to induce mdbs in the nave animal ( zatloukal et al . the reasons for this phenomenon are as yet unclear but point to a final common pathway activated by the trigger . the availability of cellular and animal models of mdb formation led to valuable insights into the mechanism of mdb formation . several pathogenic mechanisms were implicated in this process ( fig . 4 ; dobson 2004):1.enhanced oxidative stress2.disproportional k8/k18 expression together with keratin modifications3.chaperone dysfunction4.elevated p62 levels5.insufficient protein degradation enhanced oxidative stress disproportional k8/k18 expression together with keratin modifications chaperone dysfunction insufficient protein degradation ad 1 . the mdb - inducing conditions both in humans and mice cause elevated levels of oxidative stress ( tephly et al . 1981 ; mehta et al . 2002 ; dey and cederbaum 2006 ; farrell and larter 2006 ) and mdbs themselves were shown to contain misfolded keratins with increased -sheet formation ( cadrin et al . recent animal studies highlight the importance of altered methyl group metabolism and mitochondrial stress in this process ( li et al . 2008 ; zatloukal et al . , unpublished data ) . during ddc detoxification , n - methylprotoporphyrin is formed through transfer of a methyl group to heme moieties ( tephly et al . n - methylprotoporphyrin subsequently acts as a potent inhibitor of ferrochelatase , thereby causing porphyria ( tephly et al . , mdb formation and ddc effects can be effectively attenuated by feeding s - adenosylmethionine , that is a compound involved in methyl group transfer ( li et al . this is reminiscent of the situation in ash , which is also associated with disrupted methyl group metabolism ( schalinske and nieman 2005).fig . since the cytoplasm represents a hydrophilic milieu , all exposed hydrophobic molecules ( depicted by red stretches within the protein ) are predisposed to aggregation . properly folded proteins usually hide their hydrophobic stretches inside , but these get exposed in nascent protein chains or after proteins become misfolded as a consequence of oxidative stress . alternatively , damaged proteins become polyubiquitinated and degraded either by the proteasomal or autophagic system . mdb - causing agents typically generate extensive amount of oxidative stress with increased protein misfolding . dysbalanced k8/k18 expression precedes mdb formation , likely increases keratin misfolding and predisposes to posttranslational modifications , which may interfere with keratin refolding and/or repair . however , proteasomal degradation might be impaired by oxidative stress , may not be able to digest highly cross - linked protein species or might simply be overwhelmed by the excessive supply . on the other hand , autophagy is upregulated during mdb formation in mice and additional stimulation of autophagy attenuates mdb formation in certain conditions . of note , supplementation of s - adenosylmethionine or mitochondrially targeted antioxidants effectively diminishes mdb formation , thereby pointing to a central role of oxidative stress in mdb generation mdbs formation results from a complex interplay of several contributing factors . since the cytoplasm represents a hydrophilic milieu , all exposed hydrophobic molecules ( depicted by red stretches within the protein ) are predisposed to aggregation . properly folded proteins usually hide their hydrophobic stretches inside , but these get exposed in nascent protein chains or after proteins become misfolded as a consequence of oxidative stress . chaperones bind to these hydrophobic residues and facilitate protein refolding . alternatively , damaged proteins become polyubiquitinated and degraded either by the proteasomal or autophagic system . mdb - causing agents typically generate extensive amount of oxidative stress with increased protein misfolding . dysbalanced k8/k18 expression precedes mdb formation , likely increases keratin misfolding and predisposes to posttranslational modifications , which may interfere with keratin refolding and/or repair . however , proteasomal degradation might be impaired by oxidative stress , may not be able to digest highly cross - linked protein species or might simply be overwhelmed by the excessive supply . on the other hand , autophagy is upregulated during mdb formation in mice and additional stimulation of autophagy attenuates mdb formation in certain conditions . of note , supplementation of s - adenosylmethionine or mitochondrially targeted antioxidants effectively diminishes mdb formation , thereby pointing to a central role of oxidative stress in mdb generation ddc targets mitochondria , where it reacts with cytochrome p450 ( marks et al . an unbiased microarray analysis identified cytochrome p450 ( cyp ) 2a5 as a major gene induced both after ddc exposure and particularly after ddc re - challenge . , it resembles human cyp2e1 , which was implicated as a source of oxidative stress in the pathogenesis of ash and nash ( villeneuve and pichette 2004 ) . to address the role of mitochondrial stress in mdb formation , we re - fed ddc - primed mice with ddc alone or in combination with the mitochondria - targeted antioxidant mito q ( smith et al . mito q co - administration attenuated both mdb formation and ddc - induced liver damage . therefore , mitochondrial oxidative stress seems to be involved in mdb formation in mice which is in good concordance to the mitochondrial dysfunction seen both in ash and nash patients ( pessayre 2007 ; mantena et al . keratins are major constituents of mdbs and both altered k8/k18 expression and keratin modification seems to affect mdb formation ( zatloukal et al griseofulvin / ddc feeding leads to rapid induction of k8/k18 expression with disproportional k8 > k18 levels ( denk et al . 2000 ) . the elevated k8/k18 ratio is crucial for mdb formation as shown in k18-knockout and k8 overexpressing animals , who are predisposed to mdb formation already upon short exposure to ddc and even form mdbs spontaneously during aging ( magin et al . accordingly , k8-null or k18 overexpressing mice are resistant to mdb formation and the protective function of k18 is not affected by its phosphorylation status or mutation ( zatloukal et al . 2000 ; harada et al . 2007 ) . however , the exclusive mdb inducing property of k8 in vivo can not be reproduced in vitro , where aggregates resembling mdbs can also be produced by transfection of k18 ( nakamichi et al . among posttranslational modifications , mdb formation is associated with k8 hyperphosphorylation and transamidation ( zatloukal et al . 2000 ) . in a recent study , ablation of k8 s73 phosphorylation site in transgenic mice resulted in diminished mdb formation after ddc exposure ( harada et al . , unpublished results ) . k8 s73 is a well - known p38 kinase target site and p38 up - regulation induces keratin network reorganization with subsequent granule formation ( ku et al . p38 kinase - induced keratin network reorganization might be a necessary prerequisite for mdb formation , since p38 kinase inhibition prevented mdb formation in vitro ( nan et al . alternatively , k8 hyperphosphorylation may induce mdb formation through inhibition of k8 degradation , which results in increased k8/k18 ratio ( ku and omary 2000 ) . mdbs contain highly cross - linked keratins and the ablation of tissue transglutaminase effectively inhibits ddc - induced mdb formation ( zatloukal et al . since k8 is a much better in vitro transglutaminase substrate than k18 and highly cross - linked proteins found in mdb - forming mice contain k8 , but not k18 , it was suggested that excessive k8 gets preferentially transamidated and acts as a nucleus for mdb formation . this is supported by studies in transgenic mice , where k8 overexpression accelerates and k18 excess inhibits not only mdb , but also cross - link formation ( strnad et al . the protein misfolding is usually counteracted by the reparative function of chaperones ( ross and poirier 2004 ; macario and conway de macario 2005 ; bukau et al . 2006 ) , however , ddc feeding is associated with diminished chaperone expression , persistent chaperone modifications and impairment of chaperone function ( strnad et al . similarly , chaperone function is impaired in a rat model of chronic alcoholic liver disease ( carbone et al . p62 is a stress - inducible protein with multiple functions which is a constituent of multiple inclusion bodies termed sequestosomes ( shin 1998 ; kuusisto et al . it binds proteins polyubiquitinated at lysine 63 and shuttles them for proteasomal or autophagic degradation ( vadlamudi et al . 1996 ; bjorkoy et al . 2005 ; seibenhener et al . 2004 ; wooten et al . 2008 ) . p62 action is beneficial under normal conditions , since it prevents accumulation of abnormal proteins ( bjorkoy et al . 2008 ) , but may become harmful when protein degradation is inhibited ( komatsu et al . there are several lines of evidence implicating p62 ( containing the ubiquitin binding site ) in mdb formation . in cell culture experiments , protein aggregates resembling mdbs formed only after p62 co - transfection , but not when k8 , k18 , and ubiquitin were transfected alone or in combination ( stumptner et al . furthermore , p62 was rapidly induced in ddc - fed mice with p62-containing aggregates preceding the formation of mdbs ( stumptner et al . 2002 ) . furthermore , p62 inhibition attenuated , whereas p62 overexpression enhanced mdb formation in ddc - primed hepatocytes ( nan et al . accumulation of protein aggregates , as seen during mdb formation , is counteracted by proteasomal or autophagic degradation ( glickman and ciechanover 2002 ; williams et al . 2006 ) and both degradation machineries are active in the liver . for example , conditional knock - out of the autophagy - related gene 7 ( atg7 ) leads to development of ubiquitin - positive inclusions in hepatocytes ( komatsu et al . similarly , proteasomes seem to interact with mdbs and inhibition of proteasomal degradation leads to formation of mdb - like structures both in vitro and in ddc - primed mice ( harada et al . furthermore , autophagic degradation is upregulated in ddc - fed mice and its further stimulation with rapamycin attenuates spontaneous mdb formation in k8 overexpressing mice ( harada et al . in contrast , mdb formation might be facilitated by proteasomal impairment , since mdbs are preferentially seen in proteasome - depleted hepatocytes ( bardag - gorce et al . in addition , an aberrant form of ubiquitin , termed [ ubb + 1 ] arising as a consequence of molecular misreading , is observed in mdbs and may inhibit proteasomal function ( mcphaul et al . alcohol and aging , both potential inducers of mdbs can be associated with decreased proteasome function ( hayashi and goto 1998 ; fataccioli et al . however , aggregate formation must not always be caused by a general inhibition in protein degradation . alternatively , certain proteins may not be easily degradable , such as proteins with long polyglutamine stretches ( venkatraman et al . 2004 ) . the latter scenario may apply to ddc - fed mice , which exhibit an accumulation of ubiquitinated protein only in the highly insoluble protein fraction ( strnad et al . this fraction is characterized by highly cross - linked protein species generated through extensive transamidation , which may make them resistant to proteolytic degradation ( strnad et al . 2007 ) . in principle , cytoplasmic protein aggregates can be beneficial , detrimental or inert depending on the context . in many pathologic situations aggregation seems to be a protective response if the first lines of defense , that is , refolding and degradation , fail by sequestration of potentially harmful proteins ( arrasate et al . on the other hand , large protein aggregates can be detrimental either by mechanical interference with cellular transport processes ( e.g. , shown with microtubule - dependent transport in neurons ) , deprivation of the cell of vital components by aggregation or coaggregation and by overwhelming / inhibiting the capacity of the chaperone system and/or the protein degradation machinery by indigestible material ( alonso et al . 2001 ; bence et al . 2001 ; lee et al . 2004 ; grune et al . it can be expected , therefore , that in different cell systems and pathologic conditions inclusion bodies differ regarding their cellular effects and consequences . mdbs per se do not seem to compromise the viability of transfected tissue culture cells ( stumptner et al . 2007 ) or hepatocytes in vivo , who even exhibit an activated phenotype ( denk et al . 2000 ) .

intermediate filaments ( ifs ) represent the largest cytoskeletal gene family comprising ~70 genes expressed in tissue specific manner . in addition to scaffolding function , they form complex signaling platforms and interact with various kinases , adaptor , and apoptotic proteins . ifs are established cytoprotectants and if variants are associated with > 30 human diseases . furthermore , if - containing inclusion bodies are characteristic features of several neurodegenerative , muscular , and other disorders . acidic ( type i ) and basic keratins ( type ii ) build obligatory type i and type ii heteropolymers and are expressed in epithelial cells . adult hepatocytes contain k8 and k18 as their only cytoplasmic if pair , whereas cholangiocytes express k7 and k19 in addition . k8/k18-deficient animals exhibit a marked susceptibility to various toxic agents and fas - induced apoptosis . in humans , k8/k18 variants predispose to development of end - stage liver disease and acute liver failure ( alf ) . k8/k18 variants also associate with development of liver fibrosis in patients with chronic hepatitis c. mallory - denk bodies ( mdbs ) are protein aggregates consisting of ubiquitinated k8/k18 , chaperones and sequestosome1/p62 ( p62 ) as their major constituents . mdbs are found in various liver diseases including alcoholic and non - alcoholic steatohepatitis and can be formed in mice by feeding hepatotoxic substances griseofulvin and 3,5-diethoxycarbonyl-1,4-dihydrocollidine ( ddc ) . mdbs also arise in cell culture after transfection with k8/k18 , ubiquitin , and p62 . major factors that determine mdb formation in vivo are the type of stress ( with oxidative stress as a major player ) , the extent of stress - induced protein misfolding and resulting chaperone , proteasome and autophagy overload , keratin 8 excess , transglutaminase activation with transamidation of keratin 8 and p62 upregulation .

Intermediate filaments in health and disease Keratins as epithelially expressed IFs Hepatic phenotype in keratin-deficient transgenic animals Keratin variants in liver disease Keratin network alteration in steatohepatitis of the alcoholic (ASH) and the non-alcoholic (NASH) type Morphology and composition of MDBs Pathogenesis of MDBs Pathologic significance of MDBs

together with the actin microfilaments and the microtubules , intermediate filaments ( ifs ) are the components of the cytoskeleton of eukaryotic cells , which is involved in the maintenance of cell shape , locomotion , intracellular organization , and transport ( bershadsky and vasiliev 1988 ; ku et al . for an overview about the new keratin nomenclature , ( 2006 ) in addition to the posttranslational modification , if function is modified and complemented through interaction with a variety of if - associated proteins ( ifap ; table 2 ; green et al . in the case of lamin deficiencies , the impairment of nuclear composition has profound impact on many aspects of normal nuclear functions such as epigenetic changes , chromatin organization or dna transcription , and repair ( dechat et al . 2008).table 2examples of if - associated proteinstype of interactionexamplesfunctionanchoringdesmoplakin , bpag1 , -dystobrevintissue architecturecytolinkerplectin , filaggrin , cellular architecturechaperoneshsp27 , -b - crystallin , hsp70protein foldingkinasespkc , cdk5if regulation , phosphate sink , cell cycleadaptor proteins14 - 3 - 3 protein , ap-3multiple effectsmembrane proteinspolycystin-1unknownapoptotic proteinstradd , tnfr2 , c - flip , caspase3/9apoptosis regulationmotor proteinsdynein , kinesinmovement of if components examples of if - associated proteins ifs are not just simple cellular scaffolds , they rather build complex signaling platforms ( pallari and eriksson 2006 ; kim and coulombe 2007 ) . in addition to that , ifs also directly participate in apoptosis regulation through binding of several apoptosis - related molecules ( marceau et al . therefore , if variants are observed in various human diseases , which reflect their tissue specific distribution , whereas only few actin and tubulin variants have been described so far , likely due to their embryolethality ( ku et al . for example , mutations in keratins 5/14 cause a blistering skin disease termed epidermolysis bullosa simplex , whereas mutations in lamins a and c result , among others , in different diseases including premature aging , cardiomyopathy , and lipodystrophy ( omary et al . the disease relevance of ifs is also highlighted by a variety of if - containing inclusion bodies , which represent the pathological hallmarks of several neurodegenerative , muscular , and other disorders ( goebel 1998 ; ross and poirier 2004 ; omary et al . among the if - related inclusions , mallory - denk bodies ( mdbs ) are the most common and also the best studied ones due to the availability of animal mdb models ( denk et al . therefore , one focus of our review will be to describe mdb as a prototype of if - related inclusion body , which should offer useful insights into the formation of if - related aggregates in multiple human diseases . keratins represent the largest subfamily of ifs consisting of > 50 unique gene product members ( schweizer et al . based on their pi , epithelial keratins can be subdivided in types i ( acidic ) and ii ( basic ) corresponding to keratins 920 ( k9k28 ) and keratins 18 plus keratins 7180 ( k1k8 ; k71k80 ) , respectively ( table 1 ; coulombe and omary 2002 ; schweizer et al . keratins are found as obligatory type i and type ii heteropolymers ( i.e. , consisting of at least one type i and one type ii keratin ) and a homozygous disruption of a keratin results in degradation of its keratin partner at the protein level ( ku and omary 2000 ; omary et al . similarly to ifs , keratins are expressed in a tissue - specific manner , with different pairs being the major cellular ifs in different cell populations ( moll et al . however , one important caveat is the fact , that k8/k18/k19 are expressed in most simple epithelial cells and are therefore not liver - specific ( moll et al . for example , elevated serum m30 titers can distinguish simple steatosis from non - alcoholic steatohepatitis ( wieckowska et al . 2006 ) and predict several important prognostic parameters in patients with chronic hepatitis c infection ( bantel et al . adult hepatocytes are unique among simple epithelial cells in that they express exclusively k8 and k18 , whereas other glandular epithelia exhibit a more complex keratin expression pattern ( omary et al . among non - epithelial cells , stellate cells express variable amounts of gfap , desmin , vimentin , and nestin dependent on their activation status , localization , and other parameters ( table 3 ; geerts 2001).table 3ifs of liver cell populationscell typeif compositionhepatocytek8/k18oval cellsk7/k8/k18/k19cholangiocytek7/k8/k18/k19kupffer cellvimentinstellate cellgfap , vimentin , desmin , nestinendothelial cellvimentinmodified from omary et al . however , k8/k18 knockout mice exhibited mild chronic hepatitis , hepatocyte fragility and were markedly more sensitive to a variety of stress conditions ( omary et al . k18 r89c mice also predisposed to development of thioacetamide - induced liver fibrosis ( strnad et al . for example , k18 s52a variant resulted in increased sensitivity to microcystin - lr - induced liver injury , whereas k8 s73a mice were predisposed to fas - induced liver apoptosis ( ku et al . the large body of evidence from animal studies showing the importance of k8/k18 for liver homeostasis led to a search for keratin mutations in patients with liver diseases . several k8/k18 variants were found to associate with the development of cryptogenic liver disease ( ku et al . , k8/k18 were shown to represent susceptibility genes for development of end - stage liver disease of multiple etiologies ( ku et al . in particular , biologically significant k8/k18 variants were found in 44 of 467 liver explants ( 12.4% ) , but only in 11 out of 349 analyzed blood bank donors , which were used as a control group ( p < 0.0001 , prevalence or3.8 ; 95% ci = 2.17 ) . furthermore , k8/k18 variants associate with liver fibrosis progression in patients with chronic hepatitis c ( strnad et al . k8 r340h represents the most common amino acid altering k8/k18 variant and it is the only one , which was significantly associated with development of end - stage liver disease ( ku et al . , we recently analyzed a large cohort of patients with acute liver failure ( alf ) . k8/k18 variants were significantly more frequent in total alf patient cohort ( 46/345 ; 13.3% ) and in patients with acetaminophen - induced alf ( 21/169 ; 12.4% ) when compared to blood bank donors ( 11/349 ; 3.7% ; p < 0.002 for both comparisons ) . in addition , transgenic mice over - expressing k8 r340 variants displayed augmented acetaminophen - induced liver toxicity . in conclusion , k8/k18 are also susceptibility genes for development of alf and k8/k18 variants may predispose to drug - induced liver injury ( strnad et al . interestingly , we recently observed k19 g17s variant in three out of 190 patients with primary biliary cirrhosis , but none was found in control blood bank donors ( 200 samples ; zhong et al . the human k8/k18 variants described above do not cause a particular liver disease per se , they just pose a risk factor for its development . the disturbance in cytoprotective function of keratins is the likely mechanisms by which k8 and k18 variants predispose to liver disorders . k8/k18 bind to several apoptotic proteins and type i keratins are established caspase substrates ( oshima 2002 ; green et al . in addition , k8/k18 serve as physiologic kinase substrates in vitro and in transgenic mice and an ablation of the k8 s73 phosphorylation site or introduction of the naturally occurring k8 g61c variant leads to increased apoptosis through increased phosphorylation of pro - apoptotic proteins ( ku et al . the keratin - mediated anti - apoptotic function may be highly relevant given the importance of apoptosis in liver disease and the pro - fibrogenic properties of elevated rate of apoptotic cell death ( friedman 2004 ; malhi et al . k8/k18 variants may also affect organelle function , as seen in k8 knockout mice , which exhibit altered mitochondrial shape , localization , and alterations in several mitochondrial proteins ( toivola et al . for example , k18 r89c mice are predisposed to fas but not tnf - induced apoptosis and the same mice develop more pronounced liver fibrosis after thioacetamide , but not after carbon tetrachloride injection ( ku et al . steatohepatitis is characterized by hepatocyte ballooning , that is , swelling and rounding with clearing of the cytoplasm , which prevails in the perivenular zone and is often associated with pericellular fibrosis , predominantly granulocytic inflammation ( satellitosis ) , steatosis ( usually of macrovesicular type ) , and cholestasis ( brunt 2004 ; lefkowitch 2005 ) . ballooned cells are often associated with granular , rope- or clump - like cytoplasmic inclusions , called mallory - denk bodies ( mdbs ) ( originally also designated alcoholic hyalin , which is , however , a misnomer since they are not specific for alcoholic etiology ) together with derangement or even disappearance of the keratin if cytoskeleton ( mallory 1911 ; denk et al . however , ballooning ( with concomitant cytoskeletal alterations ) and mdb formation are not entirely interchangeable since not all ballooned hepatocytes contain mdbs . mdbs can be reliably detected with antibodies against k8/k18 [ green and redchannel in ( c ) and ( d ) , respectively ] or p62 [ red channel in ( c ) ] , whereas only some mdbs stain with antibodies to phosphorylated keratins such as k8 ps431 antibody [ green channel in ( d ) ] . mdbs can be reliably detected with antibodies against k8/k18 [ green and redchannel in ( c ) and ( d ) , respectively ] or p62 [ red channel in ( c ) ] , whereas only some mdbs stain with antibodies to phosphorylated keratins such as k8 ps431 antibody [ green channel in ( d ) ] . mdbs can also be detected after intestinal bypass surgery for morbid obesity , in chronic cholestasis , particularly in late stages of primary biliary cirrhosis , wilson disease and other types of copper toxicosis , various metabolic disturbances , and hepatocellular neoplasms ( mller et al . however , even in potentially mdb - forming liver diseases , mdbs are found only in a subset of patients , partially ( but not completely ) depending on the sensitivity of the detection method used . immunohistochemical staining with p62 antibody visualizes the presence of multiple irregularly shaped aggregates in patients with alcoholic steatohepatitis ( a ) , non - alcoholic steatohepatitis ( b ) , indian childhood cirrhosis and ( c ) , idiopathic copper toxicosis ( d ) mdbs are seen in various human liver diseases . immunohistochemical staining with p62 antibody visualizes the presence of multiple irregularly shaped aggregates in patients with alcoholic steatohepatitis ( a ) , non - alcoholic steatohepatitis ( b ) , indian childhood cirrhosis and ( c ) , idiopathic copper toxicosis ( d ) this suggests that mdbs require either a specific pathogenetic constellation or genetic predisposition for its formation , which is present only in a subset of patients . for example , a low percentage of hepatocytes express keratin 7 and to a lesser extent keratin 19 which indicate that these cells acquire features of precursor cells which normally express keratin 8 , 18 , 7 , and 19 during regeneration ( van eyken et al . mdbs may coincide with another type of cytoplasmic inclusions , termed intracellular hyaline bodies ( ihbs ) , which share several components with mdbs , but do not contain keratins ( stumptner et al . also in other studies , hepatocellular ballooning and mdb formation was positively correlated with disease progression , development of fibrosis , and cirrhosis and liver - related mortality ( orrego et al . small mdbs arise in association with if bundles throughout the cytoplasm , whereas larger mdbs are often seen in the perinuclear region ( denk et al . in addition , mdbs with an electron dense granular to amorphous center ( designated type 3 ) are seen predominantly in older inclusions , and filaments in parallel arrangement were also described ( designated as type i ) but seem to be exceedingly rare ( yokoo et al . keratins 8 and 18 , sequestosome 1/p62 ( p62 ) and ubiquitin are major , and low and high molecular weight heat shock proteins ( hsp 70 , hsp 25 ) , but also proteins of the protein degradation machinery , are minor constituents ( denk et al . the list of mdb components is likely to grow since , in addition to intrinsic components , proteins non - specifically incorporated in the aggregate have to be expected . mdbs can be induced under standardized conditions and their fate followed in mouse liver by chronic griseofulvin or 3,5-diethoxycarbonyl-1,4-dihydrocollidine ( ddc ) administration ( denk et al . samples from mouse fed ddc for 12 weeks ( d ) and from a patient with alcoholic steatohepatitis ( b ) exhibit multiple irregularly shaped inclusion bodies , which appear yellow due to presence of both epitopes . note that ddc feeding leads to deposition of protoporphyrin , which can be seen as occasional blue pigment in ( d ) mdbs formed in ddc - fed animals resemble inclusion bodies observed in human diseases . samples from mouse fed ddc for 12 weeks ( d ) and from a patient with alcoholic steatohepatitis ( b ) exhibit multiple irregularly shaped inclusion bodies , which appear yellow due to presence of both epitopes . note that ddc feeding leads to deposition of protoporphyrin , which can be seen as occasional blue pigment in ( d ) in patients , mdb formation is usually a chronic process requiring several years of alcohol intoxication or metabolic imbalance , that is , in association with the metabolic syndrome , wilson disease , other metabolic disorders , and chronic cholestasis . an exception is idiopathic copper toxicosis of children in which end stage liver disease associated with mdb formation occurs at very early age ( mller et al . surprisingly , in alcoholics recovered from ash , an alcohol excess may almost instantaneously lead to mdb recurrence , a situation which has been compared to an immunologic response and termed toxic memory does not only reproduce the structural and morphological features seen in humans , but also the phenomenon of toxic memory mdb formation in mice is reversible , since mdbs disappear after recovery on standard diet for 1 month . rapid mdbs formation in primed mice is rather non - specific , since it can be triggered by a variety of stress conditions including colchicine ( but not by lumicolchicine ) , bile acids , bile duct ligation , several other toxins , and proteasome inhibitors which were unable to induce mdbs in the nave animal ( zatloukal et al . the mdb - inducing conditions both in humans and mice cause elevated levels of oxidative stress ( tephly et al . , mdb formation and ddc effects can be effectively attenuated by feeding s - adenosylmethionine , that is a compound involved in methyl group transfer ( li et al . mdb - causing agents typically generate extensive amount of oxidative stress with increased protein misfolding . dysbalanced k8/k18 expression precedes mdb formation , likely increases keratin misfolding and predisposes to posttranslational modifications , which may interfere with keratin refolding and/or repair . on the other hand , autophagy is upregulated during mdb formation in mice and additional stimulation of autophagy attenuates mdb formation in certain conditions . of note , supplementation of s - adenosylmethionine or mitochondrially targeted antioxidants effectively diminishes mdb formation , thereby pointing to a central role of oxidative stress in mdb generation mdbs formation results from a complex interplay of several contributing factors . on the other hand , autophagy is upregulated during mdb formation in mice and additional stimulation of autophagy attenuates mdb formation in certain conditions . therefore , mitochondrial oxidative stress seems to be involved in mdb formation in mice which is in good concordance to the mitochondrial dysfunction seen both in ash and nash patients ( pessayre 2007 ; mantena et al . however , the exclusive mdb inducing property of k8 in vivo can not be reproduced in vitro , where aggregates resembling mdbs can also be produced by transfection of k18 ( nakamichi et al . p38 kinase - induced keratin network reorganization might be a necessary prerequisite for mdb formation , since p38 kinase inhibition prevented mdb formation in vitro ( nan et al . since k8 is a much better in vitro transglutaminase substrate than k18 and highly cross - linked proteins found in mdb - forming mice contain k8 , but not k18 , it was suggested that excessive k8 gets preferentially transamidated and acts as a nucleus for mdb formation . in cell culture experiments , protein aggregates resembling mdbs formed only after p62 co - transfection , but not when k8 , k18 , and ubiquitin were transfected alone or in combination ( stumptner et al . furthermore , p62 inhibition attenuated , whereas p62 overexpression enhanced mdb formation in ddc - primed hepatocytes ( nan et al . furthermore , autophagic degradation is upregulated in ddc - fed mice and its further stimulation with rapamycin attenuates spontaneous mdb formation in k8 overexpressing mice ( harada et al . in addition , an aberrant form of ubiquitin , termed [ ubb + 1 ] arising as a consequence of molecular misreading , is observed in mdbs and may inhibit proteasomal function ( mcphaul et al . in principle , cytoplasmic protein aggregates can be beneficial , detrimental or inert depending on the context . it can be expected , therefore , that in different cell systems and pathologic conditions inclusion bodies differ regarding their cellular effects and consequences .

copd is characterized by persistent airflow limitation related to a chronic inflammatory response in the airways and the lung derived from inhaling toxic particles or gases for a long - term exposure.1 this condition is the fourth leading cause of death worldwide , and it represents a major cause of chronic morbidity and economic and social burden.1 thus , some studies have focused on calculating the cost of treatment for copd in spain . de miguel diez et al2 showed that the total treatment cost per copd patient per year was ~2,000 . in that study , 40% of the total expenses were produced by the hospitalization component , and the drug costs represented almost the 26% . masa et al3 also conducted a study on the copd treatment costs in spain and found that although the cost per patient was not calculated , the division of expenses was similar to the one obtained by miguel diez et al , corresponding 41% to hospitalization and 37% to pharmacological treatment . in spain , 10.2% of individuals between the ages of 40 and 80 years have copd , whereas a significant proportion of the population remains undiagnosed.4 this high prevalence is worsened by the fact that ~60% of patients with copd do not adhere to the prescribed therapy.5,6 medication adherence is a complex issue that can be defined as the degree to which a patient s medication - taking behavior and/or executing lifestyle changes correspond with agreed recommendations from a health care professional with respect to timing , dosage , and frequency.7 medication adherence is a key factor for controlling the progression of chronic disease . although inhaled corticosteroids ( ics ) and long - acting 2-agonist fixed - dose combinations ( fdcs ) have shown to relieve copd symptoms similarly , inhalation technique might affect adherence and hence efficacy of pharmacological treatment . common , real - life errors during the inhalation process include holding inhaler upright , inhaling deeply and quickly , and breath - holding.8,9 delivering sufficient drug dose to the lungs is crucial to achieve a good medication efficacy so as to avoid poor health outcomes.10 as in other chronic diseases , the previous studies have demonstrated low medication adherence in copd patients . a study distributed by the world health organization reported that in patients on long - term pharmacotherapy the adherence was half or less.11 recent studies , based on the medication adherence in copd patients , additionally show a poor adherence . breekveldt - postma et al12 reported that approximately half of the patients stopped their treatment with ics within 6 months , and only 18% persevered > 1 year . bender et al13 studied the utilization of an inhaler ( fluticasone propionate / salmeterol ) and detected that only 9% of patients proceed with the treatment for more than 1 year . cramer et al14 identified that the adherence to copd medications was low over a wide range of medications , ranging from 57 to 96 mean days until discontinuation over a year of observation time . medication regimens for patients with copd are notably susceptible to adherence problems due to the chronicity of the disease , the use of multiple medications , and the periods of symptom remission and patients comorbidities . given the developments in inhaler devices ( eg , pressurized metered - dose inhalers to dry powder inhalers [ dpis ] ) , there is still a high unmet need to have more intuitive inhalers that are easier to use for patients . this is also recognized by the decision makers and physicians.15,16 the ease of use of the device according to patient comorbidities and preferences may contribute to a better medication adherence.7 the present study focused on developing a budget impact model ( bim ) in order to estimate the economic impact that arises from a growing presence in the market of duoresp spiromax for the maintenance therapy with budesonide / formoterol fdc . spiromax , a new inhalation device , helps to reduce common errors that could occur while using the device which could limit the direct drug delivery to the lungs.17 the model was elaborated from the perspective of the spanish national honor society ( nhs ) , and regional data from 5 spanish autonomous communities ( hereafter referred as regions ) were also included : andalusia , catalonia , galicia , madrid , and valencia . this study did not require any ethics committee review as the authors did not have access to patient - level data . the bim was developed in microsoft excel from the perspective of the spanish nhs with a 4-year time horizon ( 20152018 ) . the present analysis focused on the population from spain and 5 spanish regions : andalusia , catalonia , galicia , madrid , and valencia . budesonide / formoterol fdc delivered by turbuhaler was considered the relevant comparator for evaluating the budget impact of the introduction of budesonide / formoterol fdc delivered by spiromax . although fdcs such as beclomethasone / formoterol and fluticasone / salmeterol are available , only budesonide / formoterol fdc was included in the present analysis because changes in patients regimens were not hypothesized . input data on health care resources such as medical visits and length of hospitalization were obtained by expert panel consultation from various spanish hospitals . therefore , the model analyzed health care resource utilization per patient based on his / her daily maintenance treatment for copd and the number of days with events such as hospitalizations , visits to the emergency room , primary care ( pc ) visits , and specialist visits . these data were obtained from daily clinical practice , given that there is no published literature on how suboptimal adherence related to inhalation technique affects health care resource utilization . all the costs were estimated in eur 2015 , and a discount rate of 3% was assumed . it should be noted that when this study was being completed , there was a significant change in drug prices included in this analysis . from october 2015 onward , prices of both the drugs were set at the same level by the spanish ministry of health ; therefore , price effect was no longer useful to calculate the economic impact of the introduction of duoresp spiromax.18 the model focused on diagnosed patients with copd , who followed an inhaler maintenance therapy . ims health reported the proportion of patients using budesonide / formoterol fdc delivered by a dpi according to spanish national and regional sales data . therefore , the authors were able to estimate the target population , given the data on symbicort / rilast turbuhaler utilization in spain and 5 spanish regions . data on market uptake of duoresp spiromax were provided directly by teva pharma , spain . the model provided estimates for the annual costs per patient and the total direct costs of treatment from predefined market shares and other input parameters . the total cost estimated for copd patients is based on drug costs and medical resources , such as medical visits , hospitalization , emergency visits , and monitoring tests . a univariate deterministic sensitivity analysis was conducted to confirm the model robustness and to identify the parameters having the greatest influence on the results . different market shares , adherence rates , and copd severity were assessed for the analysis . furthermore , a change in the age of the target population was analyzed , considering the population older than 40 years ( greater copd prevalence ) instead of the adult population . this study did not require any ethics committee review as the authors did not have access to patient - level data . the bim was developed in microsoft excel from the perspective of the spanish nhs with a 4-year time horizon ( 20152018 ) . the present analysis focused on the population from spain and 5 spanish regions : andalusia , catalonia , galicia , madrid , and valencia . budesonide / formoterol fdc delivered by turbuhaler was considered the relevant comparator for evaluating the budget impact of the introduction of budesonide / formoterol fdc delivered by spiromax . although fdcs such as beclomethasone / formoterol and fluticasone / salmeterol are available , only budesonide / formoterol fdc was included in the present analysis because changes in patients regimens were not hypothesized . input data on health care resources such as medical visits and length of hospitalization were obtained by expert panel consultation from various spanish hospitals . therefore , the model analyzed health care resource utilization per patient based on his / her daily maintenance treatment for copd and the number of days with events such as hospitalizations , visits to the emergency room , primary care ( pc ) visits , and specialist visits . these data were obtained from daily clinical practice , given that there is no published literature on how suboptimal adherence related to inhalation technique affects health care resource utilization . all the costs were estimated in eur 2015 , and a discount rate of 3% was assumed . it should be noted that when this study was being completed , there was a significant change in drug prices included in this analysis . from october 2015 onward , prices of both the drugs were set at the same level by the spanish ministry of health ; therefore , price effect was no longer useful to calculate the economic impact of the introduction of duoresp spiromax.18 the model focused on diagnosed patients with copd , who followed an inhaler maintenance therapy . ims health reported the proportion of patients using budesonide / formoterol fdc delivered by a dpi according to spanish national and regional sales data . therefore , the authors were able to estimate the target population , given the data on symbicort / rilast turbuhaler utilization in spain and 5 spanish regions . data on market uptake of duoresp spiromax were provided directly by teva pharma , spain . the model provided estimates for the annual costs per patient and the total direct costs of treatment from predefined market shares and other input parameters . the total cost estimated for copd patients is based on drug costs and medical resources , such as medical visits , hospitalization , emergency visits , and monitoring tests . a univariate deterministic sensitivity analysis was conducted to confirm the model robustness and to identify the parameters having the greatest influence on the results . different market shares , adherence rates , and copd severity were assessed for the analysis . furthermore , a change in the age of the target population was analyzed , considering the population older than 40 years ( greater copd prevalence ) instead of the adult population . the study population was selected based on spanish national and regional prevalence of copd from the literature review.19,20 prevalence estimates of copd were extrapolated to the obtained estimates of adult population , taking into account projections performed by the spanish national institute of statistics.21 we considered that 73% of copd patients were not diagnosed.22 finally , a percentage was applied to distinguish the patients using an fdc , and among them , the number of patients who used budesonide / formoterol fdc delivered by a dpi was determined . the proxy for capturing these patients was the percentage of patients using symbicort / rilast turbuhaler ( ims health . national sales data 20132014 for copd maintenance treatment with fixed - dose - combinations for spain , unpublished data , 2015 ) . a panel of 5 clinical experts in pneumology and allergy and also a general practitioner from different spanish hospitals was asked to report whether critical inhaler misuses were observed in daily practice . the incorrect dose loading and keeping the inhaler inclined no more than 45 from the vertical axis were the most common errors in patients taking symbicort turbuhaler and rilast turbuhaler versus duoresp spiromax ( table 1 ) . these results were not used for calculations , but these were relevant to confirm that the misuse of inhaler was found in clinical practice . gathering input data on health care resource utilization related to a potential enhancement of adherence was the basis to estimate the economic impact of the introduction of spiromax in spain . the proportion of emergency room visits and hospitalizations that might be due to errors in the use of inhaler technique were reported to be 4.92% and 3.26% , respectively . moreover , according to the experts point of view , there are some differences in the number of emergency room visits and hospitalizations between patients using turbuhaler and patients using spiromax ( table 2 ) . there were some difficulties in obtaining directly the proportions of pc visits and specialist visits per patient . pc visits implies that the health care providers need to deal with different patient concerns , which might not be related only to the use of inhaler devices . such situations made it necessary to ask for the number of pc and specialist visits that a regular copd patient had undergone . on average , the number of patients who used turbuhaler should not be different from those using spiromax as they share indication with the same fdc of budesonide / formoterol.23 therefore , it is plausible to imply that deviation between health care resource utilization associated with spiromax versus turbuhaler might be related to the inhalation device . finally , the present study included other health care resources such as spirometry and thorax radiograph.3 some studies showed that a greater number of monitoring tests such as blood test and electrocardiogram were taken , causing the budget to exceed the cost of a regular monitoring test , which are most efficient to make a prognostic of copd.24 therefore , it can be concluded that the cost of monitoring tests could be underestimated in the present model . drug costs were obtained from a spanish database of pharmacists.25 cost of budesonide / formoterol fdc delivered by turbuhaler or spiromax was included exclusively as a drug cost in order to simplify the analysis . costs of health care resources included in this analysis were based on spanish regional tariff lists.2637 at the national level , costs of health care resources correspond to the mean of 12 regions , including those 5 regions included in this analysis . for each scenario , the economic impact for the time period of 20152018 was calculated based on the estimated number of patients who are treated with budesonide / formoterol fdc delivered by turbuhaler each year , the annual average cost per patient for each treatment option , the target population , and the market shares for both products included in this study . in the current scenario , duoresp spiromax this current scenario was compared with an alternative scenario in which the economic impact considered the introduction of duoresp spiromax and its effects toward medication adherence . the market uptake of duoresp spiromax increased gradually over the course of the 4 years ( table 3 ) . the study population was selected based on spanish national and regional prevalence of copd from the literature review.19,20 prevalence estimates of copd were extrapolated to the obtained estimates of adult population , taking into account projections performed by the spanish national institute of statistics.21 we considered that 73% of copd patients were not diagnosed.22 finally , a percentage was applied to distinguish the patients using an fdc , and among them , the number of patients who used budesonide / formoterol fdc delivered by a dpi was determined . the proxy for capturing these patients was the percentage of patients using symbicort / rilast turbuhaler ( ims health . national sales data 20132014 for copd maintenance treatment with fixed - dose - combinations for spain , unpublished data , 2015 ) . a panel of 5 clinical experts in pneumology and allergy and also a general practitioner from different spanish hospitals was asked to report whether critical inhaler misuses were observed in daily practice . the incorrect dose loading and keeping the inhaler inclined no more than 45 from the vertical axis were the most common errors in patients taking symbicort turbuhaler and rilast turbuhaler versus duoresp spiromax ( table 1 ) . these results were not used for calculations , but these were relevant to confirm that the misuse of inhaler was found in clinical practice . gathering input data on health care resource utilization related to a potential enhancement of adherence was the basis to estimate the economic impact of the introduction of spiromax in spain . the proportion of emergency room visits and hospitalizations that might be due to errors in the use of inhaler technique were reported to be 4.92% and 3.26% , respectively . moreover , according to the experts point of view , there are some differences in the number of emergency room visits and hospitalizations between patients using turbuhaler and patients using spiromax ( table 2 ) . there were some difficulties in obtaining directly the proportions of pc visits and specialist visits per patient . pc visits implies that the health care providers need to deal with different patient concerns , which might not be related only to the use of inhaler devices . such situations made it necessary to ask for the number of pc and specialist visits that a regular copd patient had undergone . on average , the number of patients who used turbuhaler should not be different from those using spiromax as they share indication with the same fdc of budesonide / formoterol.23 therefore , it is plausible to imply that deviation between health care resource utilization associated with spiromax versus turbuhaler might be related to the inhalation device . finally , the present study included other health care resources such as spirometry and thorax radiograph.3 some studies showed that a greater number of monitoring tests such as blood test and electrocardiogram were taken , causing the budget to exceed the cost of a regular monitoring test , which are most efficient to make a prognostic of copd.24 therefore , it can be concluded that the cost of monitoring tests could be underestimated in the present model . drug costs were obtained from a spanish database of pharmacists.25 cost of budesonide / formoterol fdc delivered by turbuhaler or spiromax was included exclusively as a drug cost in order to simplify the analysis . costs of health care resources included in this analysis were based on spanish regional tariff lists.2637 at the national level , costs of health care resources correspond to the mean of 12 regions , including those 5 regions included in this analysis . for each scenario , the economic impact for the time period of 20152018 was calculated based on the estimated number of patients who are treated with budesonide / formoterol fdc delivered by turbuhaler each year , the annual average cost per patient for each treatment option , the target population , and the market shares for both products included in this study . in the current scenario , this current scenario was compared with an alternative scenario in which the economic impact considered the introduction of duoresp spiromax and its effects toward medication adherence . the market uptake of duoresp spiromax increased gradually over the course of the 4 years ( table 3 ) . it was estimated that , in 2015 , 130,777 adults in spain suffered from copd , and they were treated with budesonide / formoterol delivered by a dpi ( in this study turbuhaler ; table 4 ) . however , this population decreased over time and will be 128,618 in 2018 . the same pattern was observed for target population in catalonia , galicia , madrid , and valencia , whereas the number of patients in andalusia increased slightly between 2015 and 2018 ( table 4 ) . based on the annual drug cost and health care resource use per patient , the total treatment cost per patient was estimated in eur 2015 . with the annual average cost per patient for each treatment option , the target population , and the market shares for both products included in this study , the overall economic impact of the management of copd for the time period of 20152018 was obtained . in the current scenario for spain , the total economic impact of treatment with budesonide / formoterol fdc delivered by turbuhaler was estimated to be 108.76 million , 110.44 million , 114.61 million , and 121.73 million for the years 2015 , 2016 , 2017 , and 2018 , respectively ( table 5 ) . in the alternative scenario for spain , with the market share of duoresp spiromax increasing annually from 2015 , matched by a reduction in the share of symbicort turbuhaler and rilast turbuhaler , the total economic impact was estimated to be 107.92 million , 109.03 million , 112.79 million , and 119.48 million for 2015 , 2016 , 2017 , and 2018 , respectively ( table 5 ) . overall , the total budget savings for spain were expected to be 6.01 million over the next 4 years ( tables 5 and 6 ) . results of the region - specific analysis vary widely . in the current scenario , the total economic impact was expected to be 65.49 million , 55.58 million , 30.45 million , 63.30 million , and 32.94 million in andalusia , catalonia , galicia , madrid , and valencia , respectively , for the time period of 20152018 ( table 5 ) . the economic impact of treating copd was reduced in all spanish regions with the introduction of spiromax , alternative scenario , over the time period of 20152018 . thus , total costs were estimated to be 64.59 million for andalusia , 54.84 million for catalonia , 29.98 million for galicia , 62.54 million for madrid , and 32.44 million for valencia ( tables 5 and 6 ) . the comparison between the current scenario and the alternative scenario led to savings in all 5 spanish regions . total savings during the time period of 20152018 were estimated to be 902,133 , 740,520 , 464,281 , 748,996 , and 495,812 for andalusia , catalonia , galicia , madrid , and valencia , respectively . results were obtained through the reduction of health care resources , especially fewer days of hospital stay and avoided pc visits and emergency room visits ( table 6 ) , the reduction in pc visits related to problems with the inhaler device being more important . finally , the univariate deterministic sensitivity analysis confirmed the robustness of the model . variations in the most sensitive parameters , such as copd severity or the adherence rate , do not lead to significant changes in the results of the analysis , since in all cases savings were obtained for both spain and the 5 regions . in addition , when changing the age of the population considered , savings were obtained with the alternative scenario compared with the current one . thus , the savings obtained ranged between 9.4 million and 606,641 for spain and 1.7 million and 50,077 for the 5 spanish regions ( table 7 ) . this study compared the costs of budesonide / formoterol fdc delivered by two different inhalation devices , spiromax and turbuhaler , to estimate the budget impact for the treatment of copd in spain and the 5 regions . results of the budget impact analysis suggest that the increasing use of spiromax would result in a 4-year budget savings for the spanish nhs of 6.01 million at the national level . results suggest that savings summed up to 902,133 , 740,520 , 464,281 , 748,996 , and 495,812 for andalusia , catalonia , galicia , madrid , and valencia , respectively . lewis et al38 conducted an assessment to determine the budget impact of using spiromax instead of turbuhaler to manage copd in adult patients in the uk . they also analyzed the potential cost benefit of the improved inhalation technique . in the lewis et al study , the model predicted total drug cost savings of 36.09 million and further savings of 3.5 million due to improvement in inhalation technique . this assessment was been carried out by torvinen et al39 on the italian copd population . their model also anticipated a total drug cost savings of 53.66 million and additional savings of 4.12 million because of the progression in inhalation technique . regarding other inhalation devices , nicolai et al40 assessed the potential economic impact of introducing an inhaler with improved features compared to spiriva handihaler to treat copd in the uk . the potential budgetary impact achieved by using the new inhaler instead of handihaler is calculated as 104.91 per patient and 16.69 million for the uk copd population per year . dealing with this chronic disease remains complex due to several problems such as the chronicity of the disease , the use of multiple medications , the periods of symptom remission , comorbidities that limit the movement such as arthritis and osteoarthritis , and the lack of adherence related to the inhalation technique . inhaled medication is typically prescribed in a maintenance therapy using fdcs , considering that single - inhaler combination regimens provide improved symptom control and slow down the progression of the disease.41,42 although current prescription guidelines contribute to improve the quality of life of copd patients , it was reported that up to 85% of the patients use their inhaler ineffectively , and this was associated with low medication adherence.41,43,44 our estimates suggest that the introduction of spiromax could result in savings by reducing health care resource utilization related to suboptimal medication adherence.45,46 this new inhalation device helps to reduce common utilization errors such as dose preparation errors , adequate flow rates , or even environmental conditions that might limit the delivery of the drug directly to the lungs , which would be closer to patients real - life situations.46 in addition , the current model can obtain region - specific results , which suggest that the highest populated regions correspond to those with greater savings for the health care budget . , these regions were expected to obtain higher savings if adherence improves in the patients diagnosed with copd . it was also explored that the breakdown of overall savings and the savings due to avoided pc visits has shown to yield significant monetary savings . copd is mostly found in a population older than 40 years , and the chronicity of the disease makes it relevant to focus effort in new strategies to solve the current problems of suboptimal adherence.43,47 when clinical data were not available , we used data from the literature . due to differences in study population , geographic area , patients health status , and additional factors , some input variables may not be generalizable to all 5 regions . moreover , the first assumption in the model was a growing market share of duoresp spiromax along with a reduction of symbicort turbuhaler and rilast turbuhaler utilization . nevertheless , the estimate of the budget impact under this scenario may not predict the real - world changes . furthermore , other formulae prescriptions different from budesonide / formoterol were not included , which could be an alternative for duoresp spiromax . regarding the study results , it was found that gains in adherence generated savings for the health care budgets . although these amounts could be considered conservative , they are adjusted only to be related with inhalation technique problems , and not with the whole adherence problem , which is linked with many factors.41,44 it is worth mentioning that difficulties in the use of inhaler devices were exacerbated in elderly patients , whose reduced inhalation effort leads to a poor drug release.48 a greater impact on the copd economic budget was expected because of the aging population in spain . furthermore , it was also observed that underdiagnosis is a current problem in spain , with up to 73% of copd potential population remain unaware of their health status.45 thus , copd health care expenditure could be even higher since a low proportion of these patients was actually treated . the results from this analysis suggest that the introduction of duoresp spiromax would result in a 6.01 million decrease in spain s overall budget in the period 20152018 associated with a lower health care resource utilization costs . region - specific data resulted in savings in 5 spanish regions of study which sum up to 902,133 , 740,520 , 464,281 , 748,996 , and 495,812 for andalusia , catalonia , galicia , madrid , and valencia , respectively . copd treatment regimens that increase the probability of higher medication adherence rates would be expected to contribute to improved disease management and to have an impact on the utilization of health care resources .

objectivethe objective of this study was to estimate the economic impact of the introduction of duoresp spiromax , budesonide / formoterol fixed - dose combination ( fdc ) , focusing on an increase in medication adherence due to an enhancement of the inhalation technique for the treatment of copd patients in spain and 5 regions including andalusia , catalonia , galicia , madrid , and valencia.methodsa 4-year budget impact model was developed for the time period of 20152018 . this study aimed at evaluating the budget impact associated with the introduction of duoresp spiromax in comparison with symbicort turbuhaler and rilast turbuhaler . national and regional data on copd prevalence were obtained from the literature . input data on health care resource utilization were obtained by clinical consultation . resource included primary care visits , specialist visits , hospitalization , and emergency room visits as well as the length of hospital stay . based on both pharmacological and health care resource costs , overall annual treatment cost per patient was estimated in eur 2015.resultsit was calculated that 130,777 adults were treated with budesonide / formoterol fdc delivered by a dry powder inhaler , turbuhaler , in spain in 2015 . however , the target population decreases over the next 4 years . this pattern was observed in 4 regions , but for andalusia , the treated population increased slightly . the overall budget savings in spain with the market share of duoresp spiromax were estimated to be 6.01 million for the time period of 20152018 . region - specific data resulted in savings of 902,133 in andalusia , 740,520 in catalonia , 464,281 in galicia , 748,996 in madrid , and 495,812 in valencia for the time period of 20152018.conclusionthe introduction of budesonide / formoterol fdc delivered by spiromax for copd treatment is likely to contribute in a reduction of health care costs for spain and in 5 spanish regions . this model forecasts that spain and these 5 spanish regions were likely to have savings , which might be due to fewer days of hospitalization , avoided emergency room , and primary care visits .

Introduction Methods Model development and structure Sensitivity analysis Model input variables Target population Adherence, medical resource utilization, and costs Budgetary impact analysis Results Discussion Limitations Conclusion

masa et al3 also conducted a study on the copd treatment costs in spain and found that although the cost per patient was not calculated , the division of expenses was similar to the one obtained by miguel diez et al , corresponding 41% to hospitalization and 37% to pharmacological treatment . in spain , 10.2% of individuals between the ages of 40 and 80 years have copd , whereas a significant proportion of the population remains undiagnosed.4 this high prevalence is worsened by the fact that ~60% of patients with copd do not adhere to the prescribed therapy.5,6 medication adherence is a complex issue that can be defined as the degree to which a patient s medication - taking behavior and/or executing lifestyle changes correspond with agreed recommendations from a health care professional with respect to timing , dosage , and frequency.7 medication adherence is a key factor for controlling the progression of chronic disease . common , real - life errors during the inhalation process include holding inhaler upright , inhaling deeply and quickly , and breath - holding.8,9 delivering sufficient drug dose to the lungs is crucial to achieve a good medication efficacy so as to avoid poor health outcomes.10 as in other chronic diseases , the previous studies have demonstrated low medication adherence in copd patients . medication regimens for patients with copd are notably susceptible to adherence problems due to the chronicity of the disease , the use of multiple medications , and the periods of symptom remission and patients comorbidities . given the developments in inhaler devices ( eg , pressurized metered - dose inhalers to dry powder inhalers [ dpis ] ) , there is still a high unmet need to have more intuitive inhalers that are easier to use for patients . this is also recognized by the decision makers and physicians.15,16 the ease of use of the device according to patient comorbidities and preferences may contribute to a better medication adherence.7 the present study focused on developing a budget impact model ( bim ) in order to estimate the economic impact that arises from a growing presence in the market of duoresp spiromax for the maintenance therapy with budesonide / formoterol fdc . spiromax , a new inhalation device , helps to reduce common errors that could occur while using the device which could limit the direct drug delivery to the lungs.17 the model was elaborated from the perspective of the spanish national honor society ( nhs ) , and regional data from 5 spanish autonomous communities ( hereafter referred as regions ) were also included : andalusia , catalonia , galicia , madrid , and valencia . the present analysis focused on the population from spain and 5 spanish regions : andalusia , catalonia , galicia , madrid , and valencia . budesonide / formoterol fdc delivered by turbuhaler was considered the relevant comparator for evaluating the budget impact of the introduction of budesonide / formoterol fdc delivered by spiromax . input data on health care resources such as medical visits and length of hospitalization were obtained by expert panel consultation from various spanish hospitals . therefore , the model analyzed health care resource utilization per patient based on his / her daily maintenance treatment for copd and the number of days with events such as hospitalizations , visits to the emergency room , primary care ( pc ) visits , and specialist visits . these data were obtained from daily clinical practice , given that there is no published literature on how suboptimal adherence related to inhalation technique affects health care resource utilization . all the costs were estimated in eur 2015 , and a discount rate of 3% was assumed . from october 2015 onward , prices of both the drugs were set at the same level by the spanish ministry of health ; therefore , price effect was no longer useful to calculate the economic impact of the introduction of duoresp spiromax.18 the model focused on diagnosed patients with copd , who followed an inhaler maintenance therapy . ims health reported the proportion of patients using budesonide / formoterol fdc delivered by a dpi according to spanish national and regional sales data . therefore , the authors were able to estimate the target population , given the data on symbicort / rilast turbuhaler utilization in spain and 5 spanish regions . the total cost estimated for copd patients is based on drug costs and medical resources , such as medical visits , hospitalization , emergency visits , and monitoring tests . furthermore , a change in the age of the target population was analyzed , considering the population older than 40 years ( greater copd prevalence ) instead of the adult population . the present analysis focused on the population from spain and 5 spanish regions : andalusia , catalonia , galicia , madrid , and valencia . budesonide / formoterol fdc delivered by turbuhaler was considered the relevant comparator for evaluating the budget impact of the introduction of budesonide / formoterol fdc delivered by spiromax . input data on health care resources such as medical visits and length of hospitalization were obtained by expert panel consultation from various spanish hospitals . therefore , the model analyzed health care resource utilization per patient based on his / her daily maintenance treatment for copd and the number of days with events such as hospitalizations , visits to the emergency room , primary care ( pc ) visits , and specialist visits . these data were obtained from daily clinical practice , given that there is no published literature on how suboptimal adherence related to inhalation technique affects health care resource utilization . all the costs were estimated in eur 2015 , and a discount rate of 3% was assumed . from october 2015 onward , prices of both the drugs were set at the same level by the spanish ministry of health ; therefore , price effect was no longer useful to calculate the economic impact of the introduction of duoresp spiromax.18 the model focused on diagnosed patients with copd , who followed an inhaler maintenance therapy . ims health reported the proportion of patients using budesonide / formoterol fdc delivered by a dpi according to spanish national and regional sales data . therefore , the authors were able to estimate the target population , given the data on symbicort / rilast turbuhaler utilization in spain and 5 spanish regions . the total cost estimated for copd patients is based on drug costs and medical resources , such as medical visits , hospitalization , emergency visits , and monitoring tests . the study population was selected based on spanish national and regional prevalence of copd from the literature review.19,20 prevalence estimates of copd were extrapolated to the obtained estimates of adult population , taking into account projections performed by the spanish national institute of statistics.21 we considered that 73% of copd patients were not diagnosed.22 finally , a percentage was applied to distinguish the patients using an fdc , and among them , the number of patients who used budesonide / formoterol fdc delivered by a dpi was determined . the incorrect dose loading and keeping the inhaler inclined no more than 45 from the vertical axis were the most common errors in patients taking symbicort turbuhaler and rilast turbuhaler versus duoresp spiromax ( table 1 ) . gathering input data on health care resource utilization related to a potential enhancement of adherence was the basis to estimate the economic impact of the introduction of spiromax in spain . the proportion of emergency room visits and hospitalizations that might be due to errors in the use of inhaler technique were reported to be 4.92% and 3.26% , respectively . on average , the number of patients who used turbuhaler should not be different from those using spiromax as they share indication with the same fdc of budesonide / formoterol.23 therefore , it is plausible to imply that deviation between health care resource utilization associated with spiromax versus turbuhaler might be related to the inhalation device . finally , the present study included other health care resources such as spirometry and thorax radiograph.3 some studies showed that a greater number of monitoring tests such as blood test and electrocardiogram were taken , causing the budget to exceed the cost of a regular monitoring test , which are most efficient to make a prognostic of copd.24 therefore , it can be concluded that the cost of monitoring tests could be underestimated in the present model . drug costs were obtained from a spanish database of pharmacists.25 cost of budesonide / formoterol fdc delivered by turbuhaler or spiromax was included exclusively as a drug cost in order to simplify the analysis . costs of health care resources included in this analysis were based on spanish regional tariff lists.2637 at the national level , costs of health care resources correspond to the mean of 12 regions , including those 5 regions included in this analysis . for each scenario , the economic impact for the time period of 20152018 was calculated based on the estimated number of patients who are treated with budesonide / formoterol fdc delivered by turbuhaler each year , the annual average cost per patient for each treatment option , the target population , and the market shares for both products included in this study . in the current scenario , duoresp spiromax this current scenario was compared with an alternative scenario in which the economic impact considered the introduction of duoresp spiromax and its effects toward medication adherence . the market uptake of duoresp spiromax increased gradually over the course of the 4 years ( table 3 ) . the study population was selected based on spanish national and regional prevalence of copd from the literature review.19,20 prevalence estimates of copd were extrapolated to the obtained estimates of adult population , taking into account projections performed by the spanish national institute of statistics.21 we considered that 73% of copd patients were not diagnosed.22 finally , a percentage was applied to distinguish the patients using an fdc , and among them , the number of patients who used budesonide / formoterol fdc delivered by a dpi was determined . the incorrect dose loading and keeping the inhaler inclined no more than 45 from the vertical axis were the most common errors in patients taking symbicort turbuhaler and rilast turbuhaler versus duoresp spiromax ( table 1 ) . gathering input data on health care resource utilization related to a potential enhancement of adherence was the basis to estimate the economic impact of the introduction of spiromax in spain . the proportion of emergency room visits and hospitalizations that might be due to errors in the use of inhaler technique were reported to be 4.92% and 3.26% , respectively . on average , the number of patients who used turbuhaler should not be different from those using spiromax as they share indication with the same fdc of budesonide / formoterol.23 therefore , it is plausible to imply that deviation between health care resource utilization associated with spiromax versus turbuhaler might be related to the inhalation device . finally , the present study included other health care resources such as spirometry and thorax radiograph.3 some studies showed that a greater number of monitoring tests such as blood test and electrocardiogram were taken , causing the budget to exceed the cost of a regular monitoring test , which are most efficient to make a prognostic of copd.24 therefore , it can be concluded that the cost of monitoring tests could be underestimated in the present model . drug costs were obtained from a spanish database of pharmacists.25 cost of budesonide / formoterol fdc delivered by turbuhaler or spiromax was included exclusively as a drug cost in order to simplify the analysis . costs of health care resources included in this analysis were based on spanish regional tariff lists.2637 at the national level , costs of health care resources correspond to the mean of 12 regions , including those 5 regions included in this analysis . for each scenario , the economic impact for the time period of 20152018 was calculated based on the estimated number of patients who are treated with budesonide / formoterol fdc delivered by turbuhaler each year , the annual average cost per patient for each treatment option , the target population , and the market shares for both products included in this study . in the current scenario , this current scenario was compared with an alternative scenario in which the economic impact considered the introduction of duoresp spiromax and its effects toward medication adherence . the market uptake of duoresp spiromax increased gradually over the course of the 4 years ( table 3 ) . it was estimated that , in 2015 , 130,777 adults in spain suffered from copd , and they were treated with budesonide / formoterol delivered by a dpi ( in this study turbuhaler ; table 4 ) . the same pattern was observed for target population in catalonia , galicia , madrid , and valencia , whereas the number of patients in andalusia increased slightly between 2015 and 2018 ( table 4 ) . based on the annual drug cost and health care resource use per patient , the total treatment cost per patient was estimated in eur 2015 . with the annual average cost per patient for each treatment option , the target population , and the market shares for both products included in this study , the overall economic impact of the management of copd for the time period of 20152018 was obtained . in the current scenario for spain , the total economic impact of treatment with budesonide / formoterol fdc delivered by turbuhaler was estimated to be 108.76 million , 110.44 million , 114.61 million , and 121.73 million for the years 2015 , 2016 , 2017 , and 2018 , respectively ( table 5 ) . in the alternative scenario for spain , with the market share of duoresp spiromax increasing annually from 2015 , matched by a reduction in the share of symbicort turbuhaler and rilast turbuhaler , the total economic impact was estimated to be 107.92 million , 109.03 million , 112.79 million , and 119.48 million for 2015 , 2016 , 2017 , and 2018 , respectively ( table 5 ) . overall , the total budget savings for spain were expected to be 6.01 million over the next 4 years ( tables 5 and 6 ) . in the current scenario , the total economic impact was expected to be 65.49 million , 55.58 million , 30.45 million , 63.30 million , and 32.94 million in andalusia , catalonia , galicia , madrid , and valencia , respectively , for the time period of 20152018 ( table 5 ) . the economic impact of treating copd was reduced in all spanish regions with the introduction of spiromax , alternative scenario , over the time period of 20152018 . thus , total costs were estimated to be 64.59 million for andalusia , 54.84 million for catalonia , 29.98 million for galicia , 62.54 million for madrid , and 32.44 million for valencia ( tables 5 and 6 ) . total savings during the time period of 20152018 were estimated to be 902,133 , 740,520 , 464,281 , 748,996 , and 495,812 for andalusia , catalonia , galicia , madrid , and valencia , respectively . results were obtained through the reduction of health care resources , especially fewer days of hospital stay and avoided pc visits and emergency room visits ( table 6 ) , the reduction in pc visits related to problems with the inhaler device being more important . variations in the most sensitive parameters , such as copd severity or the adherence rate , do not lead to significant changes in the results of the analysis , since in all cases savings were obtained for both spain and the 5 regions . thus , the savings obtained ranged between 9.4 million and 606,641 for spain and 1.7 million and 50,077 for the 5 spanish regions ( table 7 ) . this study compared the costs of budesonide / formoterol fdc delivered by two different inhalation devices , spiromax and turbuhaler , to estimate the budget impact for the treatment of copd in spain and the 5 regions . results of the budget impact analysis suggest that the increasing use of spiromax would result in a 4-year budget savings for the spanish nhs of 6.01 million at the national level . results suggest that savings summed up to 902,133 , 740,520 , 464,281 , 748,996 , and 495,812 for andalusia , catalonia , galicia , madrid , and valencia , respectively . dealing with this chronic disease remains complex due to several problems such as the chronicity of the disease , the use of multiple medications , the periods of symptom remission , comorbidities that limit the movement such as arthritis and osteoarthritis , and the lack of adherence related to the inhalation technique . inhaled medication is typically prescribed in a maintenance therapy using fdcs , considering that single - inhaler combination regimens provide improved symptom control and slow down the progression of the disease.41,42 although current prescription guidelines contribute to improve the quality of life of copd patients , it was reported that up to 85% of the patients use their inhaler ineffectively , and this was associated with low medication adherence.41,43,44 our estimates suggest that the introduction of spiromax could result in savings by reducing health care resource utilization related to suboptimal medication adherence.45,46 this new inhalation device helps to reduce common utilization errors such as dose preparation errors , adequate flow rates , or even environmental conditions that might limit the delivery of the drug directly to the lungs , which would be closer to patients real - life situations.46 in addition , the current model can obtain region - specific results , which suggest that the highest populated regions correspond to those with greater savings for the health care budget . copd is mostly found in a population older than 40 years , and the chronicity of the disease makes it relevant to focus effort in new strategies to solve the current problems of suboptimal adherence.43,47 when clinical data were not available , we used data from the literature . moreover , the first assumption in the model was a growing market share of duoresp spiromax along with a reduction of symbicort turbuhaler and rilast turbuhaler utilization . although these amounts could be considered conservative , they are adjusted only to be related with inhalation technique problems , and not with the whole adherence problem , which is linked with many factors.41,44 it is worth mentioning that difficulties in the use of inhaler devices were exacerbated in elderly patients , whose reduced inhalation effort leads to a poor drug release.48 a greater impact on the copd economic budget was expected because of the aging population in spain . the results from this analysis suggest that the introduction of duoresp spiromax would result in a 6.01 million decrease in spain s overall budget in the period 20152018 associated with a lower health care resource utilization costs . region - specific data resulted in savings in 5 spanish regions of study which sum up to 902,133 , 740,520 , 464,281 , 748,996 , and 495,812 for andalusia , catalonia , galicia , madrid , and valencia , respectively . copd treatment regimens that increase the probability of higher medication adherence rates would be expected to contribute to improved disease management and to have an impact on the utilization of health care resources .

the online version of this article ( doi:10.1007/s40261 - 015 - 0372 - 9 ) contains supplementary material , which is available to authorized users . of patients newly initiated on statin treatment , one - fifth experienced a decrease in hdl-c.this hdl - c decrease was associated with higher risk of major adverse cardiovascular events compared with unchanged hdl-c.statin induced hdl - c decrease might be more hazardous than previously recognised and patients should be monitored closely regarding potential cardiovascular risk . the role of high - density lipoprotein cholesterol ( hdl - c ) as a potential risk factor in the development of cardiovascular disease ( cvd ) is not fully understood . epidemiological studies have reported an association between hdl - c single point measurements and risk of coronary heart disease ( which forms a large proportion of cvd ) [ 13 ] . some guidelines recommend an hdl - c target above 1.0 mmol / l for men and above 1.2 mmol / l for women , but such goals have also been questioned [ 5 , 6 ] . recent studies with novel hdl - c - raising therapies have not shown a clear preventive effect of increasing hdl - c on risk of cvd . treatment with one such agent , torcetrapib , resulted in an increased risk of mortality and morbidity of unknown mechanism , whereas potential favourable effects of another agent , dalcetrapib , with respect to hdl - c were possibly offset by other unfavourable effects [ 7 , 8 ] . statins show various degrees of low - density lipoprotein cholesterol ( ldl - c)-lowering and hdl - c - raising effects , where the action on hdl - c is independent of the reduction in ldl - c . is has been indicated from a meta - analysis that among statin - treated patients , hdl - c levels are strongly and inversely associated with the risk of major cardiovascular events . notably , a large proportion of patients experienced a paradoxical decrease in hdl - c following statin treatment initiation . a recent study reported an inverse association between the paradoxical hdl - c decrease after initiation of statin therapy and major adverse cardiovascular events in patients with acute myocardial infarction . it is possible that a reduction in hdl - c is associated with suboptimal protection against cardiovascular events . the aim of this observational study was to investigate the association between paradoxical hdl - c decrease after initiation of statin therapy and major adverse cardiovascular events in a general primary care patient population . the study protocol was reviewed and approved by the regional research ethics committee in uppsala , sweden ( reference number 2012/007 ) and registered at clinicaltrials.gov ( clinical trial identifier nct01551784 ) . this study linked data from electronic patient records to hospital , drug - dispensing , and cause - of - death registers . information on blood lipids and patient characteristics was extracted from primary care medical records [ e.g. date of birth , gender , body weight , blood pressure , number of primary healthcare centre contacts , and diagnosis according to international classification of diseases , 10th revision , clinical modification ( icd-10-cm ) codes ) using an established software system . data regarding morbidity and mortality were collected from the swedish national patient register , inpatient ( admission and discharge dates , and main and secondary diagnoses ) and outpatient hospital care ( number of contacts and diagnosis according to icd-10-cm codes ) registers , and the swedish national cause - of - death register ( date and cause of death ) . the linked study database is owned and managed by the department of public health and caring sciences , uppsala university , uppsala , sweden . personal identification numbers used to identify included patients in all healthcare contacts and were anonymised prior to further data processing . the study population consisted of statin - nave patients initiating a first statin treatment at 76 primary care centres in sweden . to facilitate a representative selection of primary care centres in sweden , a mix of rural and urban areas , public and private care providers , and small , mid - sized , and large primary care centres ( all using the same electronic patient journal system ) was included , corresponding to approximately 7 % of the swedish primary care centres . men and women were eligible for inclusion if they were aged 1885 years and were prescribed statins [ anatomical therapeutic chemical ( atc ) : c10a a ] between 1 january 2004 and 31 december 2009 . patients had to have hdl - c and ldl - c measurements recorded within 12 months prior to the start of statin treatment as well as a measurement after 10 days and within 12 months on treatment ; patients with cardiovascular events before the first hdl - measurement on statin treatment were excluded . patients with an ldl - c lowering of no more than 0.5 mmol / l were also excluded due to insufficient statin effect or indication of low compliance to statin treatment . the start of the observation period for collecting endpoints was date of first hdl - c measurement on statin treatment . the end of the study observation was 31 december 2011 , the end of statin treatment , or death . if a gap of more than 90 days was observed , based on available dispensed drug data , the end of statin treatment was defined as calculated days on last available dispensed drug package plus an additional 25 % of days based on the last dispensed drug pack size . two hdl - c groups were defined based on change in between last hdl - c measurement prior statin treatment and first hdl - c measurement on at least 14 days of statin treatment : hdl - c decrease : more than 0.1 mmol / l and hdl - c unchanged group : 0.1 mmol / l . in addition , a group with more than 0.1 mmol / l increase in hdl - c was defined to explore the effect of hdl - c increase . the analysis was performed in two patient samples ; the matched sample , which included hdl - c decrease and unchanged hdl - c patients who fulfilled the inclusion and exclusion criteria and who could be propensity score matched for baseline characteristics regarding propensity of hdl - c decrease . the unmatched population used for sub - group analyses comprised all patients who fulfilled the inclusion and exclusion criteria . the major adverse cardiovascular event ( mace ) endpoint was a composite of hospitalisation for a primary diagnosis for myocardial infarction ( icd-10 , i21 ) , unstable angina pectoris ( icd-10 , i20.0 ) , ischaemic stroke ( icd-10 , i63 ) , or cardiovascular death ( all primary causes of death diagnosed with icd-10 codes i00i99 ) . differences in baseline data between the two hdl - c groups were tested by one - way anova and pearson s chi - square test according to the type of data . propensity score matching provides an alternative means to balance study groups in order to reduce confounding when randomisation is not possible [ 1620 ] . logistic regression models were included to estimate the propensity scores between the decreased and unchanged hdl - c groups , with the hdl - c decrease as the response variable and the following covariates : age , gender , baseline hdl - c , baseline ldl - c , ldl - c change on statin treatment , antihypertensive therapy , diagnosis of diabetes , heart failure , hypertension , angina pectoris , peripheral artery disease ( pad ) , and stroke . the propensity scores were matched pairwise , with exact matching for prior myocardial infarction and use of calipers of width equal to 0.1 of the standard deviation of the propensity score . the primary endpoint was analysed by a cox proportional hazards model , using a grouped jack - knife estimation of the variance to take the correlation within pairs into account . the association between hdl - c change and the primary endpoint in the decreased and increased hdl - c groups was studied in the following sub groups : gender ( men / women ) , primary / secondary prevention , with / without diabetes , and in patients above 75 years of age versus younger patients . in the sub - group analyses , cox regression with adjustment for age , gender , baseline hdl - c , baseline ldl - c , ldl - c change on statin treatment , antihypertensive therapy , diagnoses of diabetes , heart failure , hypertension , angina pectoris , pad , and stroke was used . an additional analysis was performed comparing the separate outcome of cardiovascular death or all - cause death , as well as a sensitivity analysis including patients with a ldl - c reduction of < 0.5 mmol / l . in all , 84,812 patients were initiated on statin treatment during the observation period , of whom 15,357 ( 18 % ) were eligible ( fig . 1 ) . the main reason for exclusion was lack of recorded lipid measurements before and during statin treatment . compared with the study population , the excluded patients were more often men , were older , and fewer had diabetes / more had cvd before statin treatment initiation ( table s1).fig . hdl - c high - density lipoprotein cholesterol , ldl - c low - density lipoprotein cholesterol patient flow . hdl - c high - density lipoprotein cholesterol , ldl - c low - density lipoprotein cholesterol in the full eligible study cohort , baseline mean age was 62.7 years ( range 1985 years ) and mean hdl - c was 1.48 the majority of patients ( 96 % ) were initiated on simvastatin , with a mean dose of 20 mg / day ( median 20 mg / day ) . of these patients , 20 % had a decrease in hdl - c during the observation period , 58 % were unchanged , and 22 % showed an increase ( fig . 1 ) . the patient group with a decrease in hdl - c comprised more women , had a higher hdl - c at baseline ( 1.69 mmol / l ) , less diabetes , compared with the unchanged hdl - c group ( table 1 ) . the groups were similar regarding presence of cardiovascular diagnoses ; myocardial infarction , angina pectoris , pad , stroke or heart failure . the changes in hdl - c and ldl - c did not show any correlation ( fig . s1 ) .table 1baseline characteristics for patients with a decrease in hdl - c ( 0.1 mmol / l ) ( unmatched and propensity score - matched populations)variableunmatched populationpropensity score - matched populationdecreased ( n = 3068)unchanged ( n = 8919)increased ( n = 3370)decreased ( n = 2975)unchanged ( n = 2975 ) p value women , n ( % ) 1872 ( 61.0)4840 ( 54.3)1997 ( 59.3)1803 ( 60.6)1798 ( 60.4)0.92age ( years)62.3 ( 10.2)62.6 ( 10.2)63.0 ( 9.8)62.2 ( 10.1)62.3 ( 10.2)0.64simvastatin , n ( % ) 2925 ( 95.3)8510 ( 95.4)3244 ( 96.3)2835 ( 95.3)2823 ( 94.9)0.09dose ( mg)20.8 ( 9.7)19.7 ( 8.7)20.2 ( 8.8)20.8 ( 9.7)19.7 ( 8.4)<0.01hospitalisations , number / year prior to statin start0.2 ( 0.6)0.2 ( 0.6)0.19 ( 0.6)0.2 ( 0.6)0.2 ( 0.6)0.16systolic blood pressure ( mmhg)144.6 ( 19.8)143.6 ( 18.6)144.0 ( 18.9)144.6 ( 19.8)143.3 ( 19.0)0.02diastolic blood pressure ( mmhg)82.6 ( 10.4)82.0 ( 10.1)82.0 ( 10.4)82.7 ( 10.4)81.9 ( 10.2)0.01body mass index ( kg / cm)28.6 ( 5.0)29.4 ( 5.0)28.8 ( 4.9)28.7 ( 5.0)28.6 ( 5.2)0.67hba1c ( % ) 5.5 ( 1.3)5.7 ( 1.3)5.64 ( 1.4)5.6 ( 1.3)5.6 ( 1.4)0.77hdl - c ( mol / l)1.69 ( 0.47)1.41 ( 0.40)1.44 ( 0.42)1.66 ( 0.43)1.66 ( 0.45)0.95ldl - c ( mmol / l)4.53 ( 1.00)4.45 ( 0.95)4.52 ( 0.97)4.53 ( 0.99)4.52 ( 0.96)0.71change in ldl - c ( mmol / l)1.96 ( 0.81)1.84 ( 0.70)1.86 ( 0.75)1.95 ( 0.80)1.96 ( 0.73)0.92total cholesterol ( mmol / l)6.88 ( 1.10)6.66 ( 1.04)6.77 ( 1.07)6.86 ( 1.09)6.86 ( 1.05)0.86triglycerides ( mmol / l)1.61 ( 0.45)1.37 ( 0.38)1.40 ( 0.40)1.53 ( 0.75)1.55 ( 0.75)0.23antihypertensives ( hypertension ) , n ( % ) 1426 ( 46.5)4320 ( 48.4)1530 ( 45.4)1379 ( 46.4)1410 ( 47.4)0.44diabetes , n ( % ) 691 ( 22.5)2433 ( 27.3)834 ( 24.8)678 ( 22.8)680 ( 22.9)0.98myocardial infarction , n ( % ) 107 ( 3.5)254 ( 2.9)81 ( 2.4)93 ( 3.1)93 ( 3.1)1.00unstable angina pectoris , n ( % ) 45 ( 1.5)129 ( 1.5)46 ( 1.4)44 ( 1.5)43 ( 1.5)1.00heart failure , n ( % ) 75 ( 2.4)237 ( 2.7)75 ( 2.2)73 ( 2.5)72 ( 2.4)1.00arrhythmia , n ( % ) 182 ( 5.9)480 ( 5.4)175 ( 5.2)177 ( 6.0)180 ( 6.1)0.64peripheral arterial disease , n ( % ) 54 ( 1.8)130 ( 1.5)56 ( 1.7)52 ( 1.8)44 ( 1.5)0.47cerebrovascular disease , n ( % ) 242 ( 7.9)665 ( 7.5)208 ( 6.2)181 ( 6.1)172 ( 5.8)0.66values are expressed as mean ( sd ) unless specified otherwise hba1c glycated haemoglobin , hdl high - density lipoprotein cholesterol , ldl low - density lipoprotein cholesterol t test for continuous variables and chi - square test for categorical variable baseline characteristics for patients with a decrease in hdl - c ( 0.1 mmol / l ) ( unmatched and propensity score - matched populations ) values are expressed as mean ( sd ) unless specified otherwise hba1c glycated haemoglobin , hdl high - density lipoprotein cholesterol , ldl low - density lipoprotein cholesterol t test for continuous variables and chi - square test for categorical variable the decreased and unchanged hdl - c groups showed a large degree of propensity score overlap ( 71 % ) , indicating that these groups were similar prior to the start of statin treatment . after matching , the decreased and unchanged hdl - c groups had similar baseline characteristics and ldl - c changes , with the exception of a higher simvastatin dose and lower triglyceride level in the decreased hdl - c group ( table 1 ) . the median time from hdl - c measurement to the start of statin treatment was 12 days [ interquartile range ( iqr ) 731 days ] , and the mean time from the start of statin treatment to the second hdl - c measurement was 84 days ( iqr 48148 days ) . patients were followed for up to 7 years , with a median follow - up of 2 years , including 14,198 patient - years . in the group with decreased hdl - c , the primary endpoint incidence rates ( per 1000 patient - years ) were 12.8 and 8.2 in the decreased and unchanged hdl - c groups , respectively . the risk of major cardiovascular events was 56 % higher in the decreased hdl - c group compared with the unchanged hdl - c group [ hazard ratio ( hr ) , 1.56 ; 95 % confidence interval ( ci ) , 1.122.16 ; p < 0.01 ; table 2 ; fig . the difference between the two groups was due to ischaemic stroke ( hr , 1.74 ; 95 % ci , 1.003.03 ; p = 0.05 ) , but was also driven by cardiovascular death ( hr , 1.72 ; 95 % ci , 0.863.42 ; p = 0.12).table 2exposure time ( years ) in the propensity score - matched populationsunchanged hdl - cdecreased hdl - ctotalmaximum follow - up time6.96.96.9median follow - up time1.92.02.0total patient - years7157704114,198total number of events5990149fig . 2kaplan - meier plot of time to first major cardiovascular events for the decreased and unchanged hdl - c propensity score - matched populations . mace major adverse cardiovascular events exposure time ( years ) in the propensity score - matched populations kaplan - meier plot of time to first major cardiovascular events for the decreased and unchanged hdl - c propensity score - matched populations . mace major adverse cardiovascular events the association between hdl - c change and the primary endpoint in the decreased and increased hdl - c groups showed consistent results in the sub - group analyses : gender , primary / secondary prevention , with / without diabetes , and in patients aged > 75 years of age versus younger patients ( fig . 3 ; table 3).fig . 3hazard ratio forest plot of major cardiovascular events in different sub - groupstable 3events and events rates for forest plot ( fig . 3)unchanged hdl - cno of patientsdecreased hdl - cno of patientsunchanged hdl - cno of eventsdecreased hdl - cno of eventsunchanged hdl - cevents/1000 patient - yearsdecreased hdl - cevents/1000 patient - yearstotal891930682369311.312.8female4840187298438.59.7male407911961385014.617.5primary prevention5063183862345.38.1secondary prevention385612301745918.819.0diabetes2433691933616.320.4no diabetes64862377143579.410.3age over 75 years959303723231.848.7age below 75 years79602765164618.89.2 hazard ratio forest plot of major cardiovascular events in different sub - groups events and events rates for forest plot ( fig . 3 ) no difference in risk of major cardiovascular events was observed between the hdl - c increase group compared with the unchanged hdl - c group ( hr , 1.05 ; 95 % ci , 0.821.34 ; p = 0.72 ) . ( hr , 1.61 ; 95 % ci , 0.942.75 ; p = 0.09 ) and all - cause death ( hr , 1.30 ; 95 % ci , 0.921.85 ; p = 0.14 ) showed similar results . to assess the impact of the 3161 patients with an ldl - c reduction of < 0.5 mmol / l , they were included in the analyses which showed a similar risk ( hr , 1.56 ; 95 % ci , 1.251.95 ; p < 0.01 ) . the association between hdl - c change and the primary endpoint in the decreased and increased hdl - c groups showed consistent results in the sub - group analyses : gender , primary / secondary prevention , with / without diabetes , and in patients aged > 75 years of age versus younger patients ( fig . 3 ; table 3).fig . 3hazard ratio forest plot of major cardiovascular events in different sub - groupstable 3events and events rates for forest plot ( fig . 3)unchanged hdl - cno of patientsdecreased hdl - cno of patientsunchanged hdl - cno of eventsdecreased hdl - cno of eventsunchanged hdl - cevents/1000 patient - yearsdecreased hdl - cevents/1000 patient - yearstotal891930682369311.312.8female4840187298438.59.7male407911961385014.617.5primary prevention5063183862345.38.1secondary prevention385612301745918.819.0diabetes2433691933616.320.4no diabetes64862377143579.410.3age over 75 years959303723231.848.7age below 75 years79602765164618.89.2 hazard ratio forest plot of major cardiovascular events in different sub - groups events and events rates for forest plot ( fig . 3 ) no difference in risk of major cardiovascular events was observed between the hdl - c increase group compared with the unchanged hdl - c group ( hr , 1.05 ; 95 % ci , 0.821.34 ; p = 0.72 ) . the separate outcome of cardiovascular death ( hr , 1.61 ; 95 % ci , 0.942.75 ; p = 0.09 ) and all - cause death ( hr , 1.30 ; 95 % ci , 0.921.85 ; p = 0.14 ) showed similar results . to assess the impact of the 3161 patients with an ldl - c reduction of < 0.5 mmol / l , they were included in the analyses which showed a similar risk ( hr , 1.56 ; 95 % ci , 1.251.95 ; p < 0.01 ) . in this study , two - thirds of eligible patients initiating statin treatment had a change in their hdl - c level , and the degree of change was similar to that observed in randomised clinical trials . a paradoxical decrease in hdl - c of > 0.1 mmol / l was associated with a 56 % increase in major adverse cardiovascular events compared with unchanged hdl - c levels . the results were consistent across subgroups based on age , gender , presence of diabetes , primary and secondary prevention . no association between increased hdl - c levels and risk of major adverse cardiovascular events could be observed . results from a recent meta - analysis did not demonstrate an association between statin treatment , hdl - c change , and cvd risk . our patients had a relatively high untreated hdl - c level ( 1.48 mmol / l ) , in line with observations of untreated hdl - c levels in other scandinavian studies , but in contrast with the recent publications [ 11 , 2123 ] . mmol / l ) , and the relatively small hdl - c reduction in the meta - analysis might not have been sufficient to detect cvd risk associations . furthermore , our findings are supported by a recent study which shows that a paradoxical decrease in plasma hdl - c levels after statin therapy is an important risk factor predicting long - term adverse cardiac events in patients with acute myocardial infarction . low single point measurements of hdl - c levels in patients receiving statin treatment have been reported to be associated with increased cvd risk , irrespective of the low ldl - c levels achieved . we have shown that patients with a relatively high hdl - c ( mean 1.48 mmol / l ) newly initiated on cholesterol - modifying treatment ( statin ) and who experienced a consecutive hdl - c reduction have an increased cardiovascular risk , independently of baseline ldl - c and ldl - c change on statin treatment . our findings are in line with previous observational data where a threshold for increased cardiovascular risk for hdl - c values below 1.34 mmol / l was observed . since the untreated hdl - c is relatively high in our material , this is the likely explanation for why we do not observe a reduced cardiovascular risk with increased hdl - c values . a major decrease in hdl - c level , independent of the size of the ldl - c reduction , might cause a shift in cholesterol transport . indeed , the one - third of patients initiated on statin therapy who had a paradoxical reduction in hdl - c level may have a suboptimal balance of cholesterol in / out transport to / from the inner arterial wall . other important cardiovascular risk - lowering properties of hdl - c include antioxidant , anti - apoptotic , anti - inflammatory , antithrombotic , and anti - proteolytic properties , which account for the direct protective action on endothelial cells . however , we believe that reduction of hdl - c per se is associated with increased cardiovascular risk and not necessarily a statin - specific effect . thus , we would highlight the importance of non - pharmacological efforts that will prevent hdl - c reductions , such as avoiding weight gain and/or maintaining physical activity levels . the endpoint was a composite of hospitalisation with a primary diagnose of myocardial infarction , unstable angina pectoris , or ischaemic stroke , or cardiovascular death . an analysis of the separate endpoint components showed that risk of ischaemic stroke was statistically significant . the risks of coronary events and cardiovascular death were not significant , although the trends showed indication of similar directions / patterns . this finding might be somewhat surprising , as a predominant effect of statin treatment on coronary disease would be expected . however , as more patients in sweden die outside hospital owing to coronary disease than owing to stroke , and a proportion of fatal coronary events occur in the out - of - hospital setting , stroke events were more likely to be a classified event in our study because more of these patients survived to hospitals [ 25 , 26 ] . similar results were observed when comparing outcome of separate analysis of cardiovascular death with all - cause death . interestingly , the recent study which showed that a paradoxical decrease in plasma hdl - c levels after statin therapy initiation also had results driven by significantly higher incidence of stroke in the decreased hdl - c group . eighteen percent of patients initiated on statin treatment during the observation period were included in the study . the main reason for exclusion was lack of laboratory data , as only laboratory measurements from primary care were available . this favoured the inclusion of patients with regular healthcare controls ( hypertension , diabetes , atrial fibrillation ) in primary care . a considerable proportion of secondary prevention patients with initiation of statin treatment in hospital did not have a pre - treatment hdl - c measurement available to us and were therefore not included ( table s1 ) . the exclusion of a significant proportion of patients might call into question the generalisability of the results . however , we found consistent results in all subgroup analyses , with a numerically higher risk of reaching the composite endpoint with decreased hdl - c levels for all subgroups ( older vs. younger patients , men vs. women , primary vs. secondary prevention patients , and presence of diabetes ) . however , among secondary preventive patients , a smaller numerical difference in cardiovascular risk between unchanged and decreased hdl - c groups was observed . secondary prevention , for patients recently experiencing a myocardial infarction or a stroke , might potentially a have an initial increased thrombotic risk , which is more critical than the long - term effect caused by the atherosclerosis process . altogether , this indicates that the study findings might be valid for a broad statin - treated population . a further potential limitation regarding generalisability is the fact that the absolute majority of patients in sweden are treated with relatively low doses of simvastatin . the frequent use of low - dose simvastatin might be the result of a stringent reimbursement regime , only allowing the use of high - potency statins in patients who do not reach treatment goals or in individuals who do not tolerate simvastatin . the effect on hdl - c change achieved by statins in general is reported to be independent of the reduction in ldl - c . changes in body weight , smoking pattern , or physical activity might influence levels of hdl - c , the latter two of which are not systematically recorded in primary care records . since smoking previously was reported to be associated with generally low hdl - c levels , it is likely that smokers would be in the unchanged group or increase group due to the regression to the mean effect in our study [ 10 , 27 ] . furthermore , if the increase in hdl - c was due to cessation of smoking , a decrease in hdl - c should be found more frequently in smokers . in sweden , not only is the overall smoking practice low ( < 15 % ) but the likelihood of patients starting smoking during initiation of statin therapy can also be considered to be low . furthermore , the effect of smoking cessation programmes in primary care is modest [ 28 , 29 ] . the inverse correlation between physical activity and hdl - c change is low and can therefore be considered to be of minor importance . we did not observe a marked percentage increase in body mass index in patients with a reduction in hdl - c , when compared with patients with unchanged hdl - c levels . however , patients were only included in the analyses while on statin treatment , and only if the reported ldl - c reduction was > 0.5 the risk of the results being due to low compliance and/or statin response can therefore also be considered to be low . the statin prescription pattern might be a source of confounding by indication . we found that patients with high cardiovascular risk in general had a lower untreated ldl - c , and vice versa . this correlation between ldl levels and cvd risk has been reported previously in a real - life clinical setting . however , we found no correlation between ldl - c change and hdl - c change , as also supported by a previous report . a prescription bias based on low hdl - c levels might also be a source of explanation for our findings . as low hdl - c is not a reason for initiation of statin treatment in sweden , though , it is not likely that hdl - c should be affected by confounding by indication . furthermore , we observed a mean difference of 1.1 mg of simvastatin between the decrease and unchanged groups after propensity score matching . we do not think this minimal difference in dosing had any impact on the results . biological and analytical variation of hdl - c values may be a potential source of misclassification into the different hdl - c change groups . however , we observed similar associations with baseline cholesterol parts [ hdl - c , plasma triglycerides ( tg ) , and ldl - c ] on hdl - c change pattern in our study compared to those reported in randomized clinical trials . thus , in our study , patients with high hdl - c had higher likelihood of hdl - c reduction and patients with low hdl - c and higher associated cardiovascular risk at baseline would more likely be identified for the hdl - c decrease group . in sweden , hdl - c samples are generally analysed at regional central laboratories , all of which have participated in national quality and standardisation programmes since the end of the 1980s . the analytical variation for hdl - c in the swedish external quality assurance programme is between 3 % and 4 % ( at the level of 1.68 mmol / l ) , while the biological variation of hdl - c is approximately 7 % . patients in our study had to have a decrease in hdl - c of > 0.1 our conservative estimations of the hdl - c variation support the notion that the magnitude of the observed hdl - c decrease was sufficient . second , only statin - nave patients were included in order to increase the likelihood of analysing the actual treatment effect on hdl - c levels . the observed hdl - c change pattern is similar to that observed in randomised clinical trials . third , our analyses carefully matched the patients for numerous cardiovascular diagnoses , risk factors , including baseline ldl - c , and ldl - c change on treatment , thus increasing the likelihood of similar baseline risk . finally , using swedish national health registers the follow - up was performed with basically no loss of events . a marked proportion of patients newly initiated on statin treatment experienced a decrease in hdl - c . this decrease was associated with a higher risk of major adverse cardiovascular events compared with patients in whom hdl - c levels were unchanged . statin - induced increase in hdl - c was not associated with lower risk of major adverse cardiovascular events . p hasvold , m. thuresson , g. johansson , and j. bodegrd participated equally in the study conception , design , and statistical analysis planning . m. thuresson was responsible for the statistical analyses , p. hasvold for the manuscript draft and finalization , and g. johansson for handling of data and the study database . all authors had access to study data , and had authority over manuscript preparation , approval of the final version , and the decision to submit for publication . data collection was performed by pygargus ab , stockholm , sweden , and was funded by astrazeneca . the statistical analysis was agreed on by the study steering committee , and data analysis was performed by the study database owner in collaboration with astrazeneca . as members of the study steering committee , astrazeneca took part in the interpretation of the data and the drafting of the manuscript . pl hasvold is enrolled at the phd school of the university of oslo , department of medicine . marcus thuresson is employed by an independent statistical consultant company , statisticon ab , for which astrazeneca is a client . sverre e. kjeldsen has received honoraria for lecturing / consulting and other research support from astrazeneca , bayer , hemo sapiens , medtronic , msd , novartis , pronova , serodus , and takeda .

background and objectivesstatin - induced changes in high - density lipoprotein cholesterol ( hdl - c ) and low - density lipoprotein cholesterol ( ldl - c ) are unrelated . many patients initiated on statins experience a paradoxical decrease in hdl - c . the aim of this study was to evaluate the association between a decrease in hdl - c and risk of major adverse cardiovascular events ( mace).methodsdata from 15,357 primary care patients initiated on statins during 20042009 were linked with data from mandatory national hospital , drug - dispensing , and cause - of - death registers , and were grouped according to hdl - c change : decreased 0.1 mmol / l , unchanged 0.1 or 0.1 mmol / l increased . to evaluate the association between decrease in hdl - c and risk of mace , a sample of propensity score - matched patients from the decreased and unchanged groups was created , using the latter group as reference . mace was defined as myocardial infarction , unstable angina pectoris , ischaemic stroke , or cardiovascular mortality . cox proportional hazards models were used to estimate relative risks.resultshdl-c decreased in 20 % , was unchanged in 58% , and increased in 22 % of patients initiated on statin treatment ( 96 % treated with simvastatin ) . the propensity score - matched sample comprised 5950 patients with mean baseline hdl - c and ldl - c of 1.69 and 4.53 mmol / l , respectively . hdl - c decrease was associated with 56 % higher mace risk ( hazard ratio 1.56 ; 95 % confidence interval 1.122.16 ; p < 0.01 ) compared with the unchanged hdl - c group.conclusionsparadoxical statin - induced reduction in hdl - c was relatively common and was associated with increased risk of mace.electronic supplementary materialthe online version of this article ( doi:10.1007/s40261 - 015 - 0372 - 9 ) contains supplementary material , which is available to authorized users .

Electronic supplementary material Key Points Introduction Methods Results Subgroup Analyses Sensitivity Analyses Discussion Conclusions Electronic supplementary material Contributors statements Role of the funding source Disclosures

the online version of this article ( doi:10.1007/s40261 - 015 - 0372 - 9 ) contains supplementary material , which is available to authorized users . of patients newly initiated on statin treatment , one - fifth experienced a decrease in hdl-c.this hdl - c decrease was associated with higher risk of major adverse cardiovascular events compared with unchanged hdl-c.statin induced hdl - c decrease might be more hazardous than previously recognised and patients should be monitored closely regarding potential cardiovascular risk . the role of high - density lipoprotein cholesterol ( hdl - c ) as a potential risk factor in the development of cardiovascular disease ( cvd ) is not fully understood . statins show various degrees of low - density lipoprotein cholesterol ( ldl - c)-lowering and hdl - c - raising effects , where the action on hdl - c is independent of the reduction in ldl - c . is has been indicated from a meta - analysis that among statin - treated patients , hdl - c levels are strongly and inversely associated with the risk of major cardiovascular events . notably , a large proportion of patients experienced a paradoxical decrease in hdl - c following statin treatment initiation . a recent study reported an inverse association between the paradoxical hdl - c decrease after initiation of statin therapy and major adverse cardiovascular events in patients with acute myocardial infarction . it is possible that a reduction in hdl - c is associated with suboptimal protection against cardiovascular events . the aim of this observational study was to investigate the association between paradoxical hdl - c decrease after initiation of statin therapy and major adverse cardiovascular events in a general primary care patient population . this study linked data from electronic patient records to hospital , drug - dispensing , and cause - of - death registers . data regarding morbidity and mortality were collected from the swedish national patient register , inpatient ( admission and discharge dates , and main and secondary diagnoses ) and outpatient hospital care ( number of contacts and diagnosis according to icd-10-cm codes ) registers , and the swedish national cause - of - death register ( date and cause of death ) . patients had to have hdl - c and ldl - c measurements recorded within 12 months prior to the start of statin treatment as well as a measurement after 10 days and within 12 months on treatment ; patients with cardiovascular events before the first hdl - measurement on statin treatment were excluded . patients with an ldl - c lowering of no more than 0.5 mmol / l were also excluded due to insufficient statin effect or indication of low compliance to statin treatment . two hdl - c groups were defined based on change in between last hdl - c measurement prior statin treatment and first hdl - c measurement on at least 14 days of statin treatment : hdl - c decrease : more than 0.1 mmol / l and hdl - c unchanged group : 0.1 mmol / l . in addition , a group with more than 0.1 mmol / l increase in hdl - c was defined to explore the effect of hdl - c increase . the analysis was performed in two patient samples ; the matched sample , which included hdl - c decrease and unchanged hdl - c patients who fulfilled the inclusion and exclusion criteria and who could be propensity score matched for baseline characteristics regarding propensity of hdl - c decrease . the major adverse cardiovascular event ( mace ) endpoint was a composite of hospitalisation for a primary diagnosis for myocardial infarction ( icd-10 , i21 ) , unstable angina pectoris ( icd-10 , i20.0 ) , ischaemic stroke ( icd-10 , i63 ) , or cardiovascular death ( all primary causes of death diagnosed with icd-10 codes i00i99 ) . logistic regression models were included to estimate the propensity scores between the decreased and unchanged hdl - c groups , with the hdl - c decrease as the response variable and the following covariates : age , gender , baseline hdl - c , baseline ldl - c , ldl - c change on statin treatment , antihypertensive therapy , diagnosis of diabetes , heart failure , hypertension , angina pectoris , peripheral artery disease ( pad ) , and stroke . the association between hdl - c change and the primary endpoint in the decreased and increased hdl - c groups was studied in the following sub groups : gender ( men / women ) , primary / secondary prevention , with / without diabetes , and in patients above 75 years of age versus younger patients . in the sub - group analyses , cox regression with adjustment for age , gender , baseline hdl - c , baseline ldl - c , ldl - c change on statin treatment , antihypertensive therapy , diagnoses of diabetes , heart failure , hypertension , angina pectoris , pad , and stroke was used . an additional analysis was performed comparing the separate outcome of cardiovascular death or all - cause death , as well as a sensitivity analysis including patients with a ldl - c reduction of < 0.5 mmol / l . hdl - c high - density lipoprotein cholesterol , ldl - c low - density lipoprotein cholesterol patient flow . hdl - c high - density lipoprotein cholesterol , ldl - c low - density lipoprotein cholesterol in the full eligible study cohort , baseline mean age was 62.7 years ( range 1985 years ) and mean hdl - c was 1.48 the majority of patients ( 96 % ) were initiated on simvastatin , with a mean dose of 20 mg / day ( median 20 mg / day ) . of these patients , 20 % had a decrease in hdl - c during the observation period , 58 % were unchanged , and 22 % showed an increase ( fig . the patient group with a decrease in hdl - c comprised more women , had a higher hdl - c at baseline ( 1.69 mmol / l ) , less diabetes , compared with the unchanged hdl - c group ( table 1 ) . the changes in hdl - c and ldl - c did not show any correlation ( fig . s1 ) .table 1baseline characteristics for patients with a decrease in hdl - c ( 0.1 mmol / l ) ( unmatched and propensity score - matched populations)variableunmatched populationpropensity score - matched populationdecreased ( n = 3068)unchanged ( n = 8919)increased ( n = 3370)decreased ( n = 2975)unchanged ( n = 2975 ) p value women , n ( % ) 1872 ( 61.0)4840 ( 54.3)1997 ( 59.3)1803 ( 60.6)1798 ( 60.4)0.92age ( years)62.3 ( 10.2)62.6 ( 10.2)63.0 ( 9.8)62.2 ( 10.1)62.3 ( 10.2)0.64simvastatin , n ( % ) 2925 ( 95.3)8510 ( 95.4)3244 ( 96.3)2835 ( 95.3)2823 ( 94.9)0.09dose ( mg)20.8 ( 9.7)19.7 ( 8.7)20.2 ( 8.8)20.8 ( 9.7)19.7 ( 8.4)<0.01hospitalisations , number / year prior to statin start0.2 ( 0.6)0.2 ( 0.6)0.19 ( 0.6)0.2 ( 0.6)0.2 ( 0.6)0.16systolic blood pressure ( mmhg)144.6 ( 19.8)143.6 ( 18.6)144.0 ( 18.9)144.6 ( 19.8)143.3 ( 19.0)0.02diastolic blood pressure ( mmhg)82.6 ( 10.4)82.0 ( 10.1)82.0 ( 10.4)82.7 ( 10.4)81.9 ( 10.2)0.01body mass index ( kg / cm)28.6 ( 5.0)29.4 ( 5.0)28.8 ( 4.9)28.7 ( 5.0)28.6 ( 5.2)0.67hba1c ( % ) 5.5 ( 1.3)5.7 ( 1.3)5.64 ( 1.4)5.6 ( 1.3)5.6 ( 1.4)0.77hdl - c ( mol / l)1.69 ( 0.47)1.41 ( 0.40)1.44 ( 0.42)1.66 ( 0.43)1.66 ( 0.45)0.95ldl - c ( mmol / l)4.53 ( 1.00)4.45 ( 0.95)4.52 ( 0.97)4.53 ( 0.99)4.52 ( 0.96)0.71change in ldl - c ( mmol / l)1.96 ( 0.81)1.84 ( 0.70)1.86 ( 0.75)1.95 ( 0.80)1.96 ( 0.73)0.92total cholesterol ( mmol / l)6.88 ( 1.10)6.66 ( 1.04)6.77 ( 1.07)6.86 ( 1.09)6.86 ( 1.05)0.86triglycerides ( mmol / l)1.61 ( 0.45)1.37 ( 0.38)1.40 ( 0.40)1.53 ( 0.75)1.55 ( 0.75)0.23antihypertensives ( hypertension ) , n ( % ) 1426 ( 46.5)4320 ( 48.4)1530 ( 45.4)1379 ( 46.4)1410 ( 47.4)0.44diabetes , n ( % ) 691 ( 22.5)2433 ( 27.3)834 ( 24.8)678 ( 22.8)680 ( 22.9)0.98myocardial infarction , n ( % ) 107 ( 3.5)254 ( 2.9)81 ( 2.4)93 ( 3.1)93 ( 3.1)1.00unstable angina pectoris , n ( % ) 45 ( 1.5)129 ( 1.5)46 ( 1.4)44 ( 1.5)43 ( 1.5)1.00heart failure , n ( % ) 75 ( 2.4)237 ( 2.7)75 ( 2.2)73 ( 2.5)72 ( 2.4)1.00arrhythmia , n ( % ) 182 ( 5.9)480 ( 5.4)175 ( 5.2)177 ( 6.0)180 ( 6.1)0.64peripheral arterial disease , n ( % ) 54 ( 1.8)130 ( 1.5)56 ( 1.7)52 ( 1.8)44 ( 1.5)0.47cerebrovascular disease , n ( % ) 242 ( 7.9)665 ( 7.5)208 ( 6.2)181 ( 6.1)172 ( 5.8)0.66values are expressed as mean ( sd ) unless specified otherwise hba1c glycated haemoglobin , hdl high - density lipoprotein cholesterol , ldl low - density lipoprotein cholesterol t test for continuous variables and chi - square test for categorical variable baseline characteristics for patients with a decrease in hdl - c ( 0.1 mmol / l ) ( unmatched and propensity score - matched populations ) values are expressed as mean ( sd ) unless specified otherwise hba1c glycated haemoglobin , hdl high - density lipoprotein cholesterol , ldl low - density lipoprotein cholesterol t test for continuous variables and chi - square test for categorical variable the decreased and unchanged hdl - c groups showed a large degree of propensity score overlap ( 71 % ) , indicating that these groups were similar prior to the start of statin treatment . after matching , the decreased and unchanged hdl - c groups had similar baseline characteristics and ldl - c changes , with the exception of a higher simvastatin dose and lower triglyceride level in the decreased hdl - c group ( table 1 ) . in the group with decreased hdl - c , the primary endpoint incidence rates ( per 1000 patient - years ) were 12.8 and 8.2 in the decreased and unchanged hdl - c groups , respectively . the risk of major cardiovascular events was 56 % higher in the decreased hdl - c group compared with the unchanged hdl - c group [ hazard ratio ( hr ) , 1.56 ; 95 % confidence interval ( ci ) , 1.122.16 ; p < 0.01 ; table 2 ; fig . the difference between the two groups was due to ischaemic stroke ( hr , 1.74 ; 95 % ci , 1.003.03 ; p = 0.05 ) , but was also driven by cardiovascular death ( hr , 1.72 ; 95 % ci , 0.863.42 ; p = 0.12).table 2exposure time ( years ) in the propensity score - matched populationsunchanged hdl - cdecreased hdl - ctotalmaximum follow - up time6.96.96.9median follow - up time1.92.02.0total patient - years7157704114,198total number of events5990149fig . 2kaplan - meier plot of time to first major cardiovascular events for the decreased and unchanged hdl - c propensity score - matched populations . mace major adverse cardiovascular events exposure time ( years ) in the propensity score - matched populations kaplan - meier plot of time to first major cardiovascular events for the decreased and unchanged hdl - c propensity score - matched populations . mace major adverse cardiovascular events the association between hdl - c change and the primary endpoint in the decreased and increased hdl - c groups showed consistent results in the sub - group analyses : gender , primary / secondary prevention , with / without diabetes , and in patients aged > 75 years of age versus younger patients ( fig . 3 ) no difference in risk of major cardiovascular events was observed between the hdl - c increase group compared with the unchanged hdl - c group ( hr , 1.05 ; 95 % ci , 0.821.34 ; p = 0.72 ) . to assess the impact of the 3161 patients with an ldl - c reduction of < 0.5 mmol / l , they were included in the analyses which showed a similar risk ( hr , 1.56 ; 95 % ci , 1.251.95 ; p < 0.01 ) . the association between hdl - c change and the primary endpoint in the decreased and increased hdl - c groups showed consistent results in the sub - group analyses : gender , primary / secondary prevention , with / without diabetes , and in patients aged > 75 years of age versus younger patients ( fig . 3 ) no difference in risk of major cardiovascular events was observed between the hdl - c increase group compared with the unchanged hdl - c group ( hr , 1.05 ; 95 % ci , 0.821.34 ; p = 0.72 ) . to assess the impact of the 3161 patients with an ldl - c reduction of < 0.5 mmol / l , they were included in the analyses which showed a similar risk ( hr , 1.56 ; 95 % ci , 1.251.95 ; p < 0.01 ) . a paradoxical decrease in hdl - c of > 0.1 mmol / l was associated with a 56 % increase in major adverse cardiovascular events compared with unchanged hdl - c levels . no association between increased hdl - c levels and risk of major adverse cardiovascular events could be observed . furthermore , our findings are supported by a recent study which shows that a paradoxical decrease in plasma hdl - c levels after statin therapy is an important risk factor predicting long - term adverse cardiac events in patients with acute myocardial infarction . low single point measurements of hdl - c levels in patients receiving statin treatment have been reported to be associated with increased cvd risk , irrespective of the low ldl - c levels achieved . we have shown that patients with a relatively high hdl - c ( mean 1.48 mmol / l ) newly initiated on cholesterol - modifying treatment ( statin ) and who experienced a consecutive hdl - c reduction have an increased cardiovascular risk , independently of baseline ldl - c and ldl - c change on statin treatment . indeed , the one - third of patients initiated on statin therapy who had a paradoxical reduction in hdl - c level may have a suboptimal balance of cholesterol in / out transport to / from the inner arterial wall . the endpoint was a composite of hospitalisation with a primary diagnose of myocardial infarction , unstable angina pectoris , or ischaemic stroke , or cardiovascular death . interestingly , the recent study which showed that a paradoxical decrease in plasma hdl - c levels after statin therapy initiation also had results driven by significantly higher incidence of stroke in the decreased hdl - c group . a considerable proportion of secondary prevention patients with initiation of statin treatment in hospital did not have a pre - treatment hdl - c measurement available to us and were therefore not included ( table s1 ) . furthermore , if the increase in hdl - c was due to cessation of smoking , a decrease in hdl - c should be found more frequently in smokers . we did not observe a marked percentage increase in body mass index in patients with a reduction in hdl - c , when compared with patients with unchanged hdl - c levels . however , patients were only included in the analyses while on statin treatment , and only if the reported ldl - c reduction was > 0.5 the risk of the results being due to low compliance and/or statin response can therefore also be considered to be low . the analytical variation for hdl - c in the swedish external quality assurance programme is between 3 % and 4 % ( at the level of 1.68 mmol / l ) , while the biological variation of hdl - c is approximately 7 % . patients in our study had to have a decrease in hdl - c of > 0.1 our conservative estimations of the hdl - c variation support the notion that the magnitude of the observed hdl - c decrease was sufficient . a marked proportion of patients newly initiated on statin treatment experienced a decrease in hdl - c . this decrease was associated with a higher risk of major adverse cardiovascular events compared with patients in whom hdl - c levels were unchanged . statin - induced increase in hdl - c was not associated with lower risk of major adverse cardiovascular events .

this process relies mostly on the recognition of pathogen - associated molecular patterns ( pamps ) by pattern recognition receptors ( prrs ) . among the canonical pamps are molecules such as lipopolysaccharide , peptidoglycan , bacterial lipoproteins , flagellin , and nucleic acids derived from viruses , bacteria , fungi , and protozoa ( janeway and medzhitov , 2002 ; akira et al . , 2006 ; gazzinelli and denkers , 2006 ) . upon direct or indirect ligand recognition , toll - like receptors ( tlrs ) dimerize and trigger a signaling cascade leading to the activation of proinflammatory responses ( uematsu and akira , 2006 ) . tlrs are transmembrane proteins containing an extracellular leucine - rich repeat ( lrr ) domain that facilitates pamp recognition and an intracellular domain that mediates intracellular signaling via four different adaptor proteins : tram , mal / tirap , myd88 , and trif ( o'neill , 2008 ) . depending on the nature of their specific ligands , tlrs are embedded in either the extracellular membrane ( tlr-1 , -2 , -4 , -5 , -6 , -10 , -11 ) or in the membranes of endocytic vacuoles ( tlr-3 , -7 , -8 , -9 ) . in addition to tlrs , other prr families have already been described ; these include dna / rna - sensing proteins such as the rig - i - like receptor ( rlr ) family and sensors of membrane damage and intracellular pamps such as the nod - like receptors ( nlrs ; creagh and o'neill , 2006 ) . the rna helicase domain - containing proteins retinoic acid - inducible gene i ( rig - i ) and melanoma differentiation - associated gene 5 ( mda5 ) comprise a group of cytoplasmic receptors important for the recognition of viral nucleic acids . pamp recognition by rig - i and mda5 triggers the activation of irf3 via mavs ( ips-1 ) , culminating in the production of type i interferons ( ifn ) . recent studies have demonstrated that these receptors also induce type i ifn production upon recognition of nucleic acids from intracellular bacteria ( cao , 2009 ) . in addition , some studies have shown that the recognition of dna by rig - i / mda5 is dependent on cytosolic rna polymerase iii ( pol iii ; ablasser et al . the nlrs comprise a prr family that can be classified into three sub - groups . the first sub - group is composed of receptors that trigger intracellular signaling pathways leading to the activation of transcriptional factors mediating the expression of inflammatory response genes . both nod1 and nod2 are members of this first group , and they signal via rip2 , a kinase that ubiquitinates nemo to induce the activation of nf-b and mapk ( shaw et al . , 2008 ) . the second sub - group comprises nlrs that do not require asc to trigger caspase-1 activation . among these proteins are naip5 ( birc1e ) and nlrc4 ( ipaf ) , which have been suggested to assembly a unique inflammasome ( hereafter referred to as the nlrc4 inflammasome ) . activation of this inflammasome triggers a specific form of host cell death called pyroptosis ( lightfield et al . , 2009 ; broz et al . , 2010 ; silveira and zamboni , 2010 ; whitfield et al . , the third sub - group of nlrs comprises those that trigger caspase-1 activation via the adaptor protein asc . these proteins assemble into a multimeric molecular platform known as the classical inflammasome . among the nlrs that trigger the asc - dependent inflammasome is nalp3 , which has been extensively characterized and shown to be important for the recognition of danger - associated molecular patterns ( damps ) reviewed by schroder and tschopp ( 2010 ) . in addition to tlrs , nlrs , and rlrs , previous studies have described a class of proteins that recognize cytoplasmic dna ( ishii and akira , 2006 ; stetson and medzhitov , 2006 ) . these multiple protein families include dna - dependent activators of ifn - regulatory factors ( dai ; takaoka et al . , 2007 ) , rna polymerase iii , which induces type i ifn production through the rig - i pathway ( ablasser et al . , 2009 ; chiu et al . , 2009 ) , and the recently described protein absent in melanoma ( aim2 ) , which activates inflammasomes in an asc - dependentmanner ( burckstummer et al . , 2009 ; fernandes - alnemri et al . , 2009 ; legionella pneumophila , a gram - negative bacterial pathogen that evolved infecting unicellular protozoa in freshwater reservoirs , may not have encountered strong selective pressure to avoid recognition by mammalian prrs . consequently , l. pneumophila triggers multiple prr and has been a useful model for understanding the biology of prrs and the induction of appropriate adaptive immune responses against intracellular pathogens . the successful use of l. pneumophila as a tool for studying immunology has been reviewed elsewhere ( vance , 2010 ) . here , we will review the salient findings that have contributed to our understanding of the molecular mechanisms underlying innate immune cell recognition and response to l. pneumophila infection ( figure 1 ) . furthermore , we will sumarize studies that have elucidated the importance of these processes to the outcome of l. pneumophila infection . innate immune responses of a mammalian phagocyte infected with legionella pneumophila . a schematic representation of the pathways activated in a phagocyte after infection with l. pneumophila . the red boxes indicate l. pneumophila - associated molecular patterns important for the activation of pattern recognition receptors . blue boxes indicate molecules or processes involved in the cell - autonomous restriction of l. pneumophila replication . lcv , legionella - containing vacuole ; lpn , l. pneumophila ; dot / icm , type ivb secretion system ; il , interleukin ; il-18r , il-18 receptor ; tlr , toll - like receptor ; myd88 , myeloid differentiation primary response gene 88 ; nf-b , nuclear factor kappa b ; nod , nucleotide - binding oligomerization domain - containing protein ; rip2 , receptor interacting protein 2 ; pol iii , rna polymerase iii ; rig - i , retinoic - acid - inducible protein i ; ips - i , ifn- promoter stimulator 1 ( also known as mavs , mitochondrial antiviral signaling ) ; irf3 , interferon regulatory factor 3 ; naip5 , neuronal apoptosis inhibitory protein 5 ; nlrc4 , nlr family card domain - containing protein 4 . it was originally speculated that tlr4 , a general lps sensor , would recognize l. pneumophila . however , work on non - enterobacteriaceae species has indicated that although tlr4/md2 very efficiently recognizes enterobacterial lipid a , the same is not true for other gram - negative bacteria . these non - enterobacteriaceae bacterial species often express lipid a containing long fatty acid chains that either fail to trigger tlr4 activation or antagonize the tlr4 receptor ( zamboni et al . , 2004 ) . in l. pneumophila , studies performed with tlr4-deficient or tlr4 knockout mice have confirmed that this receptor does not effectively participate in the recognition of l. pneumophila lps . even at high mois , there is no difference in l. pneumophila infection between wild - type and c3h / hej mice , which are defective for tlr4 signaling due to a missense mutation in the tlr4 gene resulting in the replacement of a proline with a histidine at position 712 ( poltorak et al . , 1998 ; the initial studies on tlr4 function using c3h / hej mice were further corroborated in tlr4 mouse experiments , which supported the hypothesis that tlr4 deficiency does not influence the outcome of l. pneumophila infection ( akamine et al . , 2005 ; archer and roy , 2006 ; fuse et al . , 2007 ) . studies by girard et al . ( 2003 ) have shown that lipid a of l. pneumophila signals via tlr2 to induce the expression of cd14 . these findings led to the suggestion that l. pneumophila lps is recognized by tlr2 , but the mechanisms underlying the recognition of lipid a by tlr2 have not been completely elucidated ; some researchers have speculated that lipid a - mediated tlr2 activation requires either a long chain fatty acid or the presence of a substituent or a branch on the penultimate carbon of a fatty acid chain ( brandenburg et al . , 1993 ) . nonetheless , future studies using a synthetic form of l. pneumophila lipid a may be required to unequivocally confirm that l. pneumophila lps is a bona fide agonist of tlr2 . regardless of the proposed role of tlr2 in lps recognition , other l. pneumophila pamps , such as lipopeptides and lipoproteins , are sufficient to activate tlr2 . activation of this receptor is critical to the outcome of l. pneumophila infection in mice . this was unequivocally demonstrated by experiments using tlr2 mice , which show impaired cytokine production and are more susceptible to bacterial multiplication in the lungs ( akamine et al . , 2005 ; archer and roy , 2006 ; hawn et al . , in addition to tlr2 , other tlrs are also important for the host response to l. pneumophila . as a flagellated bacteria , l. pneumophila is recognized by tlr5 , and a common polymorphism in the ligand - binding domain of tlr5 causes increased susceptibility to legionnaires disease in humans ( hawn et al . , 2003 ) . these data have been corroborated by studies using tlr5 mice showing that tlr5 recognition of l. pneumophila in vivo contributes to the recruitment of leukocytes to the pulmonary cavity ( hawn et al . . however , tlr5 deficiency by itself does not render mice more susceptible to infection as measured by cfu counts and cytokine production ( hawn et al . mice lacking this receptor exhibit reduced levels of cytokines when challenged with l. pneumophila and are therefore more permissive of l. pneumophila replication in the lungs ( newton et al . , 2007 ; archer et al . , 2009 ) . this observation was corroborated by experiments involving the in vivo administration of cpg oligodeoxynucleotide , a synthetic agonist of tlr9 , which protected mice that were pre - infected with l. pneumophila ( bhan et al . , 2008 ) . importantly , these studies using mice deficient for a single tlr indicate that disruption of a single tlr gene does not result in a striking susceptibility to l. pneumophila ; this is possibly due to redundancy in the signaling pathways triggered by these receptors . in contrast , the deletion of the common adaptor protein myd88 , which is important for the signaling of several tlrs , produces mice that are highly susceptible to infection . mice deficient for myd88 show impaired cytokine production in response to pulmonary infection with l. pneumophila ; they also show high numbers of cfus in the lungs and succumb to l. pneumophila infection even at low multiplicities of infection ( neild et al . , 2006 ; sporri et al . , 2006 ; archer et al . , 2009 , 2010 ) . the increased susceptibility of myd88 mice suggests that the deletion of multiple tlr genes will produce mice as susceptible to l. pneumophila infection as those lacking myd88 . to test this hypothesis , archer et al . ( 2009 ) constructed mice deficient for multiple tlrs and showed that mice lacking both tlr5 and tlr9 or deficient for tlr2 and either tlr5 or tlr9 are still able to clear l. pneumophila infection . archer and colleagues elegantly concluded that il-18 signaling via myd88 is essential for nk cell production of ifn- , a cytokine critical for the restriction of l. pneumophila infection in vivo ( archer et al . , 2009 ) . interestingly , although the authors showed that nk cells signal via il-18 to produce ifn- , they also demonstrated that mice deficient for the il-18 receptor are no more susceptible to l. pneumophila infection than wild - type animals ( archer et al . , 2009 ) . additional studies will therefore be required to further determine the importance of this pathway in vivo and its redundancy with other pathways . the first study addressing nod1 and nod2 signaling in response to l. pneumophila infection was performed by shin et al . ( 2008 ) . in this study , the authors evaluated the transcriptional responses of macrophages infected with wild - type and dota mutants of l. pneumophila to identify macrophage genes induced in a dot / icm - dependent manner . by comparing macrophages deficient for myd88 and rip2 kinase , which impairs both nod1 and nod2 signaling , or lacking both myd88 and trif , the authors identified several genes that were regulated by a rip2-dependent pathway ( shin et al . , 2008 ) . importantly , this study revealed that multiple responses occur after l. pneumophila infection of macrophages ; some of these were dependent on myd88 , others on rip2 and some responses that were induced via unknown sensors were independent of both myd88 and rip2 ( shin et al . , 2008 ) . this study was further corroborated by in vivo experiments using mice deficient for both rip2 and myd88 . the rip2/myd88 mice were significantly more susceptible to l. pneumophila than the myd88 mice , and they succumbed to infection even at low mois ( archer et al . , 2010 ) . importantly , this and another study demonstrated that although a rip2-dependent response was not critical for restricting l. pneumophila infection in vivo , a rip2-dependent response did contribute to the recruitment of phagocytes to the sites of infection ( archer et al . , 2010 ; frutuoso et al . , 2010 ) notably , the rip2-dependent responses that contributed to the recruitment of neutrophils to the lungs of infected mice were at least partially dependent on the nod1 and nod2 receptors ( berrington et al . , 2010 ; frutuoso et al . , 2010 ) . these studies confirmed that nod1 and nod2 effectively participate in the pulmonary detection of l. pneumophila infection , but nod1 and nod2 deficiency results only in a minor attenuation of bacterial growth restriction in mouse lungs ( berrington et al . importantly , these studies of l. pneumophila infection in mice deficient for tlrs and the nod / rip2 pathway demonstrate the substantial redundancy of innate immune receptors in the host response to bacterial infection . approximately 30 years ago , it was demonstrated that macrophages from a / j mice fail to restrict the intracellular replication of l. pneumophila ( yamamoto et al . , 1988 ) . these phenotypic differences between a / j and other mouse strains provided a useful model for investigating the genes responsible for the phenotypic variations . in later years , the genomic region response for l. pneumophila resistance was mapped to the autosomal recessive locus lgn1 on chromosome 13 ( beckers et al . , 1995 ; dietrich et al . , 1995 ) . in early 2003 , it was finally revealed that the susceptibility gene within the lgn1 locus was naip5 ( also known as birc1e ) , a member of the nlr proteins family ( diez et al . the mechanisms by which naip5 contributes to the host control of infection was unraveled a few years later by the discovery that naip5 interfered with caspase-1 activation in response to macrophage infection by l. pneumophila ( zamboni et al . , 2006 ) . this response is dependent on the dot / icm system and effectively contributes to the restriction of bacterial replication in macrophages in vitro and in vivo ( zamboni et al . , the naip5-dependent restriction of l. pneumophila growth required another nlr protein called nlrc4 ( ipaf ) , which contributes for caspase-1 activation upon l. pneumophila infection ( amer et al . an elegant screening experiment identified the putative agonist of this naip5/nlrc4 inflammasome : l. pneumophila deficient for the flagellin gene flaa bypassed the nlrc4 inflammasome and replicated in macrophages harboring the restrictive lgn1 allele ( molofsky et al . , 2006 ; ren et al . although the role of naip5 in caspase-1 activation was questioned ( lamkanfi et al . , 2007 ) , assays with naip5 mice unequivocally demonstrated the requirement of naip5 for caspase-1 activation in response to l. pneumophila flagellin ( lightfield et al . , 2008 ) . furthermore , both nlrc4 and naip5 were required for the detection of a carboxy - terminal domain of flagellin , a region not required for tlr5 activation ( lightfield et al . , 2008 ) . the same group has recently demonstrated that the n - terminus of l. pneumophila flagellin relieves the requirement for naip5 during activation of the nlrc4 inflammasome , which suggests that naip5 regulates the specificity of the nlrc4 inflammasome for certain species of bacteria ( lightfield et al . , 2011 ) . these data explain why for some species , such as l. pneumophila , the activation of the nlrc4 inflammasome requires naip5 , whereas for other species , such as salmonella enterica serovar typhimurium , naip5 is dispensable . strikingly , activating this nlrc4/naip5 inflammasome requires a functional dot / icm type iv secretion system . this finding led to the speculation that flagellin may leak from the legionella cell to the macrophage cytoplasm through the dot / icm . activation of these receptors triggers a caspase-1-dependent pore formation in macrophage membranes and leads to a specific form of cell death called pyroptosis ( derre and isberg , 2004 ; case et al . , 2009 ; however , the activation of naip5 and nlrc4 also facilitates a process independent of pyroptosis that culminates with the restriction of l. pneumophila multiplication within the replicative vacuole occupied by the bacteria ( swanson and molofsky , 2005 ; amer et al . , 2006 ; fortier et al . , 2007 ) . . this hypothesis has been supported by the demonstration that naip5 recognition of l. pneumophila triggers irf1- and irf8-mediated upregulation of genes important for macrophage resistance to bacterial infection ( fortier et al . , 2009 ) . the inducible nitric oxide synthase ( nos2 ) gene is a possible candidate , as the naip5- and caspase-1-dependent induction of nos2 expression and nitric oxide production has been observed in macrophages transfected with flagellin ( buzzo et al . , 2010 ) . the adaptor protein called apoptosis - associated speck - like protein containing a caspase recruitment domain ( asc / pycard ) is a small molecule composed of a pyrin and a card domain . asc bridges caspase-1 to pyrin - containing molecules , such as nalp / nlrp family members , via card / card interactions ( mariathasan et al . , 2004 ) . studies performed with macrophages from asc - deficient mice have indicated that this molecule is important for the secretion of il-1 in response to infection but is not required for controlling l. pneumophila replication in c57bl/6 macrophages ( molofsky et al . , 2006 ; ren et al . , 2006 ; zamboni et al . , 2006 ) . this finding led to the proposition that asc may participate in the nlrc4-dependent activation of caspase-1 in response to l. pneumophila ( case et al . , 2009 ; pedra et al . , 2009 ) . however , subsequent studies demonstrated that l. pneumophila triggers at least two different inflammasomes : one dependent on nlrc4 , naip5 , and flagellin ; and another dependent on asc and independent of nlrp3 ( sutterwala et al . , 2006 ; case et al . , 2009 ) . although asc is dispensable for restricting l. pneumophila replication in mice and murine macrophages , a recent report demonstrated that asc does contribute to the control of l. pneumophila infection in human monocytes ( abdelaziz et al . , 2010 ) . the proteins participating in this asc - dependent inflammasome and the molecular signals that trigger its activation have not yet been elucidated . several studies have reported the production of type i ifn in response to l. pneumophila infection ( opitz et al . , 2006 ; chiu et al . , 2009 ; monroe et al . , 2009 ) , and these experiments have contributed to our understanding of the host cell pathways responsible for type i ifn induction . although rig - i and mda5 were initially reported as sensors of viral infection , recent studies indicate that rlrs are also important for the host cell recognition and response to bacterial infection , and both rig - i and mda5 have been implicated in type i ifn production by macrophages in response to l. pneumophila infection ( opitz et al . , 2006 ; stetson and medzhitov , 2006 ; ablasser et al . , 2009 ; monroe et al . , 2009 ) . the production of type i ifn in response to l. pneumophila infection was shown to be dependent of mavs and irf3 ( opitz et al . , 2006 ; chiu et al . , 2009 ; monroe et al . , 2009 ) . furthermore , this response accounted to bacterial growth restriction , as the addition of exogenous type i ifn to macrophages lead to the inhibition of l. pneumophila replication in non - permissive macrophages ( schiavoni et al . several groups have independently demonstrated that this irf3-dependent innate immune response does not require the flagellin / naip5/nlrc4 axis and occurs in both mice and human cells ( opitz et al . activation of the irf3 pathway was found to be dependent on a functional bacterial dot / icm and to require the presence of bacterial dna in the host cell cytoplasm ( stetson and medzhitov , 2006 ) . the current hypothesis is that l. pneumophila dna leaks into the host cell cytoplasm via the dot / icm . however , a recent report has demonstrated that l. pneumophila rna , but not dna , is responsible for rig - i - dependent response to l. pneumophila ( monroe et al . , 2009 ) . a subsequent study showed that the host protein rna polymerase iii , which converts poly ( da - dt ) dna into 5-ppp rna , is important for ifn induction through the rig - i pathway ( chiu et al . , 2009 ) . future studies are required to determine the ligand and receptor responsible for dna / rna recognition . collectively , the investigations of type i ifn in l. pneumophila infection indicate that type i ifn signaling effectively restricts l. pneumophila replication in phagocytes , thus confirming the importance of this pathway for macrophage resistance to l. pneumophila . conversely , the importance of type i ifn for murine resistance to l. pneumophila infection is less pronounced . work from independent groups has demonstrated that mice deficient for ifnar , which impairs the activation by both ifn- and ifn- , show no increased susceptibility to l. pneumophila infection ( monroe et al . , 2009 ; ang et al . , 2010 ) nevertheless , further investigation may be required to determine why type i ifn affects macrophage but not mouse resistance to l. pneumophila , and future studies are necessary to identify the ligand and receptors involved in the production of type i ifn in response to l. pneumophila infection . different families of prrs , including tlrs , nlrs , and rlrs , effectively recognize l. pneumophila . this recognition leads to several events important for the outcome of infection : ( 1 ) phagocytes activate cell - autonomous mechanisms to restrict bacterial replication ; ( 2 ) phagocytes trigger the expression of hundreds of inflammatory genes , including those for cytokines and chemokines ; ( 3 ) phagocytes then express stimulatory and co - stimulatory molecules important for antigen presentation ; ( 4 ) the secreted cytokines and chemokines recruit additional cells to the sites of the infection ; ( 5 ) antigen presentation will proceed and the immune system may generate specific acquired responses that are highly protective against reinfection . the continued use of l. pneumophila to dissect these processes will aid us in understanding how the immune system fights to prevent legionnaire 's disease and will continue to provide important insight into the biological functions of the innate immune responses . 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 .

innate immune cells , such as macrophages , are highly adapted to rapidly recognize infections by distinct pathogens , including viruses , bacteria , fungi , and protozoa . this recognition is mediated by pattern recognition receptors ( prrs ) , which are found in host cell surface membranes and the host cell cytoplasm . prrs include protein families such as the toll - like receptors , nod - like receptors , rig - i - like receptors , and sensors of cytosolic dna . the activation of these prrs by pathogen - associated molecular patterns leads to transcriptional responses and specific forms of cell death . these processes effectively contribute to host resistance to infection either via cell - autonomous processes that lead to the intracellular restriction of microbial replication and/or by activating pathogen - specific adaptive immune responses . legionella pneumophila , the causative agent of legionnaires disease , is a gram - negative bacterium that triggers responses by multiple prrs . here , we review a set of studies that have contributed to our specific understanding of the molecular mechanisms by which innate immune cells recognize and respond to l. pneumophila and the importance of these processes to the outcome of infection .

Introduction Toll-Like Receptors NOD-Like Receptors: NOD1 and NOD2 NOD-Like Receptors: NLRC4 and NAIP5 ASC and the NLRP3-Independent Inflammasome Rig-I-Like Receptor and Induction of Type I Interferon Concluding Remarks Conflict of Interest Statement

this process relies mostly on the recognition of pathogen - associated molecular patterns ( pamps ) by pattern recognition receptors ( prrs ) . among the canonical pamps are molecules such as lipopolysaccharide , peptidoglycan , bacterial lipoproteins , flagellin , and nucleic acids derived from viruses , bacteria , fungi , and protozoa ( janeway and medzhitov , 2002 ; akira et al . upon direct or indirect ligand recognition , toll - like receptors ( tlrs ) dimerize and trigger a signaling cascade leading to the activation of proinflammatory responses ( uematsu and akira , 2006 ) . in addition to tlrs , other prr families have already been described ; these include dna / rna - sensing proteins such as the rig - i - like receptor ( rlr ) family and sensors of membrane damage and intracellular pamps such as the nod - like receptors ( nlrs ; creagh and o'neill , 2006 ) . the rna helicase domain - containing proteins retinoic acid - inducible gene i ( rig - i ) and melanoma differentiation - associated gene 5 ( mda5 ) comprise a group of cytoplasmic receptors important for the recognition of viral nucleic acids . pamp recognition by rig - i and mda5 triggers the activation of irf3 via mavs ( ips-1 ) , culminating in the production of type i interferons ( ifn ) . in addition , some studies have shown that the recognition of dna by rig - i / mda5 is dependent on cytosolic rna polymerase iii ( pol iii ; ablasser et al . the first sub - group is composed of receptors that trigger intracellular signaling pathways leading to the activation of transcriptional factors mediating the expression of inflammatory response genes . both nod1 and nod2 are members of this first group , and they signal via rip2 , a kinase that ubiquitinates nemo to induce the activation of nf-b and mapk ( shaw et al . among these proteins are naip5 ( birc1e ) and nlrc4 ( ipaf ) , which have been suggested to assembly a unique inflammasome ( hereafter referred to as the nlrc4 inflammasome ) . activation of this inflammasome triggers a specific form of host cell death called pyroptosis ( lightfield et al . among the nlrs that trigger the asc - dependent inflammasome is nalp3 , which has been extensively characterized and shown to be important for the recognition of danger - associated molecular patterns ( damps ) reviewed by schroder and tschopp ( 2010 ) . , 2007 ) , rna polymerase iii , which induces type i ifn production through the rig - i pathway ( ablasser et al . , 2009 ) , and the recently described protein absent in melanoma ( aim2 ) , which activates inflammasomes in an asc - dependentmanner ( burckstummer et al . , 2009 ; legionella pneumophila , a gram - negative bacterial pathogen that evolved infecting unicellular protozoa in freshwater reservoirs , may not have encountered strong selective pressure to avoid recognition by mammalian prrs . consequently , l. pneumophila triggers multiple prr and has been a useful model for understanding the biology of prrs and the induction of appropriate adaptive immune responses against intracellular pathogens . here , we will review the salient findings that have contributed to our understanding of the molecular mechanisms underlying innate immune cell recognition and response to l. pneumophila infection ( figure 1 ) . furthermore , we will sumarize studies that have elucidated the importance of these processes to the outcome of l. pneumophila infection . innate immune responses of a mammalian phagocyte infected with legionella pneumophila . a schematic representation of the pathways activated in a phagocyte after infection with l. pneumophila . the red boxes indicate l. pneumophila - associated molecular patterns important for the activation of pattern recognition receptors . blue boxes indicate molecules or processes involved in the cell - autonomous restriction of l. pneumophila replication . lcv , legionella - containing vacuole ; lpn , l. pneumophila ; dot / icm , type ivb secretion system ; il , interleukin ; il-18r , il-18 receptor ; tlr , toll - like receptor ; myd88 , myeloid differentiation primary response gene 88 ; nf-b , nuclear factor kappa b ; nod , nucleotide - binding oligomerization domain - containing protein ; rip2 , receptor interacting protein 2 ; pol iii , rna polymerase iii ; rig - i , retinoic - acid - inducible protein i ; ips - i , ifn- promoter stimulator 1 ( also known as mavs , mitochondrial antiviral signaling ) ; irf3 , interferon regulatory factor 3 ; naip5 , neuronal apoptosis inhibitory protein 5 ; nlrc4 , nlr family card domain - containing protein 4 . however , work on non - enterobacteriaceae species has indicated that although tlr4/md2 very efficiently recognizes enterobacterial lipid a , the same is not true for other gram - negative bacteria . in l. pneumophila , studies performed with tlr4-deficient or tlr4 knockout mice have confirmed that this receptor does not effectively participate in the recognition of l. pneumophila lps . even at high mois , there is no difference in l. pneumophila infection between wild - type and c3h / hej mice , which are defective for tlr4 signaling due to a missense mutation in the tlr4 gene resulting in the replacement of a proline with a histidine at position 712 ( poltorak et al . , 1998 ; the initial studies on tlr4 function using c3h / hej mice were further corroborated in tlr4 mouse experiments , which supported the hypothesis that tlr4 deficiency does not influence the outcome of l. pneumophila infection ( akamine et al . these findings led to the suggestion that l. pneumophila lps is recognized by tlr2 , but the mechanisms underlying the recognition of lipid a by tlr2 have not been completely elucidated ; some researchers have speculated that lipid a - mediated tlr2 activation requires either a long chain fatty acid or the presence of a substituent or a branch on the penultimate carbon of a fatty acid chain ( brandenburg et al . nonetheless , future studies using a synthetic form of l. pneumophila lipid a may be required to unequivocally confirm that l. pneumophila lps is a bona fide agonist of tlr2 . regardless of the proposed role of tlr2 in lps recognition , other l. pneumophila pamps , such as lipopeptides and lipoproteins , are sufficient to activate tlr2 . activation of this receptor is critical to the outcome of l. pneumophila infection in mice . , in addition to tlr2 , other tlrs are also important for the host response to l. pneumophila . as a flagellated bacteria , l. pneumophila is recognized by tlr5 , and a common polymorphism in the ligand - binding domain of tlr5 causes increased susceptibility to legionnaires disease in humans ( hawn et al . these data have been corroborated by studies using tlr5 mice showing that tlr5 recognition of l. pneumophila in vivo contributes to the recruitment of leukocytes to the pulmonary cavity ( hawn et al . mice lacking this receptor exhibit reduced levels of cytokines when challenged with l. pneumophila and are therefore more permissive of l. pneumophila replication in the lungs ( newton et al . this observation was corroborated by experiments involving the in vivo administration of cpg oligodeoxynucleotide , a synthetic agonist of tlr9 , which protected mice that were pre - infected with l. pneumophila ( bhan et al . importantly , these studies using mice deficient for a single tlr indicate that disruption of a single tlr gene does not result in a striking susceptibility to l. pneumophila ; this is possibly due to redundancy in the signaling pathways triggered by these receptors . in contrast , the deletion of the common adaptor protein myd88 , which is important for the signaling of several tlrs , produces mice that are highly susceptible to infection . mice deficient for myd88 show impaired cytokine production in response to pulmonary infection with l. pneumophila ; they also show high numbers of cfus in the lungs and succumb to l. pneumophila infection even at low multiplicities of infection ( neild et al . the increased susceptibility of myd88 mice suggests that the deletion of multiple tlr genes will produce mice as susceptible to l. pneumophila infection as those lacking myd88 . archer and colleagues elegantly concluded that il-18 signaling via myd88 is essential for nk cell production of ifn- , a cytokine critical for the restriction of l. pneumophila infection in vivo ( archer et al . interestingly , although the authors showed that nk cells signal via il-18 to produce ifn- , they also demonstrated that mice deficient for the il-18 receptor are no more susceptible to l. pneumophila infection than wild - type animals ( archer et al . additional studies will therefore be required to further determine the importance of this pathway in vivo and its redundancy with other pathways . the first study addressing nod1 and nod2 signaling in response to l. pneumophila infection was performed by shin et al . in this study , the authors evaluated the transcriptional responses of macrophages infected with wild - type and dota mutants of l. pneumophila to identify macrophage genes induced in a dot / icm - dependent manner . by comparing macrophages deficient for myd88 and rip2 kinase , which impairs both nod1 and nod2 signaling , or lacking both myd88 and trif , the authors identified several genes that were regulated by a rip2-dependent pathway ( shin et al . importantly , this study revealed that multiple responses occur after l. pneumophila infection of macrophages ; some of these were dependent on myd88 , others on rip2 and some responses that were induced via unknown sensors were independent of both myd88 and rip2 ( shin et al . the rip2/myd88 mice were significantly more susceptible to l. pneumophila than the myd88 mice , and they succumbed to infection even at low mois ( archer et al . importantly , this and another study demonstrated that although a rip2-dependent response was not critical for restricting l. pneumophila infection in vivo , a rip2-dependent response did contribute to the recruitment of phagocytes to the sites of infection ( archer et al . , 2010 ) notably , the rip2-dependent responses that contributed to the recruitment of neutrophils to the lungs of infected mice were at least partially dependent on the nod1 and nod2 receptors ( berrington et al . importantly , these studies of l. pneumophila infection in mice deficient for tlrs and the nod / rip2 pathway demonstrate the substantial redundancy of innate immune receptors in the host response to bacterial infection . approximately 30 years ago , it was demonstrated that macrophages from a / j mice fail to restrict the intracellular replication of l. pneumophila ( yamamoto et al . in later years , the genomic region response for l. pneumophila resistance was mapped to the autosomal recessive locus lgn1 on chromosome 13 ( beckers et al . in early 2003 , it was finally revealed that the susceptibility gene within the lgn1 locus was naip5 ( also known as birc1e ) , a member of the nlr proteins family ( diez et al . the mechanisms by which naip5 contributes to the host control of infection was unraveled a few years later by the discovery that naip5 interfered with caspase-1 activation in response to macrophage infection by l. pneumophila ( zamboni et al . this response is dependent on the dot / icm system and effectively contributes to the restriction of bacterial replication in macrophages in vitro and in vivo ( zamboni et al . , the naip5-dependent restriction of l. pneumophila growth required another nlr protein called nlrc4 ( ipaf ) , which contributes for caspase-1 activation upon l. pneumophila infection ( amer et al . , 2007 ) , assays with naip5 mice unequivocally demonstrated the requirement of naip5 for caspase-1 activation in response to l. pneumophila flagellin ( lightfield et al . the same group has recently demonstrated that the n - terminus of l. pneumophila flagellin relieves the requirement for naip5 during activation of the nlrc4 inflammasome , which suggests that naip5 regulates the specificity of the nlrc4 inflammasome for certain species of bacteria ( lightfield et al . these data explain why for some species , such as l. pneumophila , the activation of the nlrc4 inflammasome requires naip5 , whereas for other species , such as salmonella enterica serovar typhimurium , naip5 is dispensable . activation of these receptors triggers a caspase-1-dependent pore formation in macrophage membranes and leads to a specific form of cell death called pyroptosis ( derre and isberg , 2004 ; case et al . , 2009 ; however , the activation of naip5 and nlrc4 also facilitates a process independent of pyroptosis that culminates with the restriction of l. pneumophila multiplication within the replicative vacuole occupied by the bacteria ( swanson and molofsky , 2005 ; amer et al . this hypothesis has been supported by the demonstration that naip5 recognition of l. pneumophila triggers irf1- and irf8-mediated upregulation of genes important for macrophage resistance to bacterial infection ( fortier et al . the inducible nitric oxide synthase ( nos2 ) gene is a possible candidate , as the naip5- and caspase-1-dependent induction of nos2 expression and nitric oxide production has been observed in macrophages transfected with flagellin ( buzzo et al . the adaptor protein called apoptosis - associated speck - like protein containing a caspase recruitment domain ( asc / pycard ) is a small molecule composed of a pyrin and a card domain . asc bridges caspase-1 to pyrin - containing molecules , such as nalp / nlrp family members , via card / card interactions ( mariathasan et al . studies performed with macrophages from asc - deficient mice have indicated that this molecule is important for the secretion of il-1 in response to infection but is not required for controlling l. pneumophila replication in c57bl/6 macrophages ( molofsky et al . this finding led to the proposition that asc may participate in the nlrc4-dependent activation of caspase-1 in response to l. pneumophila ( case et al . however , subsequent studies demonstrated that l. pneumophila triggers at least two different inflammasomes : one dependent on nlrc4 , naip5 , and flagellin ; and another dependent on asc and independent of nlrp3 ( sutterwala et al . although asc is dispensable for restricting l. pneumophila replication in mice and murine macrophages , a recent report demonstrated that asc does contribute to the control of l. pneumophila infection in human monocytes ( abdelaziz et al . the proteins participating in this asc - dependent inflammasome and the molecular signals that trigger its activation have not yet been elucidated . several studies have reported the production of type i ifn in response to l. pneumophila infection ( opitz et al . , 2009 ) , and these experiments have contributed to our understanding of the host cell pathways responsible for type i ifn induction . although rig - i and mda5 were initially reported as sensors of viral infection , recent studies indicate that rlrs are also important for the host cell recognition and response to bacterial infection , and both rig - i and mda5 have been implicated in type i ifn production by macrophages in response to l. pneumophila infection ( opitz et al . furthermore , this response accounted to bacterial growth restriction , as the addition of exogenous type i ifn to macrophages lead to the inhibition of l. pneumophila replication in non - permissive macrophages ( schiavoni et al . activation of the irf3 pathway was found to be dependent on a functional bacterial dot / icm and to require the presence of bacterial dna in the host cell cytoplasm ( stetson and medzhitov , 2006 ) . the current hypothesis is that l. pneumophila dna leaks into the host cell cytoplasm via the dot / icm . however , a recent report has demonstrated that l. pneumophila rna , but not dna , is responsible for rig - i - dependent response to l. pneumophila ( monroe et al . a subsequent study showed that the host protein rna polymerase iii , which converts poly ( da - dt ) dna into 5-ppp rna , is important for ifn induction through the rig - i pathway ( chiu et al . collectively , the investigations of type i ifn in l. pneumophila infection indicate that type i ifn signaling effectively restricts l. pneumophila replication in phagocytes , thus confirming the importance of this pathway for macrophage resistance to l. pneumophila . conversely , the importance of type i ifn for murine resistance to l. pneumophila infection is less pronounced . work from independent groups has demonstrated that mice deficient for ifnar , which impairs the activation by both ifn- and ifn- , show no increased susceptibility to l. pneumophila infection ( monroe et al . , 2010 ) nevertheless , further investigation may be required to determine why type i ifn affects macrophage but not mouse resistance to l. pneumophila , and future studies are necessary to identify the ligand and receptors involved in the production of type i ifn in response to l. pneumophila infection . different families of prrs , including tlrs , nlrs , and rlrs , effectively recognize l. pneumophila . this recognition leads to several events important for the outcome of infection : ( 1 ) phagocytes activate cell - autonomous mechanisms to restrict bacterial replication ; ( 2 ) phagocytes trigger the expression of hundreds of inflammatory genes , including those for cytokines and chemokines ; ( 3 ) phagocytes then express stimulatory and co - stimulatory molecules important for antigen presentation ; ( 4 ) the secreted cytokines and chemokines recruit additional cells to the sites of the infection ; ( 5 ) antigen presentation will proceed and the immune system may generate specific acquired responses that are highly protective against reinfection . the continued use of l. pneumophila to dissect these processes will aid us in understanding how the immune system fights to prevent legionnaire 's disease and will continue to provide important insight into the biological functions of the innate immune responses .

a fractional programming problem arises in many types of optimization problems such as portfolio selection , production , information theory , and numerous decision making problems in management science . more specifically , it can be used in engineering and economics to minimize a ratio of physical or economical function or both , such as cost / time , cost / volume , and cost / benefit , in order to measure the efficiency or productivity of the system . many economic , noneconomic , and indirect applications of fractional programming problem have also been given by bector , bector and chandra , craven , mond and weir , stancu - minasian , schaible and ibaraki , ahmad et al . , ahmad and sharma , and gulati et al . . the central concept in optimization is known as the duality theory which asserts that , given a ( primal ) minimization problem , the infimum value of the primal problem can not be smaller than the supermom value of the associated ( dual ) maximization problem and the optimal values of primal and dual problems are equal . duality in fractional programming is an important class of duality theory and several contributions have been made in the past [ 1 , 5 , 8 , 1014 ] . second order duality provides a tighter bound for the value of the objective function when approximations are used . for more details , one can consult [ 15 , page 93 ] . another advantage of second order duality when applicable is that if a feasible point in the primal is given and first order duality does not apply , then we can use second order duality to provide a lower bound of the value of the primal problem ( see ) . derived fritz john and karush - kuhn tucker necessary and sufficient optimality condition for a class of nondifferentiable convex multiobjective fractional programming problems and established some duality theorems . liang et al . [ 17 , 18 ] discussed the optimality condition and duality for nonlinear fractional programming . santos et al . discussed the optimality and duality for nonsmooth multiobjective fractional programming with generalized convexity . and xu gave a mixed type duality for fractional programming , established some sufficient conditions , and obtained various duality results between the mixed dual and primal problem . zhou and wang introduced a class of mixed type dual for nonsmooth multiobjective fractional programming and established the duality results under ( v , ) invexity assumption . duality for various forms of mathematical problems involving square roots of positive semidefinite quadratic forms has been discussed by many authors [ 10 , 2325 ] . mond considered a nonlinear fractional programming problem involving square roots of positive semidefinite quadratic form in the numerator and denominator and proved the necessary and sufficient condition for optimality . kim et al . [ 26 , 27 ] formulated a nondifferentiable multiobjective fractional problem in which numerators contain support function . one of the most known approaches used for solving nonlinear fractional programming problem is called parametric approach . dinklebaeh and jagannathan introduced this approach that was used later by osuna - gomez et al . to characterize solution of a multiobjective fractional problem under generalized convexity . tripathy introduced three approaches given by dinklebaeh and jagannathan for both primal and mixed type dual of a nondifferentiable multiobjective fractional programming and established the duality results under generalized -univexity . to relax convexity assumption imposed on the function in theorems on optimality and duality , motivated by the earlier authors in this paper , we have introduced three approaches given by dinklebaeh and jagannathan for both primal and second order mixed type dual of a nondifferentiable multiobjective fractional programming problem in which the numerator and denominator of objective function contain square root of positive semidefinite quadratic form . also we have established the necessary and sufficient optimality condition and used a parameterization technique to establish duality results under generalized -univexity assumption . = ( x1 , x2 , , xn ) , y = ( y1 , y2 , , yn ) , we denote the following.(i)x > yxi > yi , for all i = 1,2 , , n.(ii)x yxi yi , for all i = 1,2 , , n.throughout the paper , let x be a nonempty open subset of . x > yxi > yi , for all i = 1,2 , , n. x yxi yi , for all i = 1,2 , , n. consider the following nondifferentiable multiobjective fractional programming problem . minimize ( 1)f(x)+(xtbx)1/2g(x)(xtcx)1/2=(k1(x),k2(x), ,kk(x ) ) , where ( 2)ki(x)=fi(x)+(xtbix)1/2gi(x)(xtcix)1/2 , i=1,2, minimize ( 3)f(x)=(f1(x),f2(x), ,fk(x ) ) , where ( 4)fi(x)=fi(x)+(xtbix)1/2i{gi(x)(xtcix)1/2},hhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhi=1,2, ,k ; i are fixed parameters.(iii ) mfp. minimize f(x ) ; is k - dimensional strictly positive vector , all subject to same constraint ( 5)h(x)0 , xxrn , where fi : , gi : , i = 1,2 , , k and h = ( h1 , , hm ) ; hj : , j = 1,2 , , , k are positive semidefinite matrices of order n. in the sequel , we assume that fi(x ) 0 and gi(x ) > 0 on for i = 1,2 , , k. mfp0 . minimize ( 1)f(x)+(xtbx)1/2g(x)(xtcx)1/2=(k1(x),k2(x), ,kk(x ) ) , where ( 2)ki(x)=fi(x)+(xtbix)1/2gi(x)(xtcix)1/2 , i=1,2, ,k . minimize ( 3)f(x)=(f1(x),f2(x), ,fk(x ) ) , where ( 4)fi(x)=fi(x)+(xtbix)1/2i{gi(x)(xtcix)1/2},hhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhi=1,2, ,k ; i are fixed parameters . mfp. minimize f(x ) ; is k - dimensional strictly positive vector , all subject to same constraint let x0 = { x x : hj(x ) 0 , j = 1,2 , , m } for all feasible solutions of mfp0 , mfp1 , and mfp and denote i = { 1,2 , 3 , , k } , m = { 1,2 , 3 , , m } , j1 = { j m : hj(x ) = 0 } , and j2 = { j m : hj(x ) < 0}. it is obvious that j1 j2 = m. throughout the paper , consider fi : x , : x x , p , . assume that : satisfying a 0(a ) 0 or (a ) 0a 0 and (a ) = (a ) , k : x x + . for x , x-x the real differentiable function fi is said to be second order -univex at x-x with respect to , , and k , if ( 6)k(x , x)[fi(x)fi(x)+12pt(2fi(x)p ) ] (x , x)t[fi(x)+2fi(x)p]+||xx||2,ssssssssssssssssssssssssssssssssssssssssssssxx . the real differentiable function fi is said to be second order -univex at x-x with respect to , , and k , if ( 6)k(x , x)[fi(x)fi(x)+12pt(2fi(x)p ) ] (x , x)t[fi(x)+2fi(x)p]+||xx||2,ssssssssssssssssssssssssssssssssssssssssssssxx . the real differentiable function fi is said to be second order -pseudounivex at x-x with respect to , , and k , if ( 7)(x , x)t[fi(x)+2fi(x)p]+||xx||20 k(x , x)[fi(x)fi(x)+12pt(2fi(x)p)]0,sssssssssssssssssssssssssssssssssssssssssssssssssssssssxx . the real differentiable function fi is said to be second order -pseudounivex at x-x with respect to , , and k , if ( 7)(x , x)t[fi(x)+2fi(x)p]+||xx||20 k(x , x)[fi(x)fi(x)+12pt(2fi(x)p)]0,sssssssssssssssssssssssssssssssssssssssssssssssssssssssxx . definition 3 . the real differentiable function fi is said to be second order -quasiunivex at x-x with respect to , , and k , if ( 8)k(x , x)[fi(x)fi(x)+12pt(2fi(x)p)]0 (x , x)t[fi(x)+2fi(x)p]+||xx||20,sssssssssssssssssssssssssssssssssssssssssssssssssssxx . the real differentiable function fi is said to be second order -quasiunivex at x-x with respect to , , and k , if ( 8)k(x , x)[fi(x)fi(x)+12pt(2fi(x)p)]0 (x , x)t[fi(x)+2fi(x)p]+||xx||20,sssssssssssssssssssssssssssssssssssssssssssssssssssxx . remark 4 . if p = 0 , the above definitions reduce to the definitions of -univex , -pseudounivex , and -quasiunivex as introduced in . if p = 0 , the above definitions reduce to the definitions of -univex , -pseudounivex , and -quasiunivex as introduced in . definition 5 . a feasible point x- is said to be efficient for mfp1 , if there exists no other feasible point x in mfp1 such that fi(x)fi(x- ) , i = 1,2 , , k , and fr(x)<fr(x- ) for some r { 1,2 , , k}. a feasible point x- is said to be efficient for mfp1 , if there exists no other feasible point x in mfp1 such that fi(x)fi(x- ) , i = 1,2 , , k , and fr(x)<fr(x- ) for some r { 1,2 , , k}. definition 6 ( see ) . a feasible point x- is said to be properly efficient for mfp1 , if it is efficient and there exist m > 0 such that , for each i { 1,2 , , k } and for all feasible point x in mfp1 satisfying fi(x)<fi(x- ) , we have fi(x-)-fi(x)m(fr(x)-fr(x- ) ) for some r { 1,2 , a feasible point x- is said to be properly efficient for mfp1 , if it is efficient and there exist m > 0 such that , for each i { 1,2 , , k } and for all feasible point x in mfp1 satisfying fi(x)<fi(x- ) , we have fi(x-)-fi(x)m(fr(x)-fr(x- ) ) for some r { 1,2 , , k } such that fr(x)>fr(x- ) . we assume that fi(x)+(xbix ) 0 , gi(x)(xcix ) > 0 , i = 1,2 , , k for all x x. definition 7 ( generalized schwarz inequality ) . let b be a positive semidefinite matrix of order n. then , for all x , w , xbw ( xbx)(wbw ) . let b be a positive semidefinite matrix of order n. then , for all x , w , xbw ( xbx)(wbw ) . m } : hj(x ) = 0 } and v - i=(fi(x-)+(xtbix)1/2)/(gi(x-)-(xtcix)1/2 ) . then define the set w(x-)={wn : wthj(x-)0,jj(x- ) } satisfying any one of the following conditions:(a)x - tbix->0 , x - tcix-=0wt(fi(x-)+bix-/(x - tbix-)1/2-v - igi(x-))+(wt(v - i2ci)w)1/20 , ww(x- ) , i = 1,2 , , k;(b)x - tbix-=0 , x - tcix->0wt(fi(x-)-v - i{gi(x-)-cix-/(x - tcix-)1/2})+(wtbiw)1/20 , ww(x- ) , i = 1,2 , , k;(c)x - tbix-=0 , x - tcix-=0wt(fi(x-)-v - igi(x-))+(wtbiw)1/2+(wt(v - i2ci)w)1/20 , ww(x- ) , i = 1,2 , , k;(d)x - tbix->0 , x - tcix->0wt(fi(x-)+bix-/(x - tbix-)1/2-v - i{gi(x-)-cix-/(x - tcix-)1/2})0 , ww(x- ) , i = 1,2 , , k. x - tbix->0 , x - tcix-=0wt(fi(x-)+bix-/(x - tbix-)1/2-v - igi(x-))+(wt(v - i2ci)w)1/20 , ww(x- ) , i = 1,2 , , k ; x - tbix-=0 , x - tcix->0wt(fi(x-)-v - i{gi(x-)-cix-/(x - tcix-)1/2})+(wtbiw)1/20 , ww(x- ) , i = 1,2 , , k ; x - tbix-=0 , x - tcix-=0wt(fi(x-)-v - igi(x-))+(wtbiw)1/2+(wt(v - i2ci)w)1/20 , ww(x- ) , i = 1,2 , , k ; x - tbix->0 , x - tcix->0wt(fi(x-)+bix-/(x - tbix-)1/2-v - i{gi(x-)-cix-/(x - tcix-)1/2})0 , ww(x- ) , i = 1,2 , if x x0 is an optimal solution of mfp , then x is properly efficient for mfp1 . if x x0 is an optimal solution of mfp , then x is properly efficient for mfp1 . x x0 is an efficient solution for mfp0 if and only if it is an efficient solution of mfp1 with f(x ) = 0 . x x0 is an efficient solution for mfp0 if and only if it is an efficient solution of mfp1 with f(x ) = 0 . lemma 10 ( see necessary optimality condition ) . if x-x0 is an optimal solution of ( mfp ) such that w(x-)= , then there exist vi + , w , z , and y such that ( 9)f(x)+yth(x ) = i=1ki[fi(x)+biwvi{gi(x)ciz } ] + j=1myjhj(x)=0,(10)fi(x)=fi(x)+(xtbix)1/2vi(gi(x)(xtcix)1/2)=0,hhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhi=1,2, ,k.(11)yth(x)=0,(12)wtbiw1 , ztciz1 , i=1,2, ,k,(13)(xtbix)1/2=xtbiw , ( xtcix)1/2=xtciz , hhhhhhhhhhhhhhhhhhhhhhhhi=1,2, ,k,(14)y0,(15)vi0 , i=1,2, ,k . if x-x0 is an optimal solution of ( mfp ) such that w(x-)= , then there exist vi + , w , z , and y such that ( 9)f(x)+yth(x ) = i=1ki[fi(x)+biwvi{gi(x)ciz } ] + j=1myjhj(x)=0,(10)fi(x)=fi(x)+(xtbix)1/2vi(gi(x)(xtcix)1/2)=0,hhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhi=1,2, ,k.(11)yth(x)=0,(12)wtbiw1 , ztciz1 , i=1,2, ,k,(13)(xtbix)1/2=xtbiw , ( xtcix)1/2=xtciz , hhhhhhhhhhhhhhhhhhhhhhhhi=1,2, let x-x0 be a feasible solution of mfp1 and there exist i + ; w , z , vi + , and y satisfying the condition in lemma 10 at x- . furthermore suppose that the following conditions hold.(i)p(x ) = i=1i[fi(x ) + ( xbix ) vi{gi(x ) ( xcix ) } ] + j=1yjhj(x ) is second order -pseudounivex with respect to , , and k at x-x0 , with ( p(x))p = 0 , where fi : x , gi : x , hj : x , i = 1,2 , m , p , : x x , k : x x + , and : satisfying (a ) 0a 0.(ii) 0 . let x-x0 be a feasible solution of mfp1 and there exist i + ; w , z , vi + , and y satisfying the condition in lemma 10 at x- . furthermore suppose that the following conditions hold.(i)p(x ) = i=1i[fi(x ) + ( xbix ) vi{gi(x ) ( xcix ) } ] + j=1yjhj(x ) is second order -pseudounivex with respect to , , and k at x-x0 , with ( p(x))p = 0 , where fi : x , gi : x , hj : x , i = 1,2 , , k ; j = 1,2 , , m , p , : x x , k : x x + , and : satisfying (a ) 0a 0.(ii) 0 . p(x ) = i=1i[fi(x ) + ( xbix ) vi{gi(x ) ( xcix ) } ] + j=1yjhj(x ) is second order -pseudounivex with respect to , , and k at x-x0 , with ( p(x))p = 0 , where fi : x , gi : x , hj : x , i = 1,2 , , k ; j = 1,2 , , m , p , : x x , k : x x + , and : satisfying (a ) 0a 0 . proofsuppose that the hypothesis holds.since the conditions of lemma 10 are satisfied , from ( 9 ) and ( 13 ) , we have ( 16)p(x ) = (j=1myjhj(x)i=1ki[fi(x)+(xtbix)1/2ssssssssssssvi{gi(x)(xtcix)1/2}]+j=1myjhj(x ) ) = (j=1myjhj(x)i=1ki[fi(x)+xtbiwvi{gi(x)xtciz}]sssssssssss+j=1myjhj(x))=0 . also from hypothesis ( i ) , we have 2p(x-)p=0so we can write p(x-)+2p(x-)p=0.now for (x , x-)n , we can write (x , x-)t(p(x-)+2p(x-)p)=0.for 0 , we have (x , x-)t(p(x-)+2p(x-)p)+||x - x-||20.since p(x ) is second order -pseudounivex with respect to , , and k at x-x0 , we have k(x , x-){p(x)-p(x-)+(1/2)pt(2p(x-))p}0 , and using the properties of k , , it gives ( 17)p(x)p(x)+12pt2p(x)p 0p(x)p(x)12pt2p(x)p . suppose that x- is not efficient solution of mfp1 ; then there exist x-x0 such that ( 19)fi(x)+(xtbix)1/2vi{gi(x)(xtcix)1/2 } fi(x)+(xtbix)1/2vi{gi(x)(xtcix)1/2},ssssssssssssssssssssssssssssssssssssssssi=1,2, ,k , and ft(x)+(xtbtx)1/2-vt{gt(x)-(xtctx)1/2}ft(x-)+(x - tbtx-)1/2-vt{gt(x-)-(xtctx)1/2 } , for some t { 1,2 , , k}.the above relation , together with the relation i > 0 , implies that ( 20)i=1ki[fi(x)+(xtbix)1/2vi{gi(x)(xtcix)1/2 } ] < i=1ki[fi(x)+(xtbix)1/2vi{gi(x)(xtcix)1/2 } ] . from the relations ( 5 ) , ( 11 ) , and ( 14 ) , we get ( 21)j=1myjhj(x)j=1myjhj(x ) . consequently ( 20 ) and ( 21 ) yield ( 22)i=1ki[fi(x)+(xtbix)1/2vi{gi(x)(xtcix)1/2 } ] + j=1myjhj(x ) < i=1ki[fi(x)+(xtbix)1/2vi{gi(x)(xtcix)1/2 } ] + j=1myjhj(x ) . since the conditions of lemma 10 are satisfied , from ( 9 ) and ( 13 ) , we have ( 16)p(x ) = (j=1myjhj(x)i=1ki[fi(x)+(xtbix)1/2ssssssssssssvi{gi(x)(xtcix)1/2}]+j=1myjhj(x ) ) = (j=1myjhj(x)i=1ki[fi(x)+xtbiwvi{gi(x)xtciz}]sssssssssss+j=1myjhj(x))=0 . also from hypothesis ( i ) , we have 2p(x-)p=0 so we can write p(x-)+2p(x-)p=0 . now for (x , x-)n , we can write (x , x-)t(p(x-)+2p(x-)p)=0 . for 0 , we have (x , x-)t(p(x-)+2p(x-)p)+||x - x-||20 . since p(x ) is second order -pseudounivex with respect to , , and k at x-x0 , we have k(x , x-){p(x)-p(x-)+(1/2)pt(2p(x-))p}0 , and using the properties of k , , it gives ( 17)p(x)p(x)+12pt2p(x)p 0p(x)p(x)12pt2p(x)p . since 2p(x-)p=0 , the above inequality implies p(x)p(x-)(18)i=1ki[fi(x)+(xtbix)1/2sssssssssssvi{gi(x)(xtcix)1/2}]+j=1myjhj(x)i=1ki[fi(x)+(xtbix)1/2sssssssssssvi{gi(x)(xtcix)1/2}]+j=1myjhj(x ) . suppose that x- is not efficient solution of mfp1 ; then there exist x-x0 such that ( 19)fi(x)+(xtbix)1/2vi{gi(x)(xtcix)1/2 } fi(x)+(xtbix)1/2vi{gi(x)(xtcix)1/2},ssssssssssssssssssssssssssssssssssssssssi=1,2, ,k , and ft(x)+(xtbtx)1/2-vt{gt(x)-(xtctx)1/2}ft(x-)+(x - tbtx-)1/2-vt{gt(x-)-(xtctx)1/2 } , for some t { 1,2 , , k}. the above relation , together with the relation i > 0 , implies that ( 20)i=1ki[fi(x)+(xtbix)1/2vi{gi(x)(xtcix)1/2 } ] < i=1ki[fi(x)+(xtbix)1/2vi{gi(x)(xtcix)1/2 } ] . from the relations ( 5 ) , ( 11 ) , and ( 14 ) , we get ( 21)j=1myjhj(x)j=1myjhj(x ) . consequently ( 20 ) and ( 21 ) yield ( 22)i=1ki[fi(x)+(xtbix)1/2vi{gi(x)(xtcix)1/2 } ] + j=1myjhj(x ) < i=1ki[fi(x)+(xtbix)1/2vi{gi(x)(xtcix)1/2 } ] + j=1myjhj(x ) . let x-x0 be a feasible solution of mfp1 and there exist i + ; w , z , vi + , and y satisfying the condition in lemma 10 at x- . furthermore suppose that the following conditions hold.(i)q(x ) = i=1i[fi(x ) + ( xbix ) vi{gi(x ) ( xcix ) } ] is second order -pseudounivex with respect to , 0 , and k at x-x0 and h(x ) = j=1yjhj(x ) is second order -quasiunivex with respect to , 1 , and k at x-x0 with ( 2q(x-))p=0 and ( 2h(x-))p=0 where fi : x , gi : x , hj : x , i = 1,2 , , k ; j = 1,2 , , m , p , : x x , k : x x + , and 0 , 1 : satisfying 0(a ) 0a 0 and 1(a ) 0a 0.(ii) + 0 . let x-x0 be a feasible solution of mfp1 and there exist i + ; w , z , vi + , and y satisfying the condition in lemma 10 at x- . furthermore suppose that the following conditions hold.(i)q(x ) = i=1i[fi(x ) + ( xbix ) vi{gi(x ) ( xcix ) } ] is second order -pseudounivex with respect to , 0 , and k at x-x0 and h(x ) = j=1yjhj(x ) is second order -quasiunivex with respect to , 1 , and k at x-x0 with ( 2q(x-))p=0 and ( 2h(x-))p=0 where fi : x , gi : x , hj : x , i = 1,2 , , k ; j = 1,2 , , m , p , : x x , k : x x + , and 0 , 1 : satisfying 0(a ) q(x ) = i=1i[fi(x ) + ( xbix ) vi{gi(x ) ( xcix ) } ] is second order -pseudounivex with respect to , 0 , and k at x-x0 and h(x ) = j=1yjhj(x ) is second order -quasiunivex with respect to , 1 , and k at x-x0 with ( 2q(x-))p=0 and ( 2h(x-))p=0 where fi : x , gi : x , hj : x , i = 1,2 , , k ; j = 1,2 , , m , p , : x x , k : x x + , and 0 , 1 : satisfying 0(a ) 0a 0 and 1(a ) 0a proofsuppose hypothesis holds.from the relations ( 5 ) , ( 11 ) , and ( 14 ) , we get ( 23)j=1myjhj(x ) j=1myjhj(x)h(x)h(x)h(x)h(x)0 . also from hypothesis ( i ) , we get ( 2h(x-))p=0.so we have the following : ( 24)h(x)h(x)+12pt(2h(x))p0 k(x , x)1{h(x)h(x)+12pt(2h(x))p}0 . hence , the -quasiunivexity of h(x ) with respect to , 1 , and k implies the following : ( 25)(x , x)t{h(x)+(2h(x))p}+||xx||20 (x , x)t{h(x)}+||xx||20 . from ( 9 ) , we get ( 26)i=1ki[fi(x)+biwvi{gi(x)ciz } ] + i=1myihi(x)=0 q(x)+h(x)=0 (x , x)t[q(x)+h(x)]=0 (x , x)tq(x)+(x , x)th(x ) + ||xx||2||xx||2=0 . using ( 25 ) in ( 26 ) , we get ( 27)(x , x)tq(x)||xx||20 . since + 0 , we get ( 28)||xx||2||xx||2 . since q(x ) is -pseudounivex with respect to , 0 , and k , we obtained ( 30)k(x , x)0[q(x)q(x)+12pt2q(x)p]0 . using the property of k and 0 , we get ( 31)q(x)q(x)+12pt2q(x)p0q(x)q(x ) i=1ki[fi(x)+(xtbix)1/2ssssssssssssssvi{gi(x)(xtcix)1/2 } ] i=1ki[fi(x)+(xtbix)1/2vi{gi(x)(xtcix)1/2 } ] . if x- were not an efficient solution to mfp1 , then , from ( 20 ) , we have ( 32)i=1ki[fi(x)+(xtbix)1/2vi{gi(x)(xtcix)1/2 } ] < i=1ki[fi(x)+(xtbix)1/2sssssssssssvi{gi(x)(xtcix)1/2 } ] . from the relations ( 5 ) , ( 11 ) , and ( 14 ) , we get ( 23)j=1myjhj(x ) j=1myjhj(x)h(x)h(x)h(x)h(x)0 . also from hypothesis ( i ) , we get ( 2h(x-))p=0 . so we have the following : ( 24)h(x)h(x)+12pt(2h(x))p0 k(x , x)1{h(x)h(x)+12pt(2h(x))p}0 . hence , the -quasiunivexity of h(x ) with respect to , 1 , and k implies the following : ( 25)(x , x)t{h(x)+(2h(x))p}+||xx||20 (x , x)t{h(x)}+||xx||20 . from ( 9 ) , we get ( 26)i=1ki[fi(x)+biwvi{gi(x)ciz } ] + i=1myihi(x)=0 q(x)+h(x)=0 (x , x)t[q(x)+h(x)]=0 (x , x)tq(x)+(x , x)th(x ) + ||xx||2||xx||2=0 . using ( 25 ) in ( 26 ) , we get ( 27)(x , x)tq(x)||xx||20 . since q(x ) is -pseudounivex with respect to , 0 , and k , we obtained ( 30)k(x , x)0[q(x)q(x)+12pt2q(x)p]0 . using the property of k and 0 , if x- were not an efficient solution to mfp1 , then , from ( 20 ) , we have ( 32)i=1ki[fi(x)+(xtbix)1/2vi{gi(x)(xtcix)1/2 } ] < i=1ki[fi(x)+(xtbix)1/2sssssssssssvi{gi(x)(xtcix)1/2 } ] . ,gk(u)12pt2gk(u)p ) , where gi(u ) = fi(u ) + yj1hj1(u ) + ubiw i{gi(u ) uciz } , i = 1,2 , , k ; i are fixed parameters.(iii ) mmfd. maximize g(u ) ; is k - dimensional strictly positive vector , all subject to same constraints ( 36)i=1ki[gi(u)+2gi(u)p]+yj2t[hj2(u)+2hj2(u)p ] = 0 , ( 37)fi(u)+yj1thj1(u)+utbiwvi{gi(u)utciz}0,hhhhhhhhhhhhhhhhhhhhhhhhhhfor i=1,2, ,k . ( 38)yj2thj2(u)12pt2(yj2thj2(u))p0,hhhhhhhhhhhhhhhhhhyj2rm|j1| , ( 39)wtbiw1 , ztciz1 , i=1,2, ,k , ( 40)y0 , vi0 , i=1,2, ,k , where fi : x , gi : x , hj : x , i = 1,2 , , k ; j = 1,2 , , m are differentiable functions , w , z , p . bi , and ci , i = 1,2 , , k are positive semidefinite matrices of order n. mmfd0 . maximize ( 33)l(u ) = ( l1(u)12pt2l1(u)p , l2(u ) sss12pt2l2(u)p, mmfd. maximize g(u ) ; is k - dimensional strictly positive vector , all subject to same constraints for the following theorems , we assume that : x x , k : x x + , and 0 , 1 : satisfying 0(a ) 0a 0 and b 01(b ) 0 and , . theorem 13 ( weak duality ) . let x be a feasible solution for the primal mfp and let ( u , y , v , w ) be feasible for dual smmfd. if ( i)i=1igi( ) is second order -pseudounivex with respect to , 0 , k , and for yj2 , yj2hj2( ) is second order -quasiunivex with respect to , 1 , and k along with(ii) + 0 , then inf(f(x ) ) sup(g(u ) ) . let x be a feasible solution for the primal mfp and let ( u , y , v , w ) be feasible for dual smmfd. if ( i)i=1igi( ) is second order -pseudounivex with respect to , 0 , k , and for yj2 , yj2hj2( ) is second order -quasiunivex with respect to , 1 , and k along with(ii) + 0 , then inf(f(x ) ) sup(g(u ) ) . i=1igi( ) is second order -pseudounivex with respect to , 0 , k , and for yj2 , yj2hj2( ) is second order -quasiunivex with respect to , 1 , and k along with + 0 , then inf(f(x ) ) sup(g(u ) ) . proofnow from the primal and dual constraints , we have ( 41)h(x)0,yj2thj2(u)12pt2(yj2thj2(u))p0 . so ( 42)yj2thj2(x)yj2thj2(u)+12pt2(yj2thj2(u))p0 k(x , u)1[12yj2thj2(x)yj2thj2(u)ssssssssssssssssssss+12pt2(yj2thj2(u))p]0 . since yj2hj2 is second order -quasiunivex with respect to , 1 , and k and in view of ( 42 ) , for x , u , we have ( 43)(x , u)t{[yj2thj2(u)]+2[yj2thj2(u)]p}+||xu||20.again from the dual constraint ( 36 ) , we have ( 44)i=1ki[gi(u)+2gi(u)p ] + yj2tt[hj2(u)+2hj2(u)p]=0 . since (x , u ) , we have ( 45)(x , u)t{i=1ki[gi(u)+2gi(u)p ] } + (x , u)t{yj2t[hj2(u)+2hj2(u)p]}=0 , (x , u)t{i=1ki[gi(u)+2gi(u)p ] } + (x , u)t{yj2t[hj2(u)+2hj2(u)p ] } + ||xu||2||xu||2=0 . using ( 43 ) in above equation , we get (x , u){i=1i[gi(u ) + gi(u)p ] } ||xu|| 0.since + 0 , we get ||xu|| ||xu||.so , we have ( 46)(x , u)t{i=1ki[gi(u)+2gi(u)p]}+||xu||20 . since i=1igi(u ) is second order -pseudounivex with respect to , 0 , and k , by definition 2 and ( 46 ) , we get k(x , u)0{i=1igi(x ) i=1igi(u ) + ( 1/2)pi=1igi(u)p } 0.using the property of 0 and k , we get ( 47)i=1kigi(x)i=1kigi(u)+12pti=1kigi(u)p0 i=1kigi(x)i=1kigi(u ) i=1ki[fi(x)+yj1thj1(x)ssssssszzssss+xtbiwvi{gi(x)xtciz } ] i=1ki[fi(u)+yj1thj1(u)sssssssssss+utbiwvi{gi(u)xtciz } ] . equation ( 5 ) gives h(x ) 0yj1hj1(x ) 0 , for yj1 0.so ( 47 ) implies that ( 48)i=1ki[fi(x)+xtbiwvi{gi(x)xtciz } ] i=1ki[fi(u)+yj1thj1(u)+utbiwssssssssssssyj1thj1vi{gi(u)utciz } ] . now by schwarz inequality and ( 39 ) , we have ( 49)xtbiw(xtbix)1/2(wtbiw)1/2(xtbix)1/2,xtciz(xtcix)1/2(ztciz)1/2(xtcix)1/2,hhhhhhhhhhhhhhhhhhhhhhhhi=1,2, ,k . so both ( 48 ) and ( 49 ) imply that ( 50)i=1ki[fi(x)+(xtbix)1/2vi{gi(x)(xtcix)1/2 } ] i=1ki[fi(u)+yj1thj1(u)+utbiwssssssssssssvi{gi(u)utciz } ] inf(f(x))sup(f(u ) ) . now from the primal and dual constraints , we have ( 41)h(x)0,yj2thj2(u)12pt2(yj2thj2(u))p0 . so ( 42)yj2thj2(x)yj2thj2(u)+12pt2(yj2thj2(u))p0 k(x , u)1[12yj2thj2(x)yj2thj2(u)ssssssssssssssssssss+12pt2(yj2thj2(u))p]0 . since yj2hj2 is second order -quasiunivex with respect to , 1 , and k and in view of ( 42 ) , for x , u , we have ( 43)(x , u)t{[yj2thj2(u)]+2[yj2thj2(u)]p}+||xu||20 . again from the dual constraint ( 36 ) , we have ( 44)i=1ki[gi(u)+2gi(u)p ] + yj2tt[hj2(u)+2hj2(u)p]=0 . since (x , u ) , we have ( 45)(x , u)t{i=1ki[gi(u)+2gi(u)p ] } + (x , u)t{yj2t[hj2(u)+2hj2(u)p]}=0 , (x , u)t{i=1ki[gi(u)+2gi(u)p ] } + (x , u)t{yj2t[hj2(u)+2hj2(u)p ] } + ||xu||2||xu||2=0 . using ( 43 ) in above equation , we get (x , u){i=1i[gi(u ) + gi(u)p ] } ||xu|| 0 . since i=1igi(u ) is second order -pseudounivex with respect to , 0 , and k , by definition 2 and ( 46 ) , we get k(x , u)0{i=1igi(x ) i=1igi(u ) + ( 1/2)pi=1igi(u)p } 0 . using the property of 0 and k , we get ( 47)i=1kigi(x)i=1kigi(u)+12pti=1kigi(u)p0 i=1kigi(x)i=1kigi(u ) i=1ki[fi(x)+yj1thj1(x)ssssssszzssss+xtbiwvi{gi(x)xtciz } ] i=1ki[fi(u)+yj1thj1(u)sssssssssss+utbiwvi{gi(u)xtciz } ] . equation ( 5 ) gives h(x ) 0yj1hj1(x ) 0 , for yj1 0 . now by schwarz inequality and ( 39 ) , we have ( 49)xtbiw(xtbix)1/2(wtbiw)1/2(xtbix)1/2,xtciz(xtcix)1/2(ztciz)1/2(xtcix)1/2,hhhhhhhhhhhhhhhhhhhhhhhhi=1,2, ,k . so both ( 48 ) and ( 49 ) imply that ( 50)i=1ki[fi(x)+(xtbix)1/2vi{gi(x)(xtcix)1/2 } ] i=1ki[fi(u)+yj1thj1(u)+utbiwssssssssssssvi{gi(u)utciz } ] inf(f(x))sup(f(u ) ) . let x- be optimal solution for mfp and let w(x-)=. then there exist i + ; w , z , vi + , and y such that ( u-,,y , v , w , z , p=0 ) is a feasible solution for dual and the objective values of both primal and dual are equal to zero . furthermore if ( i ) ( u-,,y , v , w , z ) is feasible for dual , ( ii ) i=1igi( ) is second order -pseudounivex with respect to , 0 , and k and for yj2 , yj1hj2( ) is second order -quasiunivex with respect to , 1 , and k along with ( iii ) + 0 , then ( u-,,y , v , w , z , p=0 ) is properly efficient for smmfd0 . let x- be optimal solution for mfp and let w(x-)=. then there exist i + ; w , z , vi + , and y such that ( u-,,y , v , w , z , p=0 ) is a feasible solution for dual and the objective values of both primal and dual are equal to zero . furthermore if ( i ) ( u-,,y , v , w , z ) is feasible for dual , ( ii ) i=1igi( ) is second order -pseudounivex with respect to , 0 , and k and for yj2 , yj1hj2( ) is second order -quasiunivex with respect to , 1 , and k along with ( iii ) + 0 , then ( u-,,y , v , w , z , p=0 ) is properly efficient for smmfd0 . proofsince x- is optimal solution for ( mfp ) , by lemma 10 , there exist i + ; w , z , vi + , and y such that ( 51)i=1ki[fi(x)+biwvi{gi(x)ciz}]+yth(x)=0,fi(x)+(xtbix)1/2vi(gi(x)(xtcix)1/2)=0,hhhhhhhhhhhhhhhhhhhhhhhhhhhhi=1,2, ,k , yth(x)=0,wtbiw1 , ztciz1 , i=1,2, ,k,(xtbix)1/2=xtbiw , ( xtcix)1/2=xtciz , hhhhhhhhhhhhhhhhhhhhhhhhhi=1,2, ,k , y0,vi0 , i=1,2, ,k , which can be written as ( 52)i=1ki[fi(x)+yj1thj1(x)+biwvi{gi(x)ciz } ] + yj1thj2(x)=0,fi(x)+(xtbix)1/2vi{gi(x)(xtcix)1/2 } + yj1thj1(x)=0,yj1thj2(x)=0,wtbiw1 , ztciz1 , i=1,2, ,k,(xtbix)1/2=xtbiw , ( xtcix)1/2=xtciz , hhhhhhhhhhhhhhhhhhhhhhhhhi=1,2, ,k , y0,vi0 , i=1,2, ,k . these are nothing but the dual constraints.so ( u-,,y , v , w , z , p=0 ) is feasible solution for dual problem.and the objective values of mfp and smmfd are equal to zero . it follows from theorem 13 and for any feasible solution ( u-,,y , v , w , z , p=0 ) of dual that g(u-)g(x-).so ( u-,,y , v , w , z , p=0 ) is optimal solution of smmfd. then applying lemmas 8 and 9 , we conclude that ( u-,,y , v , w , z , p=0 ) is properly efficient for ( smmfd0 ) . since x- is optimal solution for ( mfp ) , by lemma 10 , there exist i + ; w , z , vi + , and y such that ( 51)i=1ki[fi(x)+biwvi{gi(x)ciz}]+yth(x)=0,fi(x)+(xtbix)1/2vi(gi(x)(xtcix)1/2)=0,hhhhhhhhhhhhhhhhhhhhhhhhhhhhi=1,2, ,k , yth(x)=0,wtbiw1 , ztciz1 , i=1,2, ,k,(xtbix)1/2=xtbiw , ( xtcix)1/2=xtciz , hhhhhhhhhhhhhhhhhhhhhhhhhi=1,2, ,k , y0,vi0 , i=1,2, ,k , which can be written as ( 52)i=1ki[fi(x)+yj1thj1(x)+biwvi{gi(x)ciz } ] + yj1thj2(x)=0,fi(x)+(xtbix)1/2vi{gi(x)(xtcix)1/2 } + yj1thj1(x)=0,yj1thj2(x)=0,wtbiw1 , ztciz1 , i=1,2, ,k,(xtbix)1/2=xtbiw , ( xtcix)1/2=xtciz , hhhhhhhhhhhhhhhhhhhhhhhhhi=1,2, ,k , y0,vi0 , i=1,2, ,k . so ( u-,,y , v , w , z , p=0 ) is feasible solution for dual problem . and the objective values of mfp and smmfd are equal to zero . it follows from theorem 13 and for any feasible solution ( u-,,y , v , w , z , p=0 ) of dual that g(u-)g(x- ) . so ( u-,,y , v , w , z , p=0 ) is optimal solution of smmfd. then applying lemmas 8 and 9 , we conclude that ( u-,,y , v , w , z , p=0 ) is properly efficient for ( smmfd0 ) . if p = 0 , ci = 0 , i = 1,2 , , k , then our dual programming reduces to the dual programming proposed by tripathy . in this paper , three approaches given by dinklebaeh and jagannathan for both primal and second order mixed type dual of a nondifferentiable multiobjective fractional programming problem are introduced and the necessary and sufficient optimality conditions are established and a parameterization technique is used to establish duality results under generalized second order -univexity assumption . the results developed in this paper can be further extended to higher order mixed type fractional problem containing square root term . also the present work can be further extended to a class of nondifferentiable minimax mixed fractional programming problems .

three approaches of second order mixed type duality are introduced for a nondifferentiable multiobjective fractional programming problem in which the numerator and denominator of objective function contain square root of positive semidefinite quadratic form . also , the necessary and sufficient conditions of efficient solution for fractional programming are established and a parameterization technique is used to establish duality results under generalized second order -univexity assumption .

1. Introduction 2. Notations and Preliminaries 3. Second Order Mixed Type Multiobjective Fractional Duality 4. Special Case 5. Conclusion

a fractional programming problem arises in many types of optimization problems such as portfolio selection , production , information theory , and numerous decision making problems in management science . many economic , noneconomic , and indirect applications of fractional programming problem have also been given by bector , bector and chandra , craven , mond and weir , stancu - minasian , schaible and ibaraki , ahmad et al . duality in fractional programming is an important class of duality theory and several contributions have been made in the past [ 1 , 5 , 8 , 1014 ] . second order duality provides a tighter bound for the value of the objective function when approximations are used . another advantage of second order duality when applicable is that if a feasible point in the primal is given and first order duality does not apply , then we can use second order duality to provide a lower bound of the value of the primal problem ( see ) . derived fritz john and karush - kuhn tucker necessary and sufficient optimality condition for a class of nondifferentiable convex multiobjective fractional programming problems and established some duality theorems . [ 17 , 18 ] discussed the optimality condition and duality for nonlinear fractional programming . discussed the optimality and duality for nonsmooth multiobjective fractional programming with generalized convexity . and xu gave a mixed type duality for fractional programming , established some sufficient conditions , and obtained various duality results between the mixed dual and primal problem . zhou and wang introduced a class of mixed type dual for nonsmooth multiobjective fractional programming and established the duality results under ( v , ) invexity assumption . duality for various forms of mathematical problems involving square roots of positive semidefinite quadratic forms has been discussed by many authors [ 10 , 2325 ] . mond considered a nonlinear fractional programming problem involving square roots of positive semidefinite quadratic form in the numerator and denominator and proved the necessary and sufficient condition for optimality . [ 26 , 27 ] formulated a nondifferentiable multiobjective fractional problem in which numerators contain support function . one of the most known approaches used for solving nonlinear fractional programming problem is called parametric approach . to characterize solution of a multiobjective fractional problem under generalized convexity . tripathy introduced three approaches given by dinklebaeh and jagannathan for both primal and mixed type dual of a nondifferentiable multiobjective fractional programming and established the duality results under generalized -univexity . to relax convexity assumption imposed on the function in theorems on optimality and duality , motivated by the earlier authors in this paper , we have introduced three approaches given by dinklebaeh and jagannathan for both primal and second order mixed type dual of a nondifferentiable multiobjective fractional programming problem in which the numerator and denominator of objective function contain square root of positive semidefinite quadratic form . also we have established the necessary and sufficient optimality condition and used a parameterization technique to establish duality results under generalized -univexity assumption . x > yxi > yi , for all i = 1,2 , , n. x yxi yi , for all i = 1,2 , , n. consider the following nondifferentiable multiobjective fractional programming problem . for x , x-x the real differentiable function fi is said to be second order -univex at x-x with respect to , , and k , if ( 6)k(x , x)[fi(x)fi(x)+12pt(2fi(x)p ) ] (x , x)t[fi(x)+2fi(x)p]+||xx||2,ssssssssssssssssssssssssssssssssssssssssssssxx . the real differentiable function fi is said to be second order -univex at x-x with respect to , , and k , if ( 6)k(x , x)[fi(x)fi(x)+12pt(2fi(x)p ) ] (x , x)t[fi(x)+2fi(x)p]+||xx||2,ssssssssssssssssssssssssssssssssssssssssssssxx . the real differentiable function fi is said to be second order -pseudounivex at x-x with respect to , , and k , if ( 7)(x , x)t[fi(x)+2fi(x)p]+||xx||20 k(x , x)[fi(x)fi(x)+12pt(2fi(x)p)]0,sssssssssssssssssssssssssssssssssssssssssssssssssssssssxx . the real differentiable function fi is said to be second order -pseudounivex at x-x with respect to , , and k , if ( 7)(x , x)t[fi(x)+2fi(x)p]+||xx||20 k(x , x)[fi(x)fi(x)+12pt(2fi(x)p)]0,sssssssssssssssssssssssssssssssssssssssssssssssssssssssxx . let b be a positive semidefinite matrix of order n. then , for all x , w , xbw ( xbx)(wbw ) . x x0 is an efficient solution for mfp0 if and only if it is an efficient solution of mfp1 with f(x ) = 0 . x x0 is an efficient solution for mfp0 if and only if it is an efficient solution of mfp1 with f(x ) = 0 . (i)p(x ) = i=1i[fi(x ) + ( xbix ) vi{gi(x ) ( xcix ) } ] + j=1yjhj(x ) is second order -pseudounivex with respect to , , and k at x-x0 , with ( p(x))p = 0 , where fi : x , gi : x , hj : x , i = 1,2 , m , p , : x x , k : x x + , and : satisfying (a ) 0a 0. (i)p(x ) = i=1i[fi(x ) + ( xbix ) vi{gi(x ) ( xcix ) } ] + j=1yjhj(x ) is second order -pseudounivex with respect to , , and k at x-x0 , with ( p(x))p = 0 , where fi : x , gi : x , hj : x , i = 1,2 , , k ; j = 1,2 , , m , p , : x x , k : x x + , and : satisfying (a ) 0a 0. suppose that x- is not efficient solution of mfp1 ; then there exist x-x0 such that ( 19)fi(x)+(xtbix)1/2vi{gi(x)(xtcix)1/2 } fi(x)+(xtbix)1/2vi{gi(x)(xtcix)1/2},ssssssssssssssssssssssssssssssssssssssssi=1,2, ,k , and ft(x)+(xtbtx)1/2-vt{gt(x)-(xtctx)1/2}ft(x-)+(x - tbtx-)1/2-vt{gt(x-)-(xtctx)1/2 } , for some t { 1,2 , , k}.the above relation , together with the relation i > 0 , implies that ( 20)i=1ki[fi(x)+(xtbix)1/2vi{gi(x)(xtcix)1/2 } ] < i=1ki[fi(x)+(xtbix)1/2vi{gi(x)(xtcix)1/2 } ] . since the conditions of lemma 10 are satisfied , from ( 9 ) and ( 13 ) , we have ( 16)p(x ) = (j=1myjhj(x)i=1ki[fi(x)+(xtbix)1/2ssssssssssssvi{gi(x)(xtcix)1/2}]+j=1myjhj(x ) ) = (j=1myjhj(x)i=1ki[fi(x)+xtbiwvi{gi(x)xtciz}]sssssssssss+j=1myjhj(x))=0 . since 2p(x-)p=0 , the above inequality implies p(x)p(x-)(18)i=1ki[fi(x)+(xtbix)1/2sssssssssssvi{gi(x)(xtcix)1/2}]+j=1myjhj(x)i=1ki[fi(x)+(xtbix)1/2sssssssssssvi{gi(x)(xtcix)1/2}]+j=1myjhj(x ) . suppose that x- is not efficient solution of mfp1 ; then there exist x-x0 such that ( 19)fi(x)+(xtbix)1/2vi{gi(x)(xtcix)1/2 } fi(x)+(xtbix)1/2vi{gi(x)(xtcix)1/2},ssssssssssssssssssssssssssssssssssssssssi=1,2, ,k , and ft(x)+(xtbtx)1/2-vt{gt(x)-(xtctx)1/2}ft(x-)+(x - tbtx-)1/2-vt{gt(x-)-(xtctx)1/2 } , for some t { 1,2 , , k}. (i)q(x ) = i=1i[fi(x ) + ( xbix ) vi{gi(x ) ( xcix ) } ] is second order -pseudounivex with respect to , 0 , and k at x-x0 and h(x ) = j=1yjhj(x ) is second order -quasiunivex with respect to , 1 , and k at x-x0 with ( 2q(x-))p=0 and ( 2h(x-))p=0 where fi : x , gi : x , hj : x , i = 1,2 , , k ; j = 1,2 , , m , p , : x x , k : x x + , and 0 , 1 : satisfying 0(a ) 0a 0 and 1(a ) 0a 0. if x- were not an efficient solution to mfp1 , then , from ( 20 ) , we have ( 32)i=1ki[fi(x)+(xtbix)1/2vi{gi(x)(xtcix)1/2 } ] < i=1ki[fi(x)+(xtbix)1/2sssssssssssvi{gi(x)(xtcix)1/2 } ] . hence , the -quasiunivexity of h(x ) with respect to , 1 , and k implies the following : ( 25)(x , x)t{h(x)+(2h(x))p}+||xx||20 (x , x)t{h(x)}+||xx||20 . bi , and ci , i = 1,2 , , k are positive semidefinite matrices of order n. mmfd0 . if ( i)i=1igi( ) is second order -pseudounivex with respect to , 0 , k , and for yj2 , yj2hj2( ) is second order -quasiunivex with respect to , 1 , and k along with(ii) + 0 , then inf(f(x ) ) sup(g(u ) ) . let x be a feasible solution for the primal mfp and let ( u , y , v , w ) be feasible for dual smmfd. since yj2hj2 is second order -quasiunivex with respect to , 1 , and k and in view of ( 42 ) , for x , u , we have ( 43)(x , u)t{[yj2thj2(u)]+2[yj2thj2(u)]p}+||xu||20.again from the dual constraint ( 36 ) , we have ( 44)i=1ki[gi(u)+2gi(u)p ] + yj2tt[hj2(u)+2hj2(u)p]=0 . furthermore if ( i ) ( u-,,y , v , w , z ) is feasible for dual , ( ii ) i=1igi( ) is second order -pseudounivex with respect to , 0 , and k and for yj2 , yj1hj2( ) is second order -quasiunivex with respect to , 1 , and k along with ( iii ) + 0 , then ( u-,,y , v , w , z , p=0 ) is properly efficient for smmfd0 . then there exist i + ; w , z , vi + , and y such that ( u-,,y , v , w , z , p=0 ) is a feasible solution for dual and the objective values of both primal and dual are equal to zero . proofsince x- is optimal solution for ( mfp ) , by lemma 10 , there exist i + ; w , z , vi + , and y such that ( 51)i=1ki[fi(x)+biwvi{gi(x)ciz}]+yth(x)=0,fi(x)+(xtbix)1/2vi(gi(x)(xtcix)1/2)=0,hhhhhhhhhhhhhhhhhhhhhhhhhhhhi=1,2, ,k , yth(x)=0,wtbiw1 , ztciz1 , i=1,2, ,k,(xtbix)1/2=xtbiw , ( xtcix)1/2=xtciz , hhhhhhhhhhhhhhhhhhhhhhhhhi=1,2, ,k , y0,vi0 , i=1,2, ,k , which can be written as ( 52)i=1ki[fi(x)+yj1thj1(x)+biwvi{gi(x)ciz } ] + yj1thj2(x)=0,fi(x)+(xtbix)1/2vi{gi(x)(xtcix)1/2 } + yj1thj1(x)=0,yj1thj2(x)=0,wtbiw1 , ztciz1 , i=1,2, ,k,(xtbix)1/2=xtbiw , ( xtcix)1/2=xtciz , hhhhhhhhhhhhhhhhhhhhhhhhhi=1,2, ,k , y0,vi0 , i=1,2, ,k . these are nothing but the dual constraints.so ( u-,,y , v , w , z , p=0 ) is feasible solution for dual problem.and the objective values of mfp and smmfd are equal to zero . since x- is optimal solution for ( mfp ) , by lemma 10 , there exist i + ; w , z , vi + , and y such that ( 51)i=1ki[fi(x)+biwvi{gi(x)ciz}]+yth(x)=0,fi(x)+(xtbix)1/2vi(gi(x)(xtcix)1/2)=0,hhhhhhhhhhhhhhhhhhhhhhhhhhhhi=1,2, ,k , yth(x)=0,wtbiw1 , ztciz1 , i=1,2, ,k,(xtbix)1/2=xtbiw , ( xtcix)1/2=xtciz , hhhhhhhhhhhhhhhhhhhhhhhhhi=1,2, ,k , y0,vi0 , i=1,2, ,k , which can be written as ( 52)i=1ki[fi(x)+yj1thj1(x)+biwvi{gi(x)ciz } ] + yj1thj2(x)=0,fi(x)+(xtbix)1/2vi{gi(x)(xtcix)1/2 } + yj1thj1(x)=0,yj1thj2(x)=0,wtbiw1 , ztciz1 , i=1,2, ,k,(xtbix)1/2=xtbiw , ( xtcix)1/2=xtciz , hhhhhhhhhhhhhhhhhhhhhhhhhi=1,2, ,k , y0,vi0 , i=1,2, ,k . so ( u-,,y , v , w , z , p=0 ) is feasible solution for dual problem . in this paper , three approaches given by dinklebaeh and jagannathan for both primal and second order mixed type dual of a nondifferentiable multiobjective fractional programming problem are introduced and the necessary and sufficient optimality conditions are established and a parameterization technique is used to establish duality results under generalized second order -univexity assumption . the results developed in this paper can be further extended to higher order mixed type fractional problem containing square root term .

type 2 diabetes ( t2d ) is a progressive metabolic disease characterized by hyperglycemia due to a combination of insulin resistance and defective insulin secretion [ 1 , 2 ] . thus , correction of hyperglycemia to euglycemia , or near - euglycemia , has been the ultimate goal [ 35 ] . however , because intensive glycemic control increases both cardiovascular and overall mortality in t2d [ 6 , 7 ] , the latest guidelines for the management of hyperglycemia in t2d recommend a more flexible glycemic target taking into consideration an individual 's clinical characteristics . this recommendation creates a dilemma in that achieving euglycemia to prevent microvascular complications in diabetic patients [ 1 , 4 , 911 ] should then be reserved to reduce mortality in t2d , while the latest guidelines may let the patients with t2d survive longer but live with the disabilities caused by increased microvascular complications due to a relaxed glycemic target . overcoming this dilemma requires a better understanding of how euglycemia differentially affects major organs / tissues according to the accompanying insulin and/or insulin signal level . this , in turn , may explain why euglycemia that prevents microvascular complications ultimately increases cardiovascular and overall mortality in t2d . we recently reported that euglycemia achieved by diet control in diabetic rats ( fed isocalorie per body weight compared with sham - control nondiabetic rats ) is different from that in sham - control rats ; the mean liver weight in the euglycemic - diabetic rats is significantly lower than in the sham - control rats due to increased autophagy . in contrast , the hyperglycemic - diabetic rats on ad libitum diet maintained the same liver weight as the sham - control rats in the same study . therefore , we previously suggested that hyperglycemia in the presence of insulin deficiency might protect hepatocytes against excessive autophagy . however , we have not investigated the protection mechanism against excessive autophagy in the livers of the hyperglycemic - diabetic rats and the mechanism for the excessive autophagy in the livers of the euglycemic - diabetic rats at the molecular level . understanding these mechanisms may clarify whether intensive glycemic control to achieve euglycemia is more harmful than poor glycemic control in terms of the survival of hepatocytes when insulin deficiency exists , which is common in t2d , regardless of the degree of insulin resistance . importantly , chronic liver disease and/or hepatocellular carcinoma are the fourth most common causes of death among patients with t2d , so we speculated that when there is a preexisting liver ailment , euglycemia in the presence of insulin deficiency might increase liver damage , possibly leading to hepatic failure . autophagy is induced when cellular nutrient levels decrease , a process in which ampk , a major energy sensor in most cells , is activated and mediates the autophagic process . conversely , when cellular nutrient levels increase , mtor , a signaling molecule for protein synthesis , is activated and inhibits autophagy . we have observed that euglycemia in the presence of insulin deficiency increases autophagy in the liver relative to that in the presence of a normal insulin level ; therefore , we hypothesized that euglycemia differentially influences the activities of ampk and mtor according to the insulin level in the liver , where glucose uptake is not dependent upon insulin . in addition , though insulin levels did not differ significantly among the diabetic rats , the extent of liver autophagy and the activity of akt ( a major insulin signaling molecule and an activator of mtor ) were significantly different between euglycemic - diabetic and hyperglycemic - diabetic rats . therefore , we also hypothesized that the glycemic level in the presence of insulin deficiency affects the molecules that transmit insulin signals , such as akt and pten ( a major negative regulator of the pi3 kinase / akt signaling pathway , ) , in the liver . finally , because excessive autophagy induces apoptosis [ 1820 ] , we expected that increased apoptotic signaling may partially explain the significant loss of liver weight in euglycemic - diabetic rats . to test these hypotheses , molecular changes in the regulation of autophagy , insulin signaling , and apoptosis were studied in the livers of diabetic rats with different glycemic levels achieved by different diets , and these changes were compared to those of sham - operated control rats . in the present study , we added an additional calorie - controlled group with restricted protein content ( 6 kcal% versus 19 kcal% in a standard chow ) , to determine whether a protein restriction augments the effects of the calorie - controlled diet alone . in fact , protein restriction is often applied to patients with diabetes in clinical settings to prevent the progression of diabetic nephropathy [ 2123 ] . regarding the degree of protein restriction in the present study , we referred to other studies performed on protein restriction in diabetic rats [ 24 , 25 ] . eleven - week - old , specific pathogen - free male sprague - dawley rats were purchased from orient bio ( sungnam , korea ) . upon arrival , the rats were then divided into two groups and surgery was performed at 13 weeks of age as follows : five rats underwent a sham operation as the control group ( 19s ) , and 15 rats underwent subtotal pancreatectomy ( px group ) . after 7 weeks on an ad libitum diet to induce diabetes , the px group was divided into thirds and fed the following diets for another 7 weeks : ad libitum group ( 19al ) , calorie - control group ( 19r ) , and calorie and protein ( calorie / protein ) control group ( 6r ) . throughout the study , all rats except the 6r group were fed a standard chow based on ain-76a ( dyets inc . , bethlehem , pa , usa ; protein 19.4 , carbohydrate 68.8 , and lipid 11.8 kcal% ) . the 6r group was fed a low protein chow ( modified ain-76a , dyets inc . ; protein 6.0 , carbohydrate 82.2 , and lipid 11.8 kcal% ) . all rats were housed individually throughout the experiment and their daily food intake was measured . the 19r and 6r groups were pair - fed ( the same g per kg of body weight per day ) with the 19s group . all rats were housed under a 12 h light - dark cycle ( light on 08:0020:00 h ) , at 2023c with a relative humidity of 4065% . the rats had free access to tap water throughout the study . on the last day of the study after overnight fasting all animal protocols were approved by the institutional animal care and use committee at konkuk university . to generate an insulin - deficient model of diabetes in adult rats , briefly , the abdominal wall was opened under anesthesia using 0.7 mg / kg of zoletil 50 ( virbac , carros , france ) and 0.2 mg / kg of rompun ( bayer korea , ansan , korea ) . pancreatic tissue was removed carefully with a cotton tipped applicator from the spleen to 1 mm from the common bile duct without vascular injury . after surgery , the rats were placed in beds , covered , and exposed to infrared light to maintain normal body temperature . food intake was individually measured daily and an average daily food intake ( g per day ) was calculated weekly . the daily food intake per kg of body weight of each rat was calculated using the average daily food intake and body weight from the previous week . body weights were measured every weekend and on the last day of the study just before rats were euthanized . fbg levels ( mg / dl ) were measured in tail vein blood at 9 am every other week after overnight fasting using a portable glucometer ( caresens ii , gentrol co. , incheon , korea ) . immediately after co2 anesthesia , blood samples were taken from the inferior vena cava . plasma insulin and c - peptide levels were determined using radioimmunoassay kits ( millipore , billerica , ma , usa ) according to the manufacturer 's instructions . radioactivity was measured by using a counter ( beckman coulter , brea , ca , usa ) . after collecting blood samples , animals were euthanized and the liver was excised from the abdominal cavity , weighed , and immediately frozen in liquid nitrogen . frozen liver tissues were ground to powder in liquid nitrogen in a mortar and stored at 80c until use . the frozen powdered liver tissue samples were homogenized in an ice - cold buffer containing 25 mm hepes , 25 mm benzamidine , 100 mm sodium fluoride , 10 mm sodium pyrophosphate , 2 mm sodium orthovanadate , 1% triton x-100 , 4 mm edta , 5 l / ml of phosphatase inhibitor , and 5 l / ml of protease inhibitor , sonicated for 1 minute , and centrifuged at 14,000 g , for 30 minutes at 4c . total protein concentrations were quantified using a bca kit ( pierce , rockford , il , usa ) . the extracted proteins were separated on 813.5% sds polyacrylamide gels and transferred to nitrocellulose membranes . primary antibodies against total and phosphorylated ampk ( cell signaling technology , cst , denver , ma , usa ; 1 : 5000 ) , total and phosphorylated mtor ( cst ; 1 : 1000 ) , lc3b ( cst ; 1 : 1000 ) , caspase-3 ( cst ; 1 : 1000 ) , total and phosphorylated erk-1 ( cst ; 1 : 5000 ) , total and phosphorylated akt ( cst ; 1 : 5000 ) , and total and phosphorylated pten ( cst ; 1 : 1000 ) were applied overnight at 4c . the membranes were then developed using horseradish peroxidase - conjugated anti - rabbit igg ( cst ; 1 : 5000 ) followed by detection with ecl reagent ( ge healthcare , wauwatosa , wi , usa ) . the immunoreactive protein bands were quantified using multi gauge software version 3.1 ( fujifilm , tokyo , japan ) . statistical significance was evaluated using one - way anova with tukey 's post hoc test , or an unpaired t - test . to achieve euglycemia by diet control , pancreatectomized diabetic rats were fed as described in section 2 . the mean daily food intake per kg of body weight of all groups did not differ significantly during the adaptation period ( figure 2(a ) ) . however , food intake by the px groups increased by more than 2-fold during the period of diabetes induction as compared to the 19s group ( p < 0.001 ) . during the diet control period , food intake by the 19al group was unchanged from the period of diabetes induction , while intake by the 19r and 6r groups decreased to that of the 19s group due to the controlled diet ( figure 2(a ) ) . the 19al group ate about three times more per body weight than the other groups during the diet control period ( p < 0.001 ) . after the induction period while the mean body weights of all px groups were unchanged , the mean body weights of the experimental rats changed as a result of the differences in diets and food intakes ( figure 2(b ) ) . compared to the 19s group , the mean body weights of the px groups decreased significantly by about 20% during the period of diabetes induction ( p < 0.001 ) . the mean body weights of the 19r and 6r groups ( 338 16 g and 330 43 g , resp . ) decreased further during the diet control period as compared to the 19s group ( 42 and 44% for the 19r and 6r groups , resp . ; ( p = 0.017 ) and 6r ( p = 0.006 ) groups , resp . ) . the mean body weight of rats in the 19al group ( 410 26 g ) was unchanged during the diet control period compared to the induction period but was decreased by 30% as compared to the 19s group ( 588 12 g , p < 0.001 ) during the diet control period . figure 2(c ) shows sequential changes in the fbg level of all groups throughout the entire study period and confirmed that both the calorie- and calorie / protein - controlled diets achieved euglycemia in the r groups . before surgery , all groups were euglycemic ( mean fbg levels were 19s : 104 20 ; 19al : 104 18 ; 19r : 106 11 ; 6r : 93.0 10.9 mg / dl ; p = 0.586 ) . the fbg levels of the px groups increased after surgery and remained elevated throughout the period of diabetes induction ( 19al : 459 52 ; 19r : 464 68 ; 6r : 441 41 mg / dl ; p < 0.001 versus 19s at the last week of the diabetes induction period ) . however , the fbg levels of the 19r and 6r groups improved becoming euglycemic ( 113 3.7 and 109 10 mg / dl , resp . ) like the 19s group ( 115 22 mg / dl ; p = 1 versus 19s ) during the diet control period . in contrast , the 19al group continued to be hyperglycemic ( 474 55 mg / dl ; p < 0.001 versus 19s , 19r , and 6r groups ) . to confirm that pancreatectomy generated insulin - deficient diabetic rats , plasma insulin and c - peptide levels were measured at the end of study . the mean plasma insulin levels of pancreatectomized rats were 8.9 ( 19al ) , 6.5 ( 19r ) , and 5.5% ( 6r ) that of the 19s group ( p < 0.01 ) ( figure 3(a ) ) . the mean plasma c - peptide levels of pancreatectomized rats were 18.2 ( 19al ) , 6.7 ( 19r ) , and 10.4% ( 6r ) that of the 19s group ( p plasma insulin and c - peptide levels among the px groups did not differ significantly . figure 3(b ) shows that the mean liver weights of the 19r ( 9.0 0.9 g ) and 6r ( 11.5 2.9 g ) groups decreased significantly as compared to the 19s ( 16.3 2.8 g ) ( 45 ( p = 0.002 ) and 29% ( p = 0.029 ) , resp . ) or 19al ( 16.4 1.0 g ) groups ( 45 ( p = 0.002 ) and 30% ( p = 0.024 ) , resp . ) . by contrast , the liver weight of the 19al group did not differ significantly from the 19s group . the ratio of lc3 ii to i , an index of autophagy in liver tissue , is shown in figure 4(a ) . the ratio of the 19r and 6r groups increased significantly as compared to the 19s ( 1.6- ( p = 0.008 ) and 1.4-fold ( p = 0.010 ) , resp . ) and 19al groups ( 1.7- ( p = 0.003 ) and 1.6-fold ( p = 0.004 ) , resp . ) . the phosphorylation levels of ampk and mtor were examined to assess changes in factors that regulate autophagy . the phosphorylation of ampk in the 19r and 6r groups increased significantly as compared to the 19s ( 2.1- and 2.2-fold , resp . ; phosphorylation of mtor in the 19r and 6r groups decreased significantly as compared to the 19s ( 46 and 52% , resp . ; p < 0.001 ) and 19al groups ( 44 ( p = 0.001 ) and 50% ( p = 0.002 ) , resp . ) the ratio of lc3 ii to i and the levels of ampk and mtor phosphorylation did not differ significantly between the 19s and 19al groups ( figures 4(a)4(c ) ) . the phosphorylation levels of akt and pten were determined to assess the insulin signaling level , which can modulate the extent of autophagy ( figure 5 ) . the phosphorylation of akt decreased significantly in the 19r and 6r groups as compared to the 19s ( 42 ( p = 0.030 ) and 46% ( p = 0.019 ) , resp . ) and 19al groups ( 44 ( p = 0.012 ) and 48% ( p = 0.018 ) , resp . ) . the phosphorylation of pten increased significantly in the 19r and 6r groups as compared to the 19s ( 2.5- ( p < 0.001 ) and 2.3-fold ( p = 0.002 ) , resp . ) and 19al groups ( 2.9- and 2.7-fold , resp . ; the two r groups did not differ significantly ( figure 5(b ) ) , and the phosphorylation of akt and pten did not differ significantly between the 19s and 19al groups ( figures 5(a ) and 5(b ) ) . the phosphorylation of erk-1 and the cleavage ratio of caspase-3 ( the ratio of cleaved to uncleaved caspase-3 ) were measured to assess changes in factors affecting the regulation of cell growth / proliferation and apoptosis , respectively ( figure 6 ) . the phosphorylation of erk-1 in the 19r and 6r groups decreased significantly as compared to the 19s ( 72 and 75% , resp . ; the two r groups did not differ significantly , and the phosphorylation of erk-1 in the 19al group did not differ significantly from the 19s group . the cleavage ratio of caspase-3 increased in all px groups compared to the 19s group : 19al : 6.4-fold , p = 0.016 ; 19r : 9.1-fold , p = 0.001 ; and 6r : 18.3-fold , p < 0.001 ( figure 6(b ) ) . there were also significant differences in the cleavage ratio between the px groups : the ratio of the 19r and 6r groups increased significantly by 1.4- ( p = 0.003 ) and 2.9-fold ( p < 0.001 ) , respectively , compared to the 19al group . the ratio of the 6r group was highest among all the px groups ( 2-fold higher than the 19r group , p < 0.001 ) . however , the increased mortality associated with the intensive glycemic control required to achieve euglycemia has emerged as a new concern whose molecular mechanisms , and the organs involved , are still largely unknown . in the present study , we demonstrated that achieving euglycemia in insulin deficiency ( both the 19r and 6r groups ) was accompanied by a loss of a critical amount of functional mass of the liver via increased autophagy , as compared with euglycemia in the presence of normal physiologic levels of insulin ( 19s group ) . autophagy was increased in both r groups through increased ampk and decreased mtor ( via increased pten and decreased akt activation ) activation . there was also an increase in the amount of cleaved ( activated ) caspase-3 , an executor of apoptosis ( figures 46 ) . autophagy plays a major role in cell survival under stress conditions . however , based on recent studies , as well as the data presented here , autophagy can kill a cell if essential components are rapidly consumed [ 26 , 27 ] . autophagy is regulated by nutrient levels in a cell that affect the activities of ampk and mtor , the two major regulators of the autophagic process . however , our results showed that the activation level of ampk in hepatocytes differed significantly under identical euglycemic conditions ( both r groups versus the 19s group ) . this suggests that the energy level in hepatocytes may be affected by insulin as shown in skeletal muscle , despite glucose uptake being independent of insulin in liver tissue but not in skeletal muscle tissue . this may explain why the activation of ampk in both insulin - deficient r groups is greater than that of the 19s group under the same euglycemic conditions . another possibility for activating ampk in insulin - deficient hepatocytes is by 3-phosphoglycerate ( 3-pg ) , a glycolysis intermediate , via ampkk . thus , we speculate that 3-pg may accumulate because the flux of the glycolytic pathway is retarded due to insulin deficiency and because the flux of the gluconeogenic pathway is inhibited by activated ampk . taken together , euglycemia in the presence of insulin deficiency induces a starvation - like effect in the liver compared to that with physiologic insulin levels . this is shown by the activation of ampk and subsequent excessive autophagy in both r groups . the lower mtor activation in the 19r group than in the 19s group , even on the isocaloric and isoprotein diets , may be the result of insulin deficiency that decreased akt ( an activator of mtor ) phosphorylation and activated pten ( a major negative regulator of the pi3 kinase / akt signaling pathway , ) in the 19r group compared to the 19s group . furthermore , ampk is activated by energy depletion and suppresses mtor activity thus decreasing the inhibitory influence of mtor over autophagy [ 31 , 32 ] . pten phosphorylation increases the activity and stability of pten itself ; however , the total pten levels did not differ among the experimental groups . considering that pten expression is upregulated in podocytes under hyperglycemic conditions ( 30 mmol / l ) relative to under normoglycemic conditions ( 5.6 mmol / l ) , we speculated that pten in both r groups could be maintained at a similar level to those of the other two groups due to the increased stability , though the expression of pten itself might be downregulated in euglycemic conditions . because autophagy is associated with the activity of erk , a major signaling molecule for cell growth / proliferation through the inhibition of transcription factor eb ( tfeb , a master regulator of lysosomal biogenesis and autophagy , [ 34 , 35 ] ) , we investigated erk-1 activity ( figure 6(a ) ) . as expected , interestingly , while starvation - induced autophagic death , but not apoptosis , was observed in the liver of patients with anorexia nervosa that are severely undernourished [ 36 , 37 ] , both euglycemic r groups , in a comparable state of starvation because of insulin deficiency , showed increased apoptotic signaling as well as excessive autophagy ( figures 4(a ) and 6(b ) ) . this suggests that mitochondrial damage and the release of cytochrome c may occur in hepatocytes under euglycemic insulin deficiency . this , in turn , would lead to caspase-3 activation , which was not observed in anorexia nervosa without diabetes [ 36 , 37 ] . mitochondrial performance and mass were markedly reduced in the soleus muscle of stz - induced diabetic mice , but not in high fat diet induced diabetic mice with hyperinsulinemia , indicating the significance of insulin in maintaining mitochondrial function . based on these findings , we propose that , while euglycemia can be achieved without resolution of insulin deficiency , which is common even in patients with t2d , this is not beneficial to diabetic patients in terms of the survival of hepatocytes and the maintenance of the functional mass of the liver . in this study , we investigated the molecular mechanisms by which hyperglycemia in insulin deficiency ( 19al group ) protected the liver against critical weight loss compared to euglycemia in insulin deficiency ( both r groups ) . hyperglycemia due to the ad libitum diet in the 19al group resulted in an autophagy level similar to that of the 19s group through decreased ampk and increased mtor phosphorylation ( figure 4 ) . the cleavage of caspase-3 decreased as compared to both r groups ( figure 6 ) even though all px groups did not differ significantly in insulin levels ( figure 3(a ) ) . hyperphagia has been considered to be a pathologic eating habit in diabetic patients . because hyperphagia results in hyperglycemia in the presence of insulin deficiency , which is associated with increased microvascular complications , diet control has been a major strategy for diabetes care [ 8 , 38 , 39 ] . endothelial cells use glut-1 for glucose uptake from the blood [ 40 , 41 ] . the activity of glut-1 is not dependent on insulin but rather on the level of glycemia . therefore , as the glycemic level increases , endothelial cells become more exposed to glucotoxicity , and microvascular complications are more prone to develop . livers from rats in the hyperglycemic 19al group were protected against the loss of functional mass through decreased ampk and increased mtor and akt ( a major insulin receptor signaling molecule , ) phosphorylation , as compared to both r groups , in spite of insulin deficiency . this implies that hyperglycemia , which is harmful to diabetic patients , activates akt and inhibits autophagy and apoptosis like insulin [ 43 , 44 ] , but this effect is independent of the insulin level . therefore , we speculate that hyperphagia - induced hyperglycemia in the presence of insulin deficiency may not always be deleterious but rather has the beneficial effect of limiting autophagic and apoptotic cell death in the liver , but at the cost of increasing microvascular complications . our data may explain why the conventional care group ( mean hba1c , 7.5% ) showed a lower overall mortality rate than the intensive glycemic control group ( mean hba1c , 6.4% ) in the action to control cardiovascular risk study . finally , we investigated the difference in molecular changes between the two control diets ( normal or reduced protein content to achieve euglycemia ) . diabetic patients are easily undernourished , especially for protein , because of the strict diet control needed to control hyperglycemia and to prevent the progression of diabetic nephropathy [ 2123 ] . the only difference found between the two r groups was in the level of cleaved caspase-3 that was significantly higher in the 6r than in the 19r group . we speculate that , despite euglycemia in the presence of insulin deficiency , the activation of apoptotic signals may be stronger in the liver when fed a protein restricted diet than when fed a non - protein restricted diet . this finding warrants further study to determine the underlying mechanism . taken together , euglycemia achieved by feeding a calorie- or calorie / protein - controlled diet in insulin deficiency can be detrimental to the survival of hepatocytes due to energy depletion and low insulin receptor signaling in the liver parenchyma , even when the calories ingested by the diabetic rats were the same as the daily intake per body weight of sham - operated controls . in contrast , the hyperglycemia from feeding an ad libitum diet in the presence of insulin deficiency retained liver weight due to an insulin receptor signaling level that was comparable to sham - control rats . overall , we have demonstrated the molecular mechanisms by which euglycemia differentially affects the liver , depending on the insulin status . we have also shown that hyperglycemia has a protective effect on the liver in the presence of insulin deficiency . therefore , we propose that achieving euglycemia by any kinds of intervention without resolution of insulin deficiency should be avoided . it is important for care - givers of diabetic patients to understand that the hyperphagia seen in the presence of insulin deficiency is to compensate for the insulin deficiency in the liver , indicating that the present paradigm for diet control in diabetes care should be reevaluated .

euglycemia is the ultimate goal in diabetes care to prevent complications . however , the benefits of euglycemia in type 2 diabetes are controversial because near - euglycemic subjects show higher mortality than moderately hyperglycemic subjects . we previously reported that euglycemic - diabetic rats on calorie - control lose a critical liver weight ( lw ) compared with hyperglycemic rats . here , we elucidated the molecular mechanisms underlying the loss of lw in euglycemic - diabetic rats and identified a potential risk in achieving euglycemia by calorie - control . sprague - dawley diabetic rats generated by subtotal - pancreatectomy were fed a calorie - controlled diet for 7 weeks to achieve euglycemia using 19 kcal% ( 19r ) or 6 kcal% ( 6r ) protein - containing chow or fed ad libitum ( 19al ) . the diet in both r groups was isocaloric / kg body weight to the sham - operated group ( 19s ) . compared with 19s and hyperglycemic 19al , both euglycemic r groups showed lower lws , increased autophagy , and increased ampk and caspase-3 and decreased mtor activities . though degree of insulin deficiency was similar among the diabetic rats , akt activity was lower , and pten activity was higher in both r groups than in 19al whose signaling patterns were similar to 19s . in conclusion , euglycemia achieved by calorie - control is deleterious in insulin deficiency due to increased autophagy and apoptosis in the liver via ampk and caspase-3 activation .

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

type 2 diabetes ( t2d ) is a progressive metabolic disease characterized by hyperglycemia due to a combination of insulin resistance and defective insulin secretion [ 1 , 2 ] . thus , correction of hyperglycemia to euglycemia , or near - euglycemia , has been the ultimate goal [ 35 ] . however , because intensive glycemic control increases both cardiovascular and overall mortality in t2d [ 6 , 7 ] , the latest guidelines for the management of hyperglycemia in t2d recommend a more flexible glycemic target taking into consideration an individual 's clinical characteristics . this recommendation creates a dilemma in that achieving euglycemia to prevent microvascular complications in diabetic patients [ 1 , 4 , 911 ] should then be reserved to reduce mortality in t2d , while the latest guidelines may let the patients with t2d survive longer but live with the disabilities caused by increased microvascular complications due to a relaxed glycemic target . we recently reported that euglycemia achieved by diet control in diabetic rats ( fed isocalorie per body weight compared with sham - control nondiabetic rats ) is different from that in sham - control rats ; the mean liver weight in the euglycemic - diabetic rats is significantly lower than in the sham - control rats due to increased autophagy . in contrast , the hyperglycemic - diabetic rats on ad libitum diet maintained the same liver weight as the sham - control rats in the same study . therefore , we previously suggested that hyperglycemia in the presence of insulin deficiency might protect hepatocytes against excessive autophagy . however , we have not investigated the protection mechanism against excessive autophagy in the livers of the hyperglycemic - diabetic rats and the mechanism for the excessive autophagy in the livers of the euglycemic - diabetic rats at the molecular level . understanding these mechanisms may clarify whether intensive glycemic control to achieve euglycemia is more harmful than poor glycemic control in terms of the survival of hepatocytes when insulin deficiency exists , which is common in t2d , regardless of the degree of insulin resistance . importantly , chronic liver disease and/or hepatocellular carcinoma are the fourth most common causes of death among patients with t2d , so we speculated that when there is a preexisting liver ailment , euglycemia in the presence of insulin deficiency might increase liver damage , possibly leading to hepatic failure . we have observed that euglycemia in the presence of insulin deficiency increases autophagy in the liver relative to that in the presence of a normal insulin level ; therefore , we hypothesized that euglycemia differentially influences the activities of ampk and mtor according to the insulin level in the liver , where glucose uptake is not dependent upon insulin . in addition , though insulin levels did not differ significantly among the diabetic rats , the extent of liver autophagy and the activity of akt ( a major insulin signaling molecule and an activator of mtor ) were significantly different between euglycemic - diabetic and hyperglycemic - diabetic rats . therefore , we also hypothesized that the glycemic level in the presence of insulin deficiency affects the molecules that transmit insulin signals , such as akt and pten ( a major negative regulator of the pi3 kinase / akt signaling pathway , ) , in the liver . finally , because excessive autophagy induces apoptosis [ 1820 ] , we expected that increased apoptotic signaling may partially explain the significant loss of liver weight in euglycemic - diabetic rats . to test these hypotheses , molecular changes in the regulation of autophagy , insulin signaling , and apoptosis were studied in the livers of diabetic rats with different glycemic levels achieved by different diets , and these changes were compared to those of sham - operated control rats . in the present study , we added an additional calorie - controlled group with restricted protein content ( 6 kcal% versus 19 kcal% in a standard chow ) , to determine whether a protein restriction augments the effects of the calorie - controlled diet alone . regarding the degree of protein restriction in the present study , we referred to other studies performed on protein restriction in diabetic rats [ 24 , 25 ] . eleven - week - old , specific pathogen - free male sprague - dawley rats were purchased from orient bio ( sungnam , korea ) . upon arrival , the rats were then divided into two groups and surgery was performed at 13 weeks of age as follows : five rats underwent a sham operation as the control group ( 19s ) , and 15 rats underwent subtotal pancreatectomy ( px group ) . after 7 weeks on an ad libitum diet to induce diabetes , the px group was divided into thirds and fed the following diets for another 7 weeks : ad libitum group ( 19al ) , calorie - control group ( 19r ) , and calorie and protein ( calorie / protein ) control group ( 6r ) . throughout the study , all rats except the 6r group were fed a standard chow based on ain-76a ( dyets inc . , bethlehem , pa , usa ; protein 19.4 , carbohydrate 68.8 , and lipid 11.8 kcal% ) . ; protein 6.0 , carbohydrate 82.2 , and lipid 11.8 kcal% ) . to generate an insulin - deficient model of diabetes in adult rats , briefly , the abdominal wall was opened under anesthesia using 0.7 mg / kg of zoletil 50 ( virbac , carros , france ) and 0.2 mg / kg of rompun ( bayer korea , ansan , korea ) . after surgery , the rats were placed in beds , covered , and exposed to infrared light to maintain normal body temperature . plasma insulin and c - peptide levels were determined using radioimmunoassay kits ( millipore , billerica , ma , usa ) according to the manufacturer 's instructions . after collecting blood samples , animals were euthanized and the liver was excised from the abdominal cavity , weighed , and immediately frozen in liquid nitrogen . to achieve euglycemia by diet control , pancreatectomized diabetic rats were fed as described in section 2 . however , food intake by the px groups increased by more than 2-fold during the period of diabetes induction as compared to the 19s group ( p < 0.001 ) . during the diet control period , food intake by the 19al group was unchanged from the period of diabetes induction , while intake by the 19r and 6r groups decreased to that of the 19s group due to the controlled diet ( figure 2(a ) ) . the 19al group ate about three times more per body weight than the other groups during the diet control period ( p < 0.001 ) . compared to the 19s group , the mean body weights of the px groups decreased significantly by about 20% during the period of diabetes induction ( p < 0.001 ) . decreased further during the diet control period as compared to the 19s group ( 42 and 44% for the 19r and 6r groups , resp . the mean body weight of rats in the 19al group ( 410 26 g ) was unchanged during the diet control period compared to the induction period but was decreased by 30% as compared to the 19s group ( 588 12 g , p < 0.001 ) during the diet control period . figure 2(c ) shows sequential changes in the fbg level of all groups throughout the entire study period and confirmed that both the calorie- and calorie / protein - controlled diets achieved euglycemia in the r groups . the fbg levels of the px groups increased after surgery and remained elevated throughout the period of diabetes induction ( 19al : 459 52 ; 19r : 464 68 ; 6r : 441 41 mg / dl ; p < 0.001 versus 19s at the last week of the diabetes induction period ) . however , the fbg levels of the 19r and 6r groups improved becoming euglycemic ( 113 3.7 and 109 10 mg / dl , resp . ) like the 19s group ( 115 22 mg / dl ; p = 1 versus 19s ) during the diet control period . in contrast , the 19al group continued to be hyperglycemic ( 474 55 mg / dl ; p < 0.001 versus 19s , 19r , and 6r groups ) . to confirm that pancreatectomy generated insulin - deficient diabetic rats , plasma insulin and c - peptide levels were measured at the end of study . the mean plasma insulin levels of pancreatectomized rats were 8.9 ( 19al ) , 6.5 ( 19r ) , and 5.5% ( 6r ) that of the 19s group ( p < 0.01 ) ( figure 3(a ) ) . the mean plasma c - peptide levels of pancreatectomized rats were 18.2 ( 19al ) , 6.7 ( 19r ) , and 10.4% ( 6r ) that of the 19s group ( p plasma insulin and c - peptide levels among the px groups did not differ significantly . figure 3(b ) shows that the mean liver weights of the 19r ( 9.0 0.9 g ) and 6r ( 11.5 2.9 g ) groups decreased significantly as compared to the 19s ( 16.3 2.8 g ) ( 45 ( p = 0.002 ) and 29% ( p = 0.029 ) , resp . ) by contrast , the liver weight of the 19al group did not differ significantly from the 19s group . the phosphorylation of ampk in the 19r and 6r groups increased significantly as compared to the 19s ( 2.1- and 2.2-fold , resp . ; phosphorylation of mtor in the 19r and 6r groups decreased significantly as compared to the 19s ( 46 and 52% , resp . the ratio of lc3 ii to i and the levels of ampk and mtor phosphorylation did not differ significantly between the 19s and 19al groups ( figures 4(a)4(c ) ) . the phosphorylation of akt decreased significantly in the 19r and 6r groups as compared to the 19s ( 42 ( p = 0.030 ) and 46% ( p = 0.019 ) , resp . ) the phosphorylation of pten increased significantly in the 19r and 6r groups as compared to the 19s ( 2.5- ( p < 0.001 ) and 2.3-fold ( p = 0.002 ) , resp . ) ; the two r groups did not differ significantly ( figure 5(b ) ) , and the phosphorylation of akt and pten did not differ significantly between the 19s and 19al groups ( figures 5(a ) and 5(b ) ) . the phosphorylation of erk-1 in the 19r and 6r groups decreased significantly as compared to the 19s ( 72 and 75% , resp . ; the two r groups did not differ significantly , and the phosphorylation of erk-1 in the 19al group did not differ significantly from the 19s group . there were also significant differences in the cleavage ratio between the px groups : the ratio of the 19r and 6r groups increased significantly by 1.4- ( p = 0.003 ) and 2.9-fold ( p < 0.001 ) , respectively , compared to the 19al group . however , the increased mortality associated with the intensive glycemic control required to achieve euglycemia has emerged as a new concern whose molecular mechanisms , and the organs involved , are still largely unknown . in the present study , we demonstrated that achieving euglycemia in insulin deficiency ( both the 19r and 6r groups ) was accompanied by a loss of a critical amount of functional mass of the liver via increased autophagy , as compared with euglycemia in the presence of normal physiologic levels of insulin ( 19s group ) . autophagy was increased in both r groups through increased ampk and decreased mtor ( via increased pten and decreased akt activation ) activation . however , based on recent studies , as well as the data presented here , autophagy can kill a cell if essential components are rapidly consumed [ 26 , 27 ] . autophagy is regulated by nutrient levels in a cell that affect the activities of ampk and mtor , the two major regulators of the autophagic process . however , our results showed that the activation level of ampk in hepatocytes differed significantly under identical euglycemic conditions ( both r groups versus the 19s group ) . this may explain why the activation of ampk in both insulin - deficient r groups is greater than that of the 19s group under the same euglycemic conditions . another possibility for activating ampk in insulin - deficient hepatocytes is by 3-phosphoglycerate ( 3-pg ) , a glycolysis intermediate , via ampkk . thus , we speculate that 3-pg may accumulate because the flux of the glycolytic pathway is retarded due to insulin deficiency and because the flux of the gluconeogenic pathway is inhibited by activated ampk . taken together , euglycemia in the presence of insulin deficiency induces a starvation - like effect in the liver compared to that with physiologic insulin levels . this is shown by the activation of ampk and subsequent excessive autophagy in both r groups . the lower mtor activation in the 19r group than in the 19s group , even on the isocaloric and isoprotein diets , may be the result of insulin deficiency that decreased akt ( an activator of mtor ) phosphorylation and activated pten ( a major negative regulator of the pi3 kinase / akt signaling pathway , ) in the 19r group compared to the 19s group . pten phosphorylation increases the activity and stability of pten itself ; however , the total pten levels did not differ among the experimental groups . considering that pten expression is upregulated in podocytes under hyperglycemic conditions ( 30 mmol / l ) relative to under normoglycemic conditions ( 5.6 mmol / l ) , we speculated that pten in both r groups could be maintained at a similar level to those of the other two groups due to the increased stability , though the expression of pten itself might be downregulated in euglycemic conditions . because autophagy is associated with the activity of erk , a major signaling molecule for cell growth / proliferation through the inhibition of transcription factor eb ( tfeb , a master regulator of lysosomal biogenesis and autophagy , [ 34 , 35 ] ) , we investigated erk-1 activity ( figure 6(a ) ) . as expected , interestingly , while starvation - induced autophagic death , but not apoptosis , was observed in the liver of patients with anorexia nervosa that are severely undernourished [ 36 , 37 ] , both euglycemic r groups , in a comparable state of starvation because of insulin deficiency , showed increased apoptotic signaling as well as excessive autophagy ( figures 4(a ) and 6(b ) ) . mitochondrial performance and mass were markedly reduced in the soleus muscle of stz - induced diabetic mice , but not in high fat diet induced diabetic mice with hyperinsulinemia , indicating the significance of insulin in maintaining mitochondrial function . based on these findings , we propose that , while euglycemia can be achieved without resolution of insulin deficiency , which is common even in patients with t2d , this is not beneficial to diabetic patients in terms of the survival of hepatocytes and the maintenance of the functional mass of the liver . in this study , we investigated the molecular mechanisms by which hyperglycemia in insulin deficiency ( 19al group ) protected the liver against critical weight loss compared to euglycemia in insulin deficiency ( both r groups ) . hyperglycemia due to the ad libitum diet in the 19al group resulted in an autophagy level similar to that of the 19s group through decreased ampk and increased mtor phosphorylation ( figure 4 ) . the cleavage of caspase-3 decreased as compared to both r groups ( figure 6 ) even though all px groups did not differ significantly in insulin levels ( figure 3(a ) ) . because hyperphagia results in hyperglycemia in the presence of insulin deficiency , which is associated with increased microvascular complications , diet control has been a major strategy for diabetes care [ 8 , 38 , 39 ] . livers from rats in the hyperglycemic 19al group were protected against the loss of functional mass through decreased ampk and increased mtor and akt ( a major insulin receptor signaling molecule , ) phosphorylation , as compared to both r groups , in spite of insulin deficiency . this implies that hyperglycemia , which is harmful to diabetic patients , activates akt and inhibits autophagy and apoptosis like insulin [ 43 , 44 ] , but this effect is independent of the insulin level . therefore , we speculate that hyperphagia - induced hyperglycemia in the presence of insulin deficiency may not always be deleterious but rather has the beneficial effect of limiting autophagic and apoptotic cell death in the liver , but at the cost of increasing microvascular complications . our data may explain why the conventional care group ( mean hba1c , 7.5% ) showed a lower overall mortality rate than the intensive glycemic control group ( mean hba1c , 6.4% ) in the action to control cardiovascular risk study . finally , we investigated the difference in molecular changes between the two control diets ( normal or reduced protein content to achieve euglycemia ) . the only difference found between the two r groups was in the level of cleaved caspase-3 that was significantly higher in the 6r than in the 19r group . we speculate that , despite euglycemia in the presence of insulin deficiency , the activation of apoptotic signals may be stronger in the liver when fed a protein restricted diet than when fed a non - protein restricted diet . taken together , euglycemia achieved by feeding a calorie- or calorie / protein - controlled diet in insulin deficiency can be detrimental to the survival of hepatocytes due to energy depletion and low insulin receptor signaling in the liver parenchyma , even when the calories ingested by the diabetic rats were the same as the daily intake per body weight of sham - operated controls . in contrast , the hyperglycemia from feeding an ad libitum diet in the presence of insulin deficiency retained liver weight due to an insulin receptor signaling level that was comparable to sham - control rats . overall , we have demonstrated the molecular mechanisms by which euglycemia differentially affects the liver , depending on the insulin status . we have also shown that hyperglycemia has a protective effect on the liver in the presence of insulin deficiency . therefore , we propose that achieving euglycemia by any kinds of intervention without resolution of insulin deficiency should be avoided . it is important for care - givers of diabetic patients to understand that the hyperphagia seen in the presence of insulin deficiency is to compensate for the insulin deficiency in the liver , indicating that the present paradigm for diet control in diabetes care should be reevaluated .

one of the most frequently used measures of health status is the question how would you rate your health ? , a single question asking people to evaluate their overall health on a scale from excellent to poor . this single item measure has been demonstrated to be a robust predictor of health outcomes , including mortality , independent of health indicators of physical health . because of the wide range of associations with other health indicators , and the simplicity with which it is collected , self - rated health is widely used in large population surveys as a proxy measure for general health status . earlier studies assumed that the answer to this single question embodied a simple summation of all objective states , and therefore focused on the differences in prediction between the objective measures and the subjective self report . when the subjective self perception of health remained a strong predictor of mortality regardless of the statistical control of numerous objective health variables , it became clear that the simple question is not as simple as was originally thought . the attempts to understand what lies behind this measure produced studies that included mental / emotional variables in addition to physical health variables , and one explanation for the ability of the self rated health to predict mortality was that the single question of how would you rate your health is interpreted by subjects as referring to their overall health , including psychosocial aspects and not only to their physical health . two studies showed that mental health was the mediator in the relationship between physical health and self reported general health . in one case , mental health was measured by the anxiety and depression scale hads and the study was done on 449 adults undergoing hip or knee replacement in hospitals in canada . in the other case , mental health was measured by the geriatric depression scale gds in a longitudinal study of 150 older american adults . another general population study from germany found that in healthier individuals , positive affect was more important than physical functioning in predicting self rated general health , whereas among less healthy individuals the opposite was found . positive affect in that study was measured by the positive and negative affect schedule ( panas ) . the single question how would you rate your mental health ? was used in surveys much less than the single question about general health . ahmad et al . conducted a review of the literature and found fifty - seven studies that included a measurement of self rated mental health ( srmh ) but only four of these studies tried to validate the srmh against known measures of mental health . self - rated mental health was associated with use of mental health services and of medical care services . when checked against other mental health indicators the use of srmh as a proxy for mental morbidity was not recommended . flieshman and zuvekas examined the associations between srmh and measures of psychological distress , activity limitations and social role functioning . they found that the measures of emotional / psychological distress were correlated with each other much more strongly than they were with srmh , while srmh was related to physical health . their conclusion was that the self rated mental health was not a pure measure of mental morbidity and that it can not be used as the sole indicator of psychological distress in surveys . mawani and gilmour checked the associations between the srmh , mental health diagnoses , wmh - cidi diagnoses and measures of psychological distress . they found that a sizeable percentage of respondents who were classified as having mental disorders perceived their mental health as good ; on the other hand , respondents who did not pass the threshold for diagnoses perceived their mental health as fair / poor . their conclusion was that the srmh should not be used as a proxy for mental morbidity because it underestimates the prevalence of mental morbidity , and may reflect other factors as well . to our knowledge , there were no studies that checked simultaneously the self ratings of physical health , mental health and overall health . using data from a large representative sample of the adult population of israel , that included the three self rating scales as well as other objective health status indicators , we tried to understand what is the contribution of the self rating of mental health to the self rating of general health . the israel national health survey followed procedures established by the world mental health survey ( wmh ) of the world health organization ( who ) . the sample was extracted from israel s national population register and constituted non - institutionalized legal residents aged 21 and over . the sample was designed to reflect the population distribution by age , gender and three population sectors : arabs ; post-1990 jewish immigrants from the former soviet union ; and jews and others , including jewish immigrants from countries other than the former soviet union . the sample interviewed for the israel national health survey was weighted back to the total population to compensate for unequal selection probabilities resulting from disproportionate stratification , clustering effects , and non - response . the weights were adjusted to make sample totals conform to known population totals taken from reliable central bureau of statistics sources . on first personal contact with each potential survey respondent , the interviewer explained the survey and obtained verbal informed consent . overall , 73% of those contacted agreed to be interviewed ( 88% of arab - israelis and 71% of jewish - israelis ) . a total of 4859 face - to - face interviews were conducted in arabic , hebrew or russian at respondents homes from may 2003 to april 2004 . professional survey interviewers , who were trained and supervised by the central bureau of statistics , used a laptop computer and computer - assisted personal interview methods . the human subjects committee based at eitanim - kfar shaul hospital approved the survey and the field procedures in november 2000 . the diagnostic instrument used in the who - wmh was the composite international diagnostic interview ( cidi ) . the survey assessed for : anxiety disorders [ panic disorder , generalized anxiety disorder ( gad ) , agoraphobia without panic disorder , and post - traumatic stress disorder ( ptsd ) ] ; mood disorders ( major depressive disorder , dysthymia , bipolar i and ii disorders ) ; and substance abuse disorders ( alcohol abuse , alcohol dependency , drug abuse , drug dependency ) . the presence of a disorder was determined by whether respondents past or current symptoms met 30 days , 12-month or lifetime diagnostic criteria for a dsm iv disorder . for each disorder , when a respondent endorsed a specific screening item , he or she was asked all the questions in the cidi diagnostic section for that disorder to establish the presence of a current disorder . organic exclusion criteria were taken into account in the determination of the dsm - iv diagnoses . the validity of the who - wmh cidi as a diagnostic tool was assessed in france , italy , spain , and the united states by a clinical reappraisal study which found that the individual - level concordance between the scid and cidi for 12-month prevalence of any mood disorder , any anxiety disorder , and any disorder overall was substantial ( area under the curve in the range of 0.8 to 0.9 . for the analyses reported here , respondents were grouped into those with or without any mood or anxiety disorder , because of a large overlap between the two groups of diagnoses . the present analysis distinguished between respondents with and without mood or anxiety disorder in the 30 days before the interview . the conditions listed were heart attack , heart disease , stroke , high blood pressure , asthma , chronic obstructive pulmonary disorder , emphysema or other lung disease , tuberculosis , diabetes , kidney disease , neurological conditions , thyroid disease , cancer , chronic back or neck pain , arthritis or rheumatism , headaches , or any other chronic pain . in this analysis , respondents with chronic physical conditions were grouped into two categories : those reporting any one of the conditions and those reporting none . methodological research has shown that such checklists provide useful information about treated or currently untreated chronic conditions . the whodas is a disability scale that includes six scales : i ) role functioning , ii ) cognition , iii ) mobility , iv ) self - care , v ) social interaction and vi ) participation . the four questions that assessed role functioning were all frequency questions while other domains consist mostly of severity items . the calculation of the total score followed the method described in buist - bouwman : the resulting total score ranged between a score of 0 indicating no disability and 100 indicating maximum disability with intermediate scores indicating the percentage of the maximum possible score . in the present analysis , the total score was grouped into three categories : maximum disability = 100 , 9% of the entire sample , no disability = 0.47% of the entire sample and partial disability 0 > score < 100 , 44% of the entire sample . the rating of physical , mental and overall health were measured using the standard items how would you rate your . excellent , very good , good , fair , or poor ? since the connotation of good was neither very good nor bad , the present analysis followed others and grouped all three variables into two categories : positive excellent / very good and negative good / fair / poor . the control variables included the socio - demographic characteristics : age groups , gender , marital status , education , income and population groups [ classified as i ) arabs ii ) post-1990 jewish immigrants from the former soviet union iii ) jews and others , including jewish immigrants from countries other than the former soviet union ] . the data were weighted to adjust for the differential probabilities of respondents selection and non - response and for differences between the sample and the adult population in israel . cross tabulation of age groups by positive self rated physical health , positive self rated mental health , presence of chronic physical conditions and presence of any depression or anxiety disorders was done to show how these measures change with age . logistic regression analyses were conducted to estimate the association between dichotomous positive / negative outcomes of self rated health measures and the socio - demographic and health status indicators . estimates of odds ratios ( ors ) , the corresponding standard errors and 95% confidence intervals ( ci ) were also obtained from logistic regression using sudaan . the conditions listed were heart attack , heart disease , stroke , high blood pressure , asthma , chronic obstructive pulmonary disorder , emphysema or other lung disease , tuberculosis , diabetes , kidney disease , neurological conditions , thyroid disease , cancer , chronic back or neck pain , arthritis or rheumatism , headaches , or any other chronic pain . in this analysis , respondents with chronic physical conditions were grouped into two categories : those reporting any one of the conditions and those reporting none . methodological research has shown that such checklists provide useful information about treated or currently untreated chronic conditions . the whodas is a disability scale that includes six scales : i ) role functioning , ii ) cognition , iii ) mobility , iv ) self - care , v ) social interaction and vi ) participation . the four questions that assessed role functioning were all frequency questions while other domains consist mostly of severity items . the calculation of the total score followed the method described in buist - bouwman : the resulting total score ranged between a score of 0 indicating no disability and 100 indicating maximum disability with intermediate scores indicating the percentage of the maximum possible score . in the present analysis , the total score was grouped into three categories : maximum disability = 100 , 9% of the entire sample , no disability = 0.47% of the entire sample and partial disability 0 > score < 100 , 44% of the entire sample . the rating of physical , mental and overall health were measured using the standard items how would you rate your . excellent , very good , good , fair , or poor ? since the connotation of good was neither very good nor bad , the present analysis followed others and grouped all three variables into two categories : positive excellent / very good and negative good / fair / poor . the control variables included the socio - demographic characteristics : age groups , gender , marital status , education , income and population groups [ classified as i ) arabs ii ) post-1990 jewish immigrants from the former soviet union iii ) jews and others , including jewish immigrants from countries other than the former soviet union ] . the data were weighted to adjust for the differential probabilities of respondents selection and non - response and for differences between the sample and the adult population in israel . cross tabulation of age groups by positive self rated physical health , positive self rated mental health , presence of chronic physical conditions and presence of any depression or anxiety disorders was done to show how these measures change with age . logistic regression analyses were conducted to estimate the association between dichotomous positive / negative outcomes of self rated health measures and the socio - demographic and health status indicators . estimates of odds ratios ( ors ) , the corresponding standard errors and 95% confidence intervals ( ci ) were also obtained from logistic regression using sudaan . table 1 presents the distribution of the socio - demographic and health status indicators used in the analysis . we found that the 63% of the total sample rated their mental health as positive , 55% rated their overall health as positive but only 49% rated their physical health as positive . men rated their health consistently better than females : 66.5% of the males rated their mental health as positive compared to 61% of the females ( =14.7 ; p<0.001 ) , 52% of the males rated their physical health as positive compared to 48% of the females ( =11.8 ; p<0.01 ) and 58% of the males rated their overall health as positive compared to 53% of the females ( =11.2 ; p<0.01 ) . immigrants rated their health consistently lower ( 18% , 26% , 16% positive on the overall , mental and the physical health respectively ) than the arabs ( 55% , 61% , 54% ) and the jews and others ( 64% , 73% , 56% ) ( p<0.001 ) . those with high income rated their health consistently better ( 60% , 69% , 54% ) than those with low income ( 49% , 56% , 45% ) ( p<0.005 ) . those in the 21 - 34 age group rated their health consistently better ( 77% , 81% , 73% ) than those in the 65 + age group ( 18% , 35% , 13% ) ( p<0.001 ) . those who never married rated their health consistently better ( 75% , 78% , 71% ) than those who were divorced or separated ( 27% , 38% , 25% ) ( p=0.000 ) and those with high education rated their health consistently better ( 62% , 69% , 54% ) than those with low education ( 36% , 45% , 31% ) ( p<0.005 ) . table 2 presents the associations between health status indicators and the self rated physical health ( hsr ) and mental health ( mhsr ) . the odds ratios presented in the table were adjusted in the logistic regression for age , population groups , income , educational level and disability status . table 2 shows that self rated positive physical health is strongly related to chronic physical conditions but much less to mental disorders . likewise , self rated positive mental health is strongly related to mental disorders , but much less to chronic physical conditions . figure 1 shows how chronic physical conditions , mental disorders and the self rating scales of physical and mental health change with age . figure 1 shows that the percentage of those with chronic conditions goes up from 32% in the 21 - 35 age group to 78% in the 65 + group ( =653.9 ; p<0.001 ) while the poor self rated physical health follows the same pattern with a rise from 27% to 87% =1402.39 ; p<0.001 ) . on the other hand the poor self rated mental health goes up from 19.5% in the 21 - 35 age group to 64.8% in the 65 + group ( =609.98 ; p<0.001 ) while the percentage of those with any mental disorder does not change at all . table 3 presents the associations between self rated overall health ( osr ) and the self rated physical and mental health in two models : with and without the health status indicators . the odds ratios presented here were adjusted in the logistic regression for the variables population groups , age groups and disability level . table 3 shows that self rated mental and physical health are much stronger predictors of osr than the health status indicators of chronic conditions , mental disorders and disability . those with positive self rated mental health have 93 times the odds of reporting good positive overall health whereas those with positive self rated physical health have 40 times the odds of reporting positive overall health . after accounting for the self rated health measures hsr and mhsr , the predictive value of the health status indicators is much smaller : those with no mental disorders are six times more likely to have a positive osr , those with no disability have 2.5 times the odds of reporting positive osr and those with no chronic condition have only 1.33% times the odds of reporting positive osr . yet , the age group is still a significant predictor of osr beyond the self rating measures and the health status indicators . table 3 showed that after accounting for the main health variables , positive self rated mental health was twice as important as positive physical health in the prediction of positive overall health . the odds for positive overall health were 93 higher if the rating of mental health was positive compared to 40 times higher if the rating of physical health was positive . table 2 presents the associations between health status indicators and the self rated physical health ( hsr ) and mental health ( mhsr ) . the odds ratios presented in the table were adjusted in the logistic regression for age , population groups , income , educational level and disability status . table 2 shows that self rated positive physical health is strongly related to chronic physical conditions but much less to mental disorders . likewise , self rated positive mental health is strongly related to mental disorders , but much less to chronic physical conditions . figure 1 shows how chronic physical conditions , mental disorders and the self rating scales of physical and mental health change with age . figure 1 shows that the percentage of those with chronic conditions goes up from 32% in the 21 - 35 age group to 78% in the 65 + group ( =653.9 ; p<0.001 ) while the poor self rated physical health follows the same pattern with a rise from 27% to 87% =1402.39 ; p<0.001 ) . on the other hand the poor self rated mental health goes up from 19.5% in the 21 - 35 age group to 64.8% in the 65 + group ( =609.98 ; p<0.001 ) while the percentage of those with any mental disorder does not change at all . table 3 presents the associations between self rated overall health ( osr ) and the self rated physical and mental health in two models : with and without the health status indicators . the odds ratios presented here were adjusted in the logistic regression for the variables population groups , age groups and disability level . table 3 shows that self rated mental and physical health are much stronger predictors of osr than the health status indicators of chronic conditions , mental disorders and disability . those with positive self rated mental health have 93 times the odds of reporting good positive overall health whereas those with positive self rated physical health have 40 times the odds of reporting positive overall health . after accounting for the self rated health measures hsr and mhsr , the predictive value of the health status indicators is much smaller : those with no mental disorders are six times more likely to have a positive osr , those with no disability have 2.5 times the odds of reporting positive osr and those with no chronic condition have only 1.33% times the odds of reporting positive osr . yet , the age group is still a significant predictor of osr beyond the self rating measures and the health status indicators . table 3 showed that after accounting for the main health variables , positive self rated mental health was twice as important as positive physical health in the prediction of positive overall health . the odds for positive overall health were 93 higher if the rating of mental health was positive compared to 40 times higher if the rating of physical health was positive . data from the israel national health survey based on a representative sample of the adult population was used to investigate the differences between the three self rated measures of health . the general distribution of the self rated measures of health replicates results obtained in previous large population surveys showing that rates of positive self rated health are lower among females compared to males , that older individuals rate their health as less positive compared to younger individuals and that socio demographic variables such as education and income are related to the self rating of mental , physical and overall health . the main findings from the present study show that the three self rating scales represent three different domains of health : the self rating mental health and the self rating physical health maintain their distinctiveness : self rated mental health is not related to chronic physical conditions ( as does self rated physical health ) , and self rated physical health is not related to mental disorders ( as does self rated mental health ) . the self rating of physical health has a monotonic relationship with the objective indicator of physical health . the self rating of mental health does not have a monotonic relationship with the absence of mental illness . the good self rating of mental health goes down with age even though there is no age difference in mental disorders . the self rated physical health and the self rated mental health are both strong predictors of the self rated overall health , independent of the objective measures of health . this was shown when the odds of predicting the self rating of the overall health remained the same regardless of whether the objective measures of health were included in the regression equation . the self rated mental and physical health are 10 times stronger in predicting self rated overall health than any of the objective health indicators . the self rated mental health is two times more important than the self rated physical health in predicting the self rated overall health . the stronger weight of the self rated mental health implies that the mental component in the self rating of overall health is stronger than the physical one . this result reinforces conclusions of previous studies : in a cross - national survey of university students in germany , bulgaria , and poland , the overall self rated health status was assessed along with physical and psychological health as well as with social and socio demographic variables . that study found that psychosomatic complaints were the most important indicator in predicting the self rated health status . a similar finding was reported by ormel et al . from a study using a large sample of late middle - aged and older persons living independently . they found that the unique contribution of depressive symptoms on poor health perception outranked those of the medical conditions . in a survey with a follow - up period of 1 - 5 years found that those who were happier and more energetic at baseline rated their health better in the next 5 years , even after accounting for age , negative health indicators at baseline , disease status and functioning . moreover , positive indicators of functioning ; such as physical activity , work , and exercise ; did not significantly predict future self assessment of health . mental disorder was defined in our study by the presence of any mood or anxiety disorder in the past 30 days and its prevalence was the same in the four age groups . two major surveys checked the one year prevalence of any mood or anxiety and found it to be significantly less prevalent in older age groups . on the other hand , mental health which refers to hedonic well - being : feelings of happiness , satisfaction and interest in life , and to eudemonic well - being optimal functioning and self actualization , was more prevalent in younger age groups . in their review of studies on the concept of mental health , westerhof and keyes found that older age was correlated with lower positive affect , less feeling of personal growth and purpose in life , less meaning in life and less social coherence and social contribution . the empirical separation between mental health and mental illness was found also by weich et al . who used principal component analysis to describe the underlying factor structure of mental wellbeing . they found also that well - being and mental disorders are correlated but independent dimensions . the use of three separate self rating scales for mental health , physical health and overall health enabled us to observe the unique contribution of the mental health component to the self rating of overall health . further studies should investigate prospectively which of the three dimensions better predicts mortality , morbidity , hospitalizations or use of services . a key strength of this study was the utilization of a large representative sample of the entire adult population with a relatively high response rate . trends observed in this type of sample are more likely to appear in other large population samples . the main limitations of this study are that all the health information was self - reported and that the health indicators used may not have covered all the ill health domains . it is not clear whether a more comparative wording compared to your age group would have changed the clear age gradient that was found in this study . our results show a different age trajectory between mental health and mental illness . mental disorder was defined in our study by the presence of any mood or anxiety disorder in the past 30 days and two major surveys checked the one year prevalence of any mood or anxiety and found it to be significantly less prevalent in older age groups . on the other hand , mental health which refers to hedonic well - being : feelings of happiness , satisfaction and interest in life , and to eudemonic well - being optimal functioning and self actualization , was more prevalent in younger age groups . in their review of studies on the concept of mental health , westerhof and keyes found that older age was correlated with lower positive affect , less feeling of personal growth and purpose in life , less meaning in life and less social coherence and social contribution . the empirical separation between mental health and mental illness was found also by weich et al . who used principal component analysis to describe the underlying factor structure of mental wellbeing . they found also that well - being and mental disorders are correlated but independent dimensions . the use of three separate self rating scales for mental health , physical health and overall health enabled us to observe the unique contribution of the mental health component to the self rating of overall health . further studies should investigate prospectively which of the three dimensions better predicts mortality , morbidity , hospitalizations or use of services . a key strength of this study was the utilization of a large representative sample of the entire adult population with a relatively high response rate . trends observed in this type of sample are more likely to appear in other large population samples . the main limitations of this study are that all the health information was self - reported and that the health indicators used may not have covered all the ill health domains . it is not clear whether a more comparative wording compared to your age group would have changed the clear age gradient that was found in this study .

backgroundunlike the widely used self rated health , the self rated mental health was found unsuitable as a proxy for mental illness . this paper analyses the relationships between the self ratings of physical health , mental health and overall health , and their association of with the objective indicators for physical and mental health.design and methodsthe study is a secondary analysis of data from a nationwide representative sample of the non - institutionalized adult residents of israel in 2003 that was collected via computer - assisted personal interview methods [ n=4859].resultsthe self rated physical health and the self rated mental health were strongly related to each other yet the self rated mental health was not related to chronic physical conditions and the self rated physical health was not related to mental disorders . in a multiple logistic regression analysis , those with positive self rated mental health had 93 times the odds of reporting positive overall health whereas those with positive self rated physical health had 40 times the odds of reporting positive overall health.conclusionsthe self rating of mental health presents a qualitatively different dimension from mental illness . the self rated mental health is two times more important than the self rated physical health in predicting the self rated overall healthsignificance for public healththe present study is an original study on the self rated physical , mental and overall health measures . because of the wide range of associations with other health indicators , and the simplicity with which they are collected , self - rated health measures are widely used in large population surveys.the present study questions the automatic assumption that the self rated mental health functions as a proxy measure of psychiatric morbidity , and suggests that the self rated mental health is more closely related to subjective well - being . the results show that self rated mental health predicts self rated general health better than self rated physical health .

Introduction Design and methods Diagnostic assessment Chronic general medical conditions Disability measure Self report of physical health, mental health and general health Statistical analysis Results The relationship between health conditions, and self rated physical and mental health The difference between self rated physical health and self rated mental health The relationship of self rated overall health with the self rated physical and mental health Discussion and conclusions Mental health Strengths and limitations

this single item measure has been demonstrated to be a robust predictor of health outcomes , including mortality , independent of health indicators of physical health . because of the wide range of associations with other health indicators , and the simplicity with which it is collected , self - rated health is widely used in large population surveys as a proxy measure for general health status . earlier studies assumed that the answer to this single question embodied a simple summation of all objective states , and therefore focused on the differences in prediction between the objective measures and the subjective self report . when the subjective self perception of health remained a strong predictor of mortality regardless of the statistical control of numerous objective health variables , it became clear that the simple question is not as simple as was originally thought . the attempts to understand what lies behind this measure produced studies that included mental / emotional variables in addition to physical health variables , and one explanation for the ability of the self rated health to predict mortality was that the single question of how would you rate your health is interpreted by subjects as referring to their overall health , including psychosocial aspects and not only to their physical health . two studies showed that mental health was the mediator in the relationship between physical health and self reported general health . in one case , mental health was measured by the anxiety and depression scale hads and the study was done on 449 adults undergoing hip or knee replacement in hospitals in canada . in the other case , mental health was measured by the geriatric depression scale gds in a longitudinal study of 150 older american adults . another general population study from germany found that in healthier individuals , positive affect was more important than physical functioning in predicting self rated general health , whereas among less healthy individuals the opposite was found . was used in surveys much less than the single question about general health . conducted a review of the literature and found fifty - seven studies that included a measurement of self rated mental health ( srmh ) but only four of these studies tried to validate the srmh against known measures of mental health . self - rated mental health was associated with use of mental health services and of medical care services . when checked against other mental health indicators the use of srmh as a proxy for mental morbidity was not recommended . they found that the measures of emotional / psychological distress were correlated with each other much more strongly than they were with srmh , while srmh was related to physical health . their conclusion was that the self rated mental health was not a pure measure of mental morbidity and that it can not be used as the sole indicator of psychological distress in surveys . mawani and gilmour checked the associations between the srmh , mental health diagnoses , wmh - cidi diagnoses and measures of psychological distress . they found that a sizeable percentage of respondents who were classified as having mental disorders perceived their mental health as good ; on the other hand , respondents who did not pass the threshold for diagnoses perceived their mental health as fair / poor . their conclusion was that the srmh should not be used as a proxy for mental morbidity because it underestimates the prevalence of mental morbidity , and may reflect other factors as well . to our knowledge , there were no studies that checked simultaneously the self ratings of physical health , mental health and overall health . using data from a large representative sample of the adult population of israel , that included the three self rating scales as well as other objective health status indicators , we tried to understand what is the contribution of the self rating of mental health to the self rating of general health . the israel national health survey followed procedures established by the world mental health survey ( wmh ) of the world health organization ( who ) . the sample was extracted from israel s national population register and constituted non - institutionalized legal residents aged 21 and over . the sample interviewed for the israel national health survey was weighted back to the total population to compensate for unequal selection probabilities resulting from disproportionate stratification , clustering effects , and non - response . professional survey interviewers , who were trained and supervised by the central bureau of statistics , used a laptop computer and computer - assisted personal interview methods . the validity of the who - wmh cidi as a diagnostic tool was assessed in france , italy , spain , and the united states by a clinical reappraisal study which found that the individual - level concordance between the scid and cidi for 12-month prevalence of any mood disorder , any anxiety disorder , and any disorder overall was substantial ( area under the curve in the range of 0.8 to 0.9 . for the analyses reported here , respondents were grouped into those with or without any mood or anxiety disorder , because of a large overlap between the two groups of diagnoses . in this analysis , respondents with chronic physical conditions were grouped into two categories : those reporting any one of the conditions and those reporting none . the whodas is a disability scale that includes six scales : i ) role functioning , ii ) cognition , iii ) mobility , iv ) self - care , v ) social interaction and vi ) participation . in the present analysis , the total score was grouped into three categories : maximum disability = 100 , 9% of the entire sample , no disability = 0.47% of the entire sample and partial disability 0 > score < 100 , 44% of the entire sample . the rating of physical , mental and overall health were measured using the standard items how would you rate your . since the connotation of good was neither very good nor bad , the present analysis followed others and grouped all three variables into two categories : positive excellent / very good and negative good / fair / poor . the data were weighted to adjust for the differential probabilities of respondents selection and non - response and for differences between the sample and the adult population in israel . cross tabulation of age groups by positive self rated physical health , positive self rated mental health , presence of chronic physical conditions and presence of any depression or anxiety disorders was done to show how these measures change with age . logistic regression analyses were conducted to estimate the association between dichotomous positive / negative outcomes of self rated health measures and the socio - demographic and health status indicators . estimates of odds ratios ( ors ) , the corresponding standard errors and 95% confidence intervals ( ci ) were also obtained from logistic regression using sudaan . in this analysis , respondents with chronic physical conditions were grouped into two categories : those reporting any one of the conditions and those reporting none . the whodas is a disability scale that includes six scales : i ) role functioning , ii ) cognition , iii ) mobility , iv ) self - care , v ) social interaction and vi ) participation . in the present analysis , the total score was grouped into three categories : maximum disability = 100 , 9% of the entire sample , no disability = 0.47% of the entire sample and partial disability 0 > score < 100 , 44% of the entire sample . the rating of physical , mental and overall health were measured using the standard items how would you rate your . since the connotation of good was neither very good nor bad , the present analysis followed others and grouped all three variables into two categories : positive excellent / very good and negative good / fair / poor . the data were weighted to adjust for the differential probabilities of respondents selection and non - response and for differences between the sample and the adult population in israel . cross tabulation of age groups by positive self rated physical health , positive self rated mental health , presence of chronic physical conditions and presence of any depression or anxiety disorders was done to show how these measures change with age . logistic regression analyses were conducted to estimate the association between dichotomous positive / negative outcomes of self rated health measures and the socio - demographic and health status indicators . estimates of odds ratios ( ors ) , the corresponding standard errors and 95% confidence intervals ( ci ) were also obtained from logistic regression using sudaan . table 1 presents the distribution of the socio - demographic and health status indicators used in the analysis . we found that the 63% of the total sample rated their mental health as positive , 55% rated their overall health as positive but only 49% rated their physical health as positive . men rated their health consistently better than females : 66.5% of the males rated their mental health as positive compared to 61% of the females ( =14.7 ; p<0.001 ) , 52% of the males rated their physical health as positive compared to 48% of the females ( =11.8 ; p<0.01 ) and 58% of the males rated their overall health as positive compared to 53% of the females ( =11.2 ; p<0.01 ) . immigrants rated their health consistently lower ( 18% , 26% , 16% positive on the overall , mental and the physical health respectively ) than the arabs ( 55% , 61% , 54% ) and the jews and others ( 64% , 73% , 56% ) ( p<0.001 ) . table 2 presents the associations between health status indicators and the self rated physical health ( hsr ) and mental health ( mhsr ) . the odds ratios presented in the table were adjusted in the logistic regression for age , population groups , income , educational level and disability status . table 2 shows that self rated positive physical health is strongly related to chronic physical conditions but much less to mental disorders . likewise , self rated positive mental health is strongly related to mental disorders , but much less to chronic physical conditions . figure 1 shows how chronic physical conditions , mental disorders and the self rating scales of physical and mental health change with age . figure 1 shows that the percentage of those with chronic conditions goes up from 32% in the 21 - 35 age group to 78% in the 65 + group ( =653.9 ; p<0.001 ) while the poor self rated physical health follows the same pattern with a rise from 27% to 87% =1402.39 ; p<0.001 ) . on the other hand the poor self rated mental health goes up from 19.5% in the 21 - 35 age group to 64.8% in the 65 + group ( =609.98 ; p<0.001 ) while the percentage of those with any mental disorder does not change at all . table 3 presents the associations between self rated overall health ( osr ) and the self rated physical and mental health in two models : with and without the health status indicators . the odds ratios presented here were adjusted in the logistic regression for the variables population groups , age groups and disability level . table 3 shows that self rated mental and physical health are much stronger predictors of osr than the health status indicators of chronic conditions , mental disorders and disability . those with positive self rated mental health have 93 times the odds of reporting good positive overall health whereas those with positive self rated physical health have 40 times the odds of reporting positive overall health . after accounting for the self rated health measures hsr and mhsr , the predictive value of the health status indicators is much smaller : those with no mental disorders are six times more likely to have a positive osr , those with no disability have 2.5 times the odds of reporting positive osr and those with no chronic condition have only 1.33% times the odds of reporting positive osr . yet , the age group is still a significant predictor of osr beyond the self rating measures and the health status indicators . table 3 showed that after accounting for the main health variables , positive self rated mental health was twice as important as positive physical health in the prediction of positive overall health . the odds for positive overall health were 93 higher if the rating of mental health was positive compared to 40 times higher if the rating of physical health was positive . table 2 presents the associations between health status indicators and the self rated physical health ( hsr ) and mental health ( mhsr ) . the odds ratios presented in the table were adjusted in the logistic regression for age , population groups , income , educational level and disability status . table 2 shows that self rated positive physical health is strongly related to chronic physical conditions but much less to mental disorders . likewise , self rated positive mental health is strongly related to mental disorders , but much less to chronic physical conditions . figure 1 shows how chronic physical conditions , mental disorders and the self rating scales of physical and mental health change with age . figure 1 shows that the percentage of those with chronic conditions goes up from 32% in the 21 - 35 age group to 78% in the 65 + group ( =653.9 ; p<0.001 ) while the poor self rated physical health follows the same pattern with a rise from 27% to 87% =1402.39 ; p<0.001 ) . on the other hand the poor self rated mental health goes up from 19.5% in the 21 - 35 age group to 64.8% in the 65 + group ( =609.98 ; p<0.001 ) while the percentage of those with any mental disorder does not change at all . table 3 presents the associations between self rated overall health ( osr ) and the self rated physical and mental health in two models : with and without the health status indicators . the odds ratios presented here were adjusted in the logistic regression for the variables population groups , age groups and disability level . table 3 shows that self rated mental and physical health are much stronger predictors of osr than the health status indicators of chronic conditions , mental disorders and disability . those with positive self rated mental health have 93 times the odds of reporting good positive overall health whereas those with positive self rated physical health have 40 times the odds of reporting positive overall health . after accounting for the self rated health measures hsr and mhsr , the predictive value of the health status indicators is much smaller : those with no mental disorders are six times more likely to have a positive osr , those with no disability have 2.5 times the odds of reporting positive osr and those with no chronic condition have only 1.33% times the odds of reporting positive osr . yet , the age group is still a significant predictor of osr beyond the self rating measures and the health status indicators . table 3 showed that after accounting for the main health variables , positive self rated mental health was twice as important as positive physical health in the prediction of positive overall health . the odds for positive overall health were 93 higher if the rating of mental health was positive compared to 40 times higher if the rating of physical health was positive . data from the israel national health survey based on a representative sample of the adult population was used to investigate the differences between the three self rated measures of health . the general distribution of the self rated measures of health replicates results obtained in previous large population surveys showing that rates of positive self rated health are lower among females compared to males , that older individuals rate their health as less positive compared to younger individuals and that socio demographic variables such as education and income are related to the self rating of mental , physical and overall health . the main findings from the present study show that the three self rating scales represent three different domains of health : the self rating mental health and the self rating physical health maintain their distinctiveness : self rated mental health is not related to chronic physical conditions ( as does self rated physical health ) , and self rated physical health is not related to mental disorders ( as does self rated mental health ) . the self rating of physical health has a monotonic relationship with the objective indicator of physical health . the self rating of mental health does not have a monotonic relationship with the absence of mental illness . the good self rating of mental health goes down with age even though there is no age difference in mental disorders . the self rated physical health and the self rated mental health are both strong predictors of the self rated overall health , independent of the objective measures of health . this was shown when the odds of predicting the self rating of the overall health remained the same regardless of whether the objective measures of health were included in the regression equation . the self rated mental and physical health are 10 times stronger in predicting self rated overall health than any of the objective health indicators . the self rated mental health is two times more important than the self rated physical health in predicting the self rated overall health . the stronger weight of the self rated mental health implies that the mental component in the self rating of overall health is stronger than the physical one . this result reinforces conclusions of previous studies : in a cross - national survey of university students in germany , bulgaria , and poland , the overall self rated health status was assessed along with physical and psychological health as well as with social and socio demographic variables . that study found that psychosomatic complaints were the most important indicator in predicting the self rated health status . from a study using a large sample of late middle - aged and older persons living independently . they found that the unique contribution of depressive symptoms on poor health perception outranked those of the medical conditions . in a survey with a follow - up period of 1 - 5 years found that those who were happier and more energetic at baseline rated their health better in the next 5 years , even after accounting for age , negative health indicators at baseline , disease status and functioning . on the other hand , mental health which refers to hedonic well - being : feelings of happiness , satisfaction and interest in life , and to eudemonic well - being optimal functioning and self actualization , was more prevalent in younger age groups . in their review of studies on the concept of mental health , westerhof and keyes found that older age was correlated with lower positive affect , less feeling of personal growth and purpose in life , less meaning in life and less social coherence and social contribution . the empirical separation between mental health and mental illness was found also by weich et al . they found also that well - being and mental disorders are correlated but independent dimensions . the use of three separate self rating scales for mental health , physical health and overall health enabled us to observe the unique contribution of the mental health component to the self rating of overall health . further studies should investigate prospectively which of the three dimensions better predicts mortality , morbidity , hospitalizations or use of services . a key strength of this study was the utilization of a large representative sample of the entire adult population with a relatively high response rate . the main limitations of this study are that all the health information was self - reported and that the health indicators used may not have covered all the ill health domains . it is not clear whether a more comparative wording compared to your age group would have changed the clear age gradient that was found in this study . our results show a different age trajectory between mental health and mental illness . on the other hand , mental health which refers to hedonic well - being : feelings of happiness , satisfaction and interest in life , and to eudemonic well - being optimal functioning and self actualization , was more prevalent in younger age groups . in their review of studies on the concept of mental health , westerhof and keyes found that older age was correlated with lower positive affect , less feeling of personal growth and purpose in life , less meaning in life and less social coherence and social contribution . the empirical separation between mental health and mental illness was found also by weich et al . they found also that well - being and mental disorders are correlated but independent dimensions . the use of three separate self rating scales for mental health , physical health and overall health enabled us to observe the unique contribution of the mental health component to the self rating of overall health . further studies should investigate prospectively which of the three dimensions better predicts mortality , morbidity , hospitalizations or use of services . a key strength of this study was the utilization of a large representative sample of the entire adult population with a relatively high response rate . the main limitations of this study are that all the health information was self - reported and that the health indicators used may not have covered all the ill health domains . it is not clear whether a more comparative wording compared to your age group would have changed the clear age gradient that was found in this study .

oral cancer is one of the most common noncommunicable diseases worldwide with an estimated increase of 275,000 new cases each year . oral cancer is the term used for cancers that form in tissues of the oral cavity ( the mouth ) or the oropharynx ( the part of the throat at the back of the mouth ) . these along with other head and neck cancers are the sixth most prevalent type of cancer in the world [ 3 , 4 ] and one of the leading causes of death in developing countries . the countries of south asian region including india , pakistan , afghanistan , bangladesh , sri lanka , bhutan , nepal , iran , and maldives are particularly affected , with oral cancer ranking either first or second with regard to different types of cancer prevalence in these countries . the reasons for the high prevalence of head and neck cancers in south asia have been investigated to some extent but , as is the case with most developing countries , a lack of research infrastructure has put constraints on studying the epidemiology of these conditions in the context of south asia . one of the major risk factors associated with the high prevalence of head and neck cancer and oral potentially malignant diseases ( opmd ) in this region is smokeless tobacco ( slt ) . it is estimated that over 90% of the global smokeless tobacco use burden is in south east asia ; around 100 million people use smokeless tobacco in india and pakistan alone . slt is used in many forms varying from chewing tobacco not mixed with any other ingredient to a mixture of tobacco with other ingredients such as in betel quid , areca nut with tobacco , naswar , paan - masala with tobacco , gutkha , khaini , and mishri [ 12 , 13 ] . these include the nonvolatile alkaloid - derived tobacco - specific n - nitrosamine and n - nitrosamino acids as the major group while volatile tobacco - specific nitrosamines , volatile aldehydes , and some poly nuclear agents have also been shown to be present in smokeless tobacco . with such a high prevalence of both slt use and oral cancer in the south asian region , it is of utmost importance that epidemiological research is carried out to carefully assess their detailed relationship . two published reviews coauthored by iarc researchers have focused on overall associations found in studies worldwide [ 15 , 16 ] . several overviews originating from south asia have been published on oral cancer and smokeless tobacco [ 15 , 1726 ] but to date no systematic review of the published literature on association of oral cancer with different forms of smokeless tobacco focusing specifically on south asia has been conducted . this paper aims to address the issue by systematically reviewing publications reporting epidemiological observational studies carried out / published in the south asian region during the last 30 years , that is , published after 1984 on the use of all forms of slt and its relationship with oral cancer . an electronic search was carried out in medline and isi web of knowledge in august 2013 . various combinations of the terms oral premalignant disease , oral precancer , leukoplakia , erythroplakia , submucous fibrosis , lichen planus , oral cancer , oral carcinoma , mouth neoplasm , head and neck cancer , squamous cell carcinoma of the oral cavity , carcinoma lip , carcinoma tongue , oral neoplasms , and head and neck neoplasms ; smokeless tobacco , naswar , paan , snuff , oral snuff , chewing tobacco , betel quid , areca nut , and gutkha ; pakistan , india , bangladesh , iran , sri lanka , afghanistan , bhutan , nepal , the terms for different opmds were included in the search process as sometimes these conditions are studied together with oral cancer . the following selection criteria were applied to all the publications returned by the electronic searches to be included in the review . inclusion criteria are as follows : papers published after 1984,epidemiological observational study in humans of cohort or case - control design , studies carried out in south asia according to the united nations geographical region classification ( including the following countries : india , pakistan , afghanistan , bangladesh , sri lanka , bhutan , nepal , iran , and maldives),reported outcome or one of the reported outcomes is oral cancer or head and neck cancer , andexposure to paan , gutkha , betel nut , areca nut , or any other type of smokeless tobacco . papers published after 1984 , epidemiological observational study in humans of cohort or case - control design , studies carried out in south asia according to the united nations geographical region classification ( including the following countries : india , pakistan , afghanistan , bangladesh , sri lanka , bhutan , nepal , iran , and maldives ) , reported outcome or one of the reported outcomes is oral cancer or head and neck cancer , and exposure to paan , gutkha , betel nut , areca nut , or any other type of smokeless tobacco . exclusion criteria are as follows.studies reporting oesophageal , base of the tongue , and salivary glands cancers were excluded.studies involving laboratory research and molecular / genetic epidemiology were excluded . studies reporting oesophageal , base of the tongue , and salivary glands cancers were excluded . the selection process was done in three steps : first , the titles of all publications were scanned and relevant publications selected . the next step involved reading the abstracts of the publications selected in the first step . the selected publications were then divided into three groups according to their reported outcomes : ( 1 ) opmd as an outcome , ( 2 ) oral cancer as an outcome , and ( 3 ) opmd and oral cancer as an outcome ; publications reporting only opmds as an outcome were excluded at this stage . all selected publications were assessed for their quality on the basis of the effective public health practice project quality assessment tool for quantitative studies . studies were ranked as strong , moderate , and weak after being assessed on six parameters , that is , selection bias , study design , confounding , blinding , data collection methods , and withdrawals and dropouts . quality assessment was carried out by two authors independently and the results were later compared . any differences were discussed in the presence of all three authors and a final decision was reached by mutual consensus . first , data regarding the study type , location of the conducted study , sample size , year of publication , exposure , outcome , and the effect size were tabulated separately by two authors and later compared in the presence of all three authors . the data were then divided into two broad groups according to the difference in the type of slt exposure , that is , paan or betel quid with tobacco and chewable tobacco which included all types of smokeless tobacco other than paan . adjusted odds ratios ( or ) along with their 95% confidence intervals ( 95% ci ) , if reported , were recorded . in studies , where or were not reported but the data required to calculate them were available , or were calculated using a mantel - haenszel ( mh ) approach , thus providing us with weighted or across the different strata reported . however , if the paper did not report an adjusted or and the data given were too scarce to calculate mh - or , then the crude or as reported in the paper or calculated from the given data was recorded . or were also recorded or calculated for male and females separately , total duration of the habit in years , and frequency of daily use . standard errors of the natural logs of the or were calculated either from the 95% ci of the respective log or or by using the formula se(lnormh)=(bici / ni)vi/(bici / ni) when a mh - or was calculated . during the data extraction stage it had become obvious that there was major heterogeneity regarding methodological and other parameters among the selected publications . nevertheless all data were entered into rev man 5.2 and meta - analyses performed across all exposure categories , and their effect on oral cancer separately and combined was recorded . the i estimate was used to assess heterogeneity as it provides a better estimate for quantifying heterogeneity . heterogeneity was considered low if the i estimate was below 25% , moderate if it was between 25 and 50% , high if it was between 50 and 75% , and very high above the value of 75% . due to a very high level of heterogeneity , random effect meta - analysis has been used for this review . sensitivity and influence analysis were done by excluding one study at a time and checking its effect on the pooled estimate and the heterogeneity , but this had little effect on lowering the i statistic . meta - analyses were performed for overall estimates , case - control studies , studies with hospital controls , cohort studies , studies from india only , studies from southern india only , studies for maharashtra state , studies adjusting for smoking and/or alcohol , studies with moderate quality , and studies involving only men . for the categories where the data was incomplete , unavailable , or calculated using different methods , for example , the exposure response categories , a narrative synthesis was done . the synthesis highlights the highest and lowest estimates in general , according to gender and for studies that had done adjustment for alcohol and/or smoking . a total of 734 publications were identified from both database searches ( medline , isi web of knowledge ) ( figure 1 ) . one more paper was identified from a supplementary web search but it just reported the findings from one of the included studies and hence was excluded . after the first round of exclusion 137 publications remained ; after reading the abstracts , 38 publications were selected and their full text versions obtained . 21 publications reported oral cancer as the outcome or one of the outcomes and 13 publications reported just opmd as the outcome . the 21 publications [ 2949 ] for oral cancer included in this review correspond to 19 different studies and three studies were of cohort design while the remaining were of case - control design ( table 1 ) . 13 publications were published in or after the year 2000 while the remaining publications were published before the year 2000 , the oldest publication being from 1989 . 11 of the selected publications reported or contained data on paan with tobacco ( betel quid ) as a risk factor whereas 14 publications reported or contained data on chewing tobacco other than paan or without specifying any particular type of slt . data regarding daily frequencies of smokeless tobacco use were reported in 14 publications , while data on the total duration of the habit was reported in 10 publications . table 1 includes all selected studies for oral cancer and their features along with the quality assessment result for each study . the values for i statistic ranged from 77% to 96% when pooling studies across different strata . additional characteristics of the included studies are presented in the supplementary table 1 available online at http://dx.doi.org/10.1155/2014/394696 . for the purpose of clarity and taking into consideration the considerable difference between the outcome estimates related with the use of betel quid and other forms of slt , we reviewed the relationship of oral cancer with slt in two groups : ( 1 ) chewing tobacco of all kinds excluding betel quid or paan with tobacco and ( 2 ) betel quid or paan with tobacco . overall 14 publications reported different forms of chewing tobacco , predominantly gutkha and chewing tobacco leafs ( table 2 ) . five publications reported or that had been adjusted for smoking among other confounders . the adjusted or ranged from 3.6 [ 2.55.6 ] to 8.3 [ 5.413 ] . the or ranged from 1.2 [ 1.01.4 ] to 12.9 [ 7.522.3 ] among the publications in which either crude odds ratios were mentioned or a mh - or was calculated from the given data . the pooled or for chewing tobacco and risk of oral cancer was 4.7 [ 3.17.1 ] ( figure 2 ) . the studies where adjustment for alcohol and/or smoking had been done , when combined , provided a pooled or of 4.3 [ 3.15.8 ] . the pooled or from combining only case - control studies was 5.4 [ 4.17.1 ] . case - control studies having hospitals as a source of controls when combined gave a pooled estimate of 4.2 [ 2.56.9 ] . cohort studies when combined provided a pooled or of 2.9 [ 1.18.3 ] . for studies carried out in india for studies carried out in south india , which comprises of the states of andhra pradesh , kerala , karnataka , and tamil nadu , the pooled or was 5.1 [ 3.38.1 ] . the pooled or for studies carried out in the state of maharashtra was 4.8 [ 1.713.5 ] . when studies of moderate quality were combined , the pooled estimate came out to be 4.5 [ 2.87.3 ] . the pooled estimate for studies ranked as weak was 5.2 [ 2.610.3 ] . three publications reported or contained data from which or for men and/or women could be calculated separately ( table 2 ) . among men the or ranged from 1.2 [ 1.01.4 ] to 5.8 [ 3.69.5 ] . only two studies reported or separately for women ranging from 6.4 [ 3.39.0 ] to 25.3 [ 11.257.3 ] . studies carried out with only men taken as study subjects when combined provided a pooled or of 4.0 [ 2.95.7 ] . a total of seven publications provided dose response relationships according to the intensity of daily usage as exposure metric ( table 2 ) . these or varied from 1.1 [ 1.01.4 ] for chewing tobacco or chewable products containing tobacco for less than 5 times a day to 20.0 [ 8.148.9 ] for more than 10 times a day compared to nonchewers ; among studies adjusted for smoking and/or alcohol the corresponding values were 2.0 [ 13.8 ] and 13.9 [ 7.127.2 ] , both coming from the same study done by diskshit et al . . six publications described the effect of chewing tobacco on developing oral cancer in terms of the total duration of the habit ( table 2 ) . the or varied from 0.8 [ 0.41.7 ] for the total duration of the habit being less than 10 years , compared to nonchewers , to 10.9 [ 5.920.0 ] for a usage duration of 20 years or more compared to nonchewers . a total of nine publications included in this review reported or or contained data from which or could be calculated for the risk of chewing paan / betel quid and oral cancer ( table 3 ) . six publications [ 2931 , 38 , 41 , 44 ] reported overall or which were adjusted for confounding factors such as smoking and/or alcohol . overall , the or ( both adjusted and unadjusted ) varied from 3.1 to 15.7 [ 11.022.1 ] . the pooled or for chewing paan / betel quid and risk of oral cancer was 7.1 [ 4.511.1 ] ( figure 3 ) . the studies where adjustment for alcohol and/or smoking had been done , when combined , provided a pooled or of 6.3 [ 3.910.2 ] . case - control studies having hospitals as a source of controls when combined gave a pooled estimate of 7.4 [ 4.412.4 ] . for studies carried out in india the pooled estimate was 7.0 [ 4.411.1 ] . for studies carried out in south india only one study was carried out in the state of maharashtra where the or was 9.3 [ 5.117.2 ] . when the one weak study , for which the or was 3.9 [ 2.46.4 ] , was excluded , the pooled estimate came out to be 7.6 [ 4.712.3 ] . similarly the pooled risk estimates from studies carried out in south india were comparatively higher than the overall pooled estimate . six studies reported or contained data from which or could be calculated separately from men and/or women ( table 3 ) . for men the or for chewing betel quid with tobacco ranged from 1.5 [ 0.753.02 ] to 10.9 ; among women the or ranged between 6.5 and 45.8 [ 2584.1 ] . five studies reported the effect of frequency of daily use of paan with tobacco on oral cancer ( table 3 ) . the or varied from 3.3 [ 1.66.9 ] for chewing paan with tobacco , for less than 5 times a day compared to nonchewers , to 24.7 [ 12.548.7 ] for someone chewing it more than 10 times a day compared to nonchewers ; for studies adjusted for smoking and/or alcohol the corresponding values were 3.3 [ 1.66.9 ] and 15.7 . four studies reported or for the total duration of habit and oral cancer ( table 3 , last column ) . the or for chewing habit duration varied from 3.4 for a chewing habit of less than 10 years to 14.6 for a chewing habit persisting for 20 years or more ; the corresponding values for studies adjusting for smoking and/or alcohol were 3.4 and 14.6 both from the same study by sankarnarayanan et al . . the results of this systematic review suggest a strong link between different forms of smokeless tobacco ( slt ) and oral cancer and further strengthens and supports the iarc 's take on slt that it is a risk factor for oral cancer [ 15 , 16 ] . users of betel quid with tobacco have a sevenfold higher risk for developing oral cancer as compared to nonchewers , or 7.1 [ 4.511.1 ] . this finding is consistent with findings from the earlier reviews [ 15 , 16 ] . similarly , people using other forms of slt than betel quid with tobacco have an almost five - time higher risk of developing oral cancer as compared to nonchewers , or 4.7 [ 3.17.1 ] . these increased risks were consistently significant even after adjustment for other risks factors such as alcohol and smoking ; that is , pooled or for betel quid with tobacco after adjustment for alcohol and smoking was 6.3 [ 3.910.2 ] and the corresponding value for the chewing tobacco group was 4.3 [ 3.15.8 ] . these pooled estimates , however , should be dealt with caution because of the high levels of heterogeneity present among the studies but , despite indications of heterogeneity , even the lowest effect estimates among the individual studies are above the value of 1 , pointing towards a causal link between slt and oral cancer . the large variability of the effect estimates among individual studies may be attributed to differences in the composition of the products and population characteristics across the region . additionally , although most studies are case - control design , there are differences between the sources and ratio of controls to the number of cases . in general , the three cohort studies provide relatively conservative estimates as compared to the case - control studies ( table 1 ) . for the chewing tobacco category , case - control studies provided a pooled estimate , or 5.46 [ 4.17.1 ] , which was significantly higher than that of the cohort studies , or 2.9 [ 1.08.3 ] ; albeit the pooled estimate for the cohort studies had an increased width of the confidence intervals . this finding is in contrast with the review done by guha et al . , where they reported a higher pooled estimate for cohort studies as compared to the case - control ones . this may be explained by a difference in the selected cohort studies , as this review has only one cohort study in common with that review . they have included two cohort studies published prior to 1983 , which might have reported considerably higher risk estimates . the source of controls had only a slight bearing on the pooled estimates , with the pooled or for combining studies where controls were taken from hospitals , being slightly lower as compared to studies where population controls were recruited . the pooled or for studies carried out in south india and the state of maharashtra are relatively higher than the overall pooled estimate and this might be explained by the relatively high prevalence of slt use in these geographic locations [ 9 , 13 , 50 ] and incidence of oral cancer . the quality of the combined studies had minimal effect on the overall summary estimate , that is , or 4.5 [ 2.87.3 ] , compared to the overall pooled or of 4.7 [ 3.17.1 ] ; however , in the chewing tobacco group exclusion of the weak studies ( n = 4 ) lowered the pooled estimate , while in the betel quid group , where there was just one weak study , the overall estimate increased when the weak study was excluded . the studies which were ranked as weak did not play any role in the narrative synthesis either ; most had not reported any results or suitable data for calculation of ors , in the exposure response categories of frequency / intensity and duration . paan with tobacco appears to have a higher risk as compared to chewing other tobacco products ; the overall pooled or for paan as well as the pooled or across different exposure strata are significantly higher in comparison with the other forms of chewing tobacco ( figure 3 and tables 2 and 3 ) . a possible reason for this could be the use of areca nut in paan , as it has been shown to have carcinogenic properties on its own and thus might have a synergistic effect with the carcinogenicity of slt , resulting in a higher risk of oral cancer as compared to other forms of slt use . similarly another ingredient , slaked lime , used in betel quid preparation has been shown to have carcinogenic potential . it facilitates the production of reactive oxygen species ( ros ) in the saliva of chewers and also facilitates the hydrolysis of arecoline into arecaidine which in turn facilitates increased fibroblast proliferation and collagen synthesis , which are essential for premalignant and malignant transformation of the affected tissues . the betel quid with tobacco group analysis included only case - control studies and therefore a formal comparison of the risk estimates among case - control and cohort studies could not be done . however , similar to the chewing tobacco group , the studies which recruited hospital controls had a relatively higher pooled risk estimate , that is , or 7.4 [ 4.412.2 ] , compared to the overall estimate . an interesting observation is the risk differences among males and females , with females being at a significantly higher risk of oral cancer from slt use as compared to men ( tables 2 and 3 ) . this may be attributed to increased susceptibility of the female oral mucosa to damage by tobacco products and relative lack of education and poverty , all of which have been shown to be significant risk factors on their own [ 9 , 22 ] . also it may be due to a lower background risk for oral cancer among women of this region because of a lower prevalence of smoking and alcohol drinking . also a high prevalence of cervical cancer among women in india may be suggestive of the presence of human papilloma virus ( hpv ) , which is an established risk factor for oral cancer as well . there is , however , significant inconsistency in effect estimates among the case - control studies regarding risks in women and also between the case - control and the cohort studies , which might have led to an overestimation of the risk estimates among women . in the study carried out by jayalekshmi et al . where cohorts of men and women were analyzed separately [ 45 , 46 ] , the authors found that the risk estimates were almost similar among both sexes , which underscores the argument that the true effect size for the relationship between slt and oral cancer in women may be overestimated . however , it should be clear that , regardless of the magnitude of effect size , all included studies that provide sex - specific estimates provide evidence that slt is a major risk factor for oral cancer among women in the south asian region . these results may warrant future research to specifically focus on sex differences and provide reliable risk estimates among men and women using slt . the results of our review suggest that there is an exposure - response causal relationship between slt use and oral cancer , for both the intensity and duration of use . this effect is somewhat linear in case of the chewing tobacco group but for the betel quid group , though the data suggests a possible relationship , it is a nonlinear one . this result is consistent with the iarc reviews but differs from findings of some other reviews [ 5658 ] carried out on published literature from north america and europe . in these reviews no dose response relationships were identified . this may be explained by the difference in the types of slt used in south asia compared to north america and europe . differences in ethnicity and socioeconomic status and environmental differences may be additional reasons for these conflicting findings . it may also be noted that the effect sizes reported in the reviews of studies carried out on slt and oral cancer in europe and north america report significantly smaller observed effect estimates as compared to the studies included in the present review . the synthesis of the reviewed publications suggests that the total duration of exposure to slt increases the risk of oral cancer ; that is , subjects who used slt ( chewing tobacco , paan / betel quid ) for more than 10 years were at a higher risk of oral cancer than those who used slt for less than 10 years . parallels can be drawn here with the habit of smoking and alcohol use , where the risk for developing oral cancer increases with an increase in the total duration of exposure to these substances . furthermore , the mean age of oral cancer cases in the included studies was mostly in the fifth decade of life ( table 1 ) . given that usually habits like slt use are generally taken up in early adolescence , this might suggest that prolonged exposure to slt increases the risk of oral cancer , although age itself has been shown to be a risk factor for oral cancer . some of the limitations are inherent to the observational study designs included in this systematic review , such as recall and selection bias , under-/overreporting of exposure status , retrospective exposure assessment , and uncontrolled confounding . our electronic search included terms for all countries comprised in the south asian region , but only publications from india and pakistan were included because no case - control or cohort studies could be found for other countries . due to a lack of resources a metaregression analysis could not be performed to identify the sources of heterogeneity ; however , in the most recent review done by iarc researchers , which includes most of the studies included in our review , metaregression analysis did not lower heterogeneity to moderate or low levels . given the various types of slt used in the indian subcontinent and its increasing popularity in the neighboring countries , it is of great importance that the general public be made aware of slt use as a major risk factor for oral cancer . most of the tobacco control initiatives around the world have been aimed towards cessation of smoking , where the main strategy to decrease smoking prevalence is the high amount of taxes levied on smoking products . although this might be productive for smoking cessation , this strategy may facilitate an unintentional push towards smokeless tobacco use and increasing prevalence because slt is cheaper compared to smoking . additionally , big tobacco companies revert to manufacturing smokeless tobacco products and advertising them as less harmful than smoking . all these scenarios may potentially lead to a surge in the use of smokeless tobacco products and subsequent increased risks for oral cancer for the general public . the governments and general public should be made aware of the potential dangers related to such approaches and may consider new programs for smokeless tobacco cessation or incorporate the risks of slt consumption into smoking cessation programs . from the published literature it appears that various forms of smokeless tobacco used in south asia should be considered as strong risk factors for oral cancer . public health policies in affected countries should consider slt cessation programs in addition to campaigns and activities to inform the general public about slt use and oral cancer risks .

introduction . smokeless tobacco is considered one of the major risk factors for oral cancer . it is estimated that over 90% of the global smokeless tobacco use burden is in south asia . this paper aims to systematically review publications reporting epidemiological observational studies published in south asia from 1984 till 2013 . methods . an electronic search in medline and isi web of knowledge yielded 734 publications out of which 21 were included in this review . all publications were assessed for quality using a standard quality assessment tool . effect estimates ( odds ratios ( or ) ) were abstracted or calculated from the given data . a random effects meta - analysis was performed to assess the risk of oral cancer with the use of different forms of smokeless tobacco . results and conclusion . the pooled or for chewing tobacco and risk of oral cancer was 4.7 [ 3.17.1 ] and for paan with tobacco and risk of oral cancer was 7.1 [ 4.511.1 ] . the findings of this study suggest a strong causal link between oral cancer and various forms of smokeless tobacco . public health policies in affected countries should consider slt specific cessation programs in addition to campaigns and activities incorporated into smoking cessation programs .

1. Introduction 2. Methods 3. Results 4. Discussion 5. Limitations of the Review 6. Policy Implications 7. Conclusion

oral cancer is one of the most common noncommunicable diseases worldwide with an estimated increase of 275,000 new cases each year . oral cancer is the term used for cancers that form in tissues of the oral cavity ( the mouth ) or the oropharynx ( the part of the throat at the back of the mouth ) . these along with other head and neck cancers are the sixth most prevalent type of cancer in the world [ 3 , 4 ] and one of the leading causes of death in developing countries . one of the major risk factors associated with the high prevalence of head and neck cancer and oral potentially malignant diseases ( opmd ) in this region is smokeless tobacco ( slt ) . it is estimated that over 90% of the global smokeless tobacco use burden is in south east asia ; around 100 million people use smokeless tobacco in india and pakistan alone . slt is used in many forms varying from chewing tobacco not mixed with any other ingredient to a mixture of tobacco with other ingredients such as in betel quid , areca nut with tobacco , naswar , paan - masala with tobacco , gutkha , khaini , and mishri [ 12 , 13 ] . these include the nonvolatile alkaloid - derived tobacco - specific n - nitrosamine and n - nitrosamino acids as the major group while volatile tobacco - specific nitrosamines , volatile aldehydes , and some poly nuclear agents have also been shown to be present in smokeless tobacco . several overviews originating from south asia have been published on oral cancer and smokeless tobacco [ 15 , 1726 ] but to date no systematic review of the published literature on association of oral cancer with different forms of smokeless tobacco focusing specifically on south asia has been conducted . this paper aims to address the issue by systematically reviewing publications reporting epidemiological observational studies carried out / published in the south asian region during the last 30 years , that is , published after 1984 on the use of all forms of slt and its relationship with oral cancer . an electronic search was carried out in medline and isi web of knowledge in august 2013 . various combinations of the terms oral premalignant disease , oral precancer , leukoplakia , erythroplakia , submucous fibrosis , lichen planus , oral cancer , oral carcinoma , mouth neoplasm , head and neck cancer , squamous cell carcinoma of the oral cavity , carcinoma lip , carcinoma tongue , oral neoplasms , and head and neck neoplasms ; smokeless tobacco , naswar , paan , snuff , oral snuff , chewing tobacco , betel quid , areca nut , and gutkha ; pakistan , india , bangladesh , iran , sri lanka , afghanistan , bhutan , nepal , the terms for different opmds were included in the search process as sometimes these conditions are studied together with oral cancer . inclusion criteria are as follows : papers published after 1984,epidemiological observational study in humans of cohort or case - control design , studies carried out in south asia according to the united nations geographical region classification ( including the following countries : india , pakistan , afghanistan , bangladesh , sri lanka , bhutan , nepal , iran , and maldives),reported outcome or one of the reported outcomes is oral cancer or head and neck cancer , andexposure to paan , gutkha , betel nut , areca nut , or any other type of smokeless tobacco . papers published after 1984 , epidemiological observational study in humans of cohort or case - control design , studies carried out in south asia according to the united nations geographical region classification ( including the following countries : india , pakistan , afghanistan , bangladesh , sri lanka , bhutan , nepal , iran , and maldives ) , reported outcome or one of the reported outcomes is oral cancer or head and neck cancer , and exposure to paan , gutkha , betel nut , areca nut , or any other type of smokeless tobacco . the selection process was done in three steps : first , the titles of all publications were scanned and relevant publications selected . the selected publications were then divided into three groups according to their reported outcomes : ( 1 ) opmd as an outcome , ( 2 ) oral cancer as an outcome , and ( 3 ) opmd and oral cancer as an outcome ; publications reporting only opmds as an outcome were excluded at this stage . all selected publications were assessed for their quality on the basis of the effective public health practice project quality assessment tool for quantitative studies . the data were then divided into two broad groups according to the difference in the type of slt exposure , that is , paan or betel quid with tobacco and chewable tobacco which included all types of smokeless tobacco other than paan . adjusted odds ratios ( or ) along with their 95% confidence intervals ( 95% ci ) , if reported , were recorded . however , if the paper did not report an adjusted or and the data given were too scarce to calculate mh - or , then the crude or as reported in the paper or calculated from the given data was recorded . or were also recorded or calculated for male and females separately , total duration of the habit in years , and frequency of daily use . standard errors of the natural logs of the or were calculated either from the 95% ci of the respective log or or by using the formula se(lnormh)=(bici / ni)vi/(bici / ni) when a mh - or was calculated . due to a very high level of heterogeneity , random effect meta - analysis has been used for this review . a total of 734 publications were identified from both database searches ( medline , isi web of knowledge ) ( figure 1 ) . one more paper was identified from a supplementary web search but it just reported the findings from one of the included studies and hence was excluded . 21 publications reported oral cancer as the outcome or one of the outcomes and 13 publications reported just opmd as the outcome . the 21 publications [ 2949 ] for oral cancer included in this review correspond to 19 different studies and three studies were of cohort design while the remaining were of case - control design ( table 1 ) . 11 of the selected publications reported or contained data on paan with tobacco ( betel quid ) as a risk factor whereas 14 publications reported or contained data on chewing tobacco other than paan or without specifying any particular type of slt . data regarding daily frequencies of smokeless tobacco use were reported in 14 publications , while data on the total duration of the habit was reported in 10 publications . table 1 includes all selected studies for oral cancer and their features along with the quality assessment result for each study . for the purpose of clarity and taking into consideration the considerable difference between the outcome estimates related with the use of betel quid and other forms of slt , we reviewed the relationship of oral cancer with slt in two groups : ( 1 ) chewing tobacco of all kinds excluding betel quid or paan with tobacco and ( 2 ) betel quid or paan with tobacco . overall 14 publications reported different forms of chewing tobacco , predominantly gutkha and chewing tobacco leafs ( table 2 ) . the or ranged from 1.2 [ 1.01.4 ] to 12.9 [ 7.522.3 ] among the publications in which either crude odds ratios were mentioned or a mh - or was calculated from the given data . the pooled or for chewing tobacco and risk of oral cancer was 4.7 [ 3.17.1 ] ( figure 2 ) . the studies where adjustment for alcohol and/or smoking had been done , when combined , provided a pooled or of 4.3 [ 3.15.8 ] . the pooled or from combining only case - control studies was 5.4 [ 4.17.1 ] . for studies carried out in india for studies carried out in south india , which comprises of the states of andhra pradesh , kerala , karnataka , and tamil nadu , the pooled or was 5.1 [ 3.38.1 ] . the pooled or for studies carried out in the state of maharashtra was 4.8 [ 1.713.5 ] . these or varied from 1.1 [ 1.01.4 ] for chewing tobacco or chewable products containing tobacco for less than 5 times a day to 20.0 [ 8.148.9 ] for more than 10 times a day compared to nonchewers ; among studies adjusted for smoking and/or alcohol the corresponding values were 2.0 [ 13.8 ] and 13.9 [ 7.127.2 ] , both coming from the same study done by diskshit et al . six publications described the effect of chewing tobacco on developing oral cancer in terms of the total duration of the habit ( table 2 ) . a total of nine publications included in this review reported or or contained data from which or could be calculated for the risk of chewing paan / betel quid and oral cancer ( table 3 ) . the pooled or for chewing paan / betel quid and risk of oral cancer was 7.1 [ 4.511.1 ] ( figure 3 ) . the studies where adjustment for alcohol and/or smoking had been done , when combined , provided a pooled or of 6.3 [ 3.910.2 ] . for studies carried out in india the pooled estimate was 7.0 [ 4.411.1 ] . when the one weak study , for which the or was 3.9 [ 2.46.4 ] , was excluded , the pooled estimate came out to be 7.6 [ 4.712.3 ] . similarly the pooled risk estimates from studies carried out in south india were comparatively higher than the overall pooled estimate . for men the or for chewing betel quid with tobacco ranged from 1.5 [ 0.753.02 ] to 10.9 ; among women the or ranged between 6.5 and 45.8 [ 2584.1 ] . five studies reported the effect of frequency of daily use of paan with tobacco on oral cancer ( table 3 ) . the or varied from 3.3 [ 1.66.9 ] for chewing paan with tobacco , for less than 5 times a day compared to nonchewers , to 24.7 [ 12.548.7 ] for someone chewing it more than 10 times a day compared to nonchewers ; for studies adjusted for smoking and/or alcohol the corresponding values were 3.3 [ 1.66.9 ] and 15.7 . the or for chewing habit duration varied from 3.4 for a chewing habit of less than 10 years to 14.6 for a chewing habit persisting for 20 years or more ; the corresponding values for studies adjusting for smoking and/or alcohol were 3.4 and 14.6 both from the same study by sankarnarayanan et al . the results of this systematic review suggest a strong link between different forms of smokeless tobacco ( slt ) and oral cancer and further strengthens and supports the iarc 's take on slt that it is a risk factor for oral cancer [ 15 , 16 ] . users of betel quid with tobacco have a sevenfold higher risk for developing oral cancer as compared to nonchewers , or 7.1 [ 4.511.1 ] . similarly , people using other forms of slt than betel quid with tobacco have an almost five - time higher risk of developing oral cancer as compared to nonchewers , or 4.7 [ 3.17.1 ] . these increased risks were consistently significant even after adjustment for other risks factors such as alcohol and smoking ; that is , pooled or for betel quid with tobacco after adjustment for alcohol and smoking was 6.3 [ 3.910.2 ] and the corresponding value for the chewing tobacco group was 4.3 [ 3.15.8 ] . these pooled estimates , however , should be dealt with caution because of the high levels of heterogeneity present among the studies but , despite indications of heterogeneity , even the lowest effect estimates among the individual studies are above the value of 1 , pointing towards a causal link between slt and oral cancer . the large variability of the effect estimates among individual studies may be attributed to differences in the composition of the products and population characteristics across the region . for the chewing tobacco category , case - control studies provided a pooled estimate , or 5.46 [ 4.17.1 ] , which was significantly higher than that of the cohort studies , or 2.9 [ 1.08.3 ] ; albeit the pooled estimate for the cohort studies had an increased width of the confidence intervals . this finding is in contrast with the review done by guha et al . the source of controls had only a slight bearing on the pooled estimates , with the pooled or for combining studies where controls were taken from hospitals , being slightly lower as compared to studies where population controls were recruited . the pooled or for studies carried out in south india and the state of maharashtra are relatively higher than the overall pooled estimate and this might be explained by the relatively high prevalence of slt use in these geographic locations [ 9 , 13 , 50 ] and incidence of oral cancer . the quality of the combined studies had minimal effect on the overall summary estimate , that is , or 4.5 [ 2.87.3 ] , compared to the overall pooled or of 4.7 [ 3.17.1 ] ; however , in the chewing tobacco group exclusion of the weak studies ( n = 4 ) lowered the pooled estimate , while in the betel quid group , where there was just one weak study , the overall estimate increased when the weak study was excluded . paan with tobacco appears to have a higher risk as compared to chewing other tobacco products ; the overall pooled or for paan as well as the pooled or across different exposure strata are significantly higher in comparison with the other forms of chewing tobacco ( figure 3 and tables 2 and 3 ) . a possible reason for this could be the use of areca nut in paan , as it has been shown to have carcinogenic properties on its own and thus might have a synergistic effect with the carcinogenicity of slt , resulting in a higher risk of oral cancer as compared to other forms of slt use . the betel quid with tobacco group analysis included only case - control studies and therefore a formal comparison of the risk estimates among case - control and cohort studies could not be done . however , similar to the chewing tobacco group , the studies which recruited hospital controls had a relatively higher pooled risk estimate , that is , or 7.4 [ 4.412.2 ] , compared to the overall estimate . an interesting observation is the risk differences among males and females , with females being at a significantly higher risk of oral cancer from slt use as compared to men ( tables 2 and 3 ) . this may be attributed to increased susceptibility of the female oral mucosa to damage by tobacco products and relative lack of education and poverty , all of which have been shown to be significant risk factors on their own [ 9 , 22 ] . also it may be due to a lower background risk for oral cancer among women of this region because of a lower prevalence of smoking and alcohol drinking . also a high prevalence of cervical cancer among women in india may be suggestive of the presence of human papilloma virus ( hpv ) , which is an established risk factor for oral cancer as well . there is , however , significant inconsistency in effect estimates among the case - control studies regarding risks in women and also between the case - control and the cohort studies , which might have led to an overestimation of the risk estimates among women . where cohorts of men and women were analyzed separately [ 45 , 46 ] , the authors found that the risk estimates were almost similar among both sexes , which underscores the argument that the true effect size for the relationship between slt and oral cancer in women may be overestimated . however , it should be clear that , regardless of the magnitude of effect size , all included studies that provide sex - specific estimates provide evidence that slt is a major risk factor for oral cancer among women in the south asian region . this effect is somewhat linear in case of the chewing tobacco group but for the betel quid group , though the data suggests a possible relationship , it is a nonlinear one . this result is consistent with the iarc reviews but differs from findings of some other reviews [ 5658 ] carried out on published literature from north america and europe . it may also be noted that the effect sizes reported in the reviews of studies carried out on slt and oral cancer in europe and north america report significantly smaller observed effect estimates as compared to the studies included in the present review . the synthesis of the reviewed publications suggests that the total duration of exposure to slt increases the risk of oral cancer ; that is , subjects who used slt ( chewing tobacco , paan / betel quid ) for more than 10 years were at a higher risk of oral cancer than those who used slt for less than 10 years . parallels can be drawn here with the habit of smoking and alcohol use , where the risk for developing oral cancer increases with an increase in the total duration of exposure to these substances . furthermore , the mean age of oral cancer cases in the included studies was mostly in the fifth decade of life ( table 1 ) . given that usually habits like slt use are generally taken up in early adolescence , this might suggest that prolonged exposure to slt increases the risk of oral cancer , although age itself has been shown to be a risk factor for oral cancer . some of the limitations are inherent to the observational study designs included in this systematic review , such as recall and selection bias , under-/overreporting of exposure status , retrospective exposure assessment , and uncontrolled confounding . our electronic search included terms for all countries comprised in the south asian region , but only publications from india and pakistan were included because no case - control or cohort studies could be found for other countries . due to a lack of resources a metaregression analysis could not be performed to identify the sources of heterogeneity ; however , in the most recent review done by iarc researchers , which includes most of the studies included in our review , metaregression analysis did not lower heterogeneity to moderate or low levels . given the various types of slt used in the indian subcontinent and its increasing popularity in the neighboring countries , it is of great importance that the general public be made aware of slt use as a major risk factor for oral cancer . although this might be productive for smoking cessation , this strategy may facilitate an unintentional push towards smokeless tobacco use and increasing prevalence because slt is cheaper compared to smoking . all these scenarios may potentially lead to a surge in the use of smokeless tobacco products and subsequent increased risks for oral cancer for the general public . the governments and general public should be made aware of the potential dangers related to such approaches and may consider new programs for smokeless tobacco cessation or incorporate the risks of slt consumption into smoking cessation programs . from the published literature it appears that various forms of smokeless tobacco used in south asia should be considered as strong risk factors for oral cancer . public health policies in affected countries should consider slt cessation programs in addition to campaigns and activities to inform the general public about slt use and oral cancer risks .

patients with right cerebral hemisphere lesions often show a reduced tendency to respond to stimuli and to search actively for them in the contralateral part of space . this condition described as left spatial neglect is typically demonstrated by clinical observation and simple perceptual motor tests such as a line bisection or cancellation test . left spatial neglect occurs in about 2530% of all stroke patients , and although some degrees of spontaneous recovery occurs , the disorder persists chronically in many cases . frequently associated with contralesional motor or somatosensory deficit , left spatial neglect is recognized as one of the main factors associated with poor functional outcome [ 68 ] . for these reasons , the improvement of left spatial neglect over and above spontaneous recovery represents a challenge in the area of neurological rehabilitation . over the past 60 years , many different attempts to alleviate this impairment have been developed ( for a review see ) . among these , prism adaptation has been widely used since the end of the nineteenth century as a paradigm to demonstrate visuomotor short - term plasticity . exposure to prisms produces a lateral shift of the visual field so that the visual target appears at a displaced position . adaptation to such an optical induced shift critically requires a set of successive perceptual - motor pointing movements . while the initial movements tend to approximate to the virtual position of the target , subsequent pointing movements ensure that the pointing error rapidly decreases so that subjects can readily point towards the real target position . this initial error reduction comprises a strategic component of the reaction to prisms and does not necessarily produce adaptation at this stage . to obtain robust compensatory after - effects following removal of prisms , further pointing movements are required . these reinforce the sensory - motor adaptation and are considered characteristic of the real or true after - effects result from a compensatory shift in manual straightahead pointing in a direction opposite to the original visual shift produced by prisms . rossetti et al . proposed that the adaptation to right - deviating prisms with leftward compensatory after - effects ( using the intact right hand ) improved left neglect symptoms . in this study , a significant improvement was demonstrated across a variety of different standard paper and pencil tests ( line bisection , line cancellation , copying a scene , and reading a simple text ) . subsequent studies have shown that these clinical effects could extend to numerous neglect - related processes ( for a review see ) such as straightahead pointing , visual exploration toward the left hemispace , postural control , contralesional somatosensory perception [ 2022 ] , temporal order judgment , and mental representation [ 2426 ] . from a rehabilitation perspective , the long - term beneficial effect on several functional measures set this intervention apart from the other attempts . ( i ) farn et al . found a reduction of spatial dyslexia still present one day after a single session of prism adaptation in a group of 6 patients with left spatial neglect . ( ii ) rode et al . reported a positive effect of prism adaptation on spatial dysgraphia the improvement concerned the right - page preference and was maintained up to 4 days after a single session of prism adaptation . watanabe and amimoto reported an improvement of wheelchair navigation after a single session of prism adaptation in two single - case studies . ( iv ) a long - lasting amelioration , up to five weeks , was reported on several functional tasks following a twice - daily adaptation program during a period of two weeks [ 31 , 32 ] or one - daily prism - adaptation session during two weeks [ 33 , 34 ] . this impressive generalization and long - standing effects of prism adaptation have revived interest in the neuro - cognitive mechanisms by which it has been achieved . the most two basic questions about the mechanisms of action of prism adaptation are ( i ) whether adaptation per se is necessary to produce cognitive after - effects or whether simple visuomanual pointing could produce similar effects , and ( ii ) whether this adaptation is specific in terms of its direction . as a matter of facts , such specificity has been demonstrated in healthy individuals ( see for review ) , in patients with complex regional pain syndrome , but no data is available on neglect patients . the purpose of the present study was to evaluate the directional specificity of prism adaptation in neglect patients . given that the effect of right - deviating prisms on left spatial neglect is already well documented ( cf . supra ) , this work was designed to evaluate the effect of left - deviating prisms on left spatial neglect . since it has been shown that adaptation to right - deviating prism may affect differently straightahead pointing movements and attentional tasks , the effect of left - deviating prism was measured both on straightahead pointing movements and several attentional tasks classically used to assess left spatial neglect . patients were selected from the neuro - rehabilitation department of the hospices civils de lyon , france . inclusion criteria were right - handed patients with left spatial neglect after right hemispheric ischemic or hemorrhagic stroke . patients with previous history of stroke , psychiatric diseases , global cognitive deterioration , or any impairment that could compromise comprehension and compliance with the tasks were excluded . for all patients screened , hand preference was assessed by the edinburgh inventory . left spatial neglect was assessed using a battery of six paper and pencil tests : line cancellation , balloon test , line bisection , copy of a scene , drawing from memory , simple text reading ( cf . a cerebral computerized tomography ( ct ) or mri scan was performed for each patient in order to specify the type of lesion ( ischemic or hemorrhagic ) , to rule out any other relevant prestroke lesions and to determine the anatomic location of the lesion . this study was conducted with the informed consent of the participants , in agreement with the french law ( march 2002 ) and the helsinki declaration relative to patient 's rights . the sample comprised six healthy subjects and five patients aged between 67 and 80 years old ( see table 1 for clinical profiles of each patient ) . the mean time period between stroke onset and inclusion was 1.5 months ( range : 1 to 2.5 months ) . one patient had a hemorrhagic stroke ( patient 2 ) ; the four others had an ischemic stroke : two in the posterior part of the superficial middle cerebral artery territory ( patients 1 and 3 ) , one in the anterior part of the superficial middle cerebral artery territory ( patient 5 ) , and one in the deep part of the middle cerebral artery territory ( patient 4 ) . lesion analysis showed the involvement of the inferior , middle , and superior temporal gyri in three patients ( patient 1 , patient 3 , and patient 5 ) ; the temporoparietooccipital junction was damaged in two patients ( patient 1 and patient 3 ) . lesions of other brain structures involved the somatosensory parietal cortex ( patient 1 and patient 5 ) , the primary motor cortex ( patient 1 and patient 5 ) , the occipital cortex ( patient 1 and patient 3 ) , the prefrontal and the orbito - frontal cortex ( patient 5 ) , the insula ( patient 1 , patient 2 , patient 4 , and patient 5 ) , the thalamus ( patient 2 and patient 4 ) , the putamen and pallidum ( patient 2 , patient 4 , and patient 5 ) , the internal capsule ( patient 2 and patient 4 ) , the caudate nucleus , the hippocampus , and parahippocampus ( patient 4 ) . patients ' performance was investigated in sessions that took place before prism adaptation ( referred to as pre ) , immediately after ( post ) , and 2 hours after ( late ) . healthy subjects performed the same tasks as patients before ( pre ) and immediately after ( post ) prism adaptation . during each session , left spatial neglect was assessed using line cancellation , balloon test , line bisection , copy of a scene , drawing from memory , and a simple text reading ( cf . section 2.2.2 . for description ) . in order to check whether healthy subjects and patients correctly adapted to prisms , a measure of straightahead pointing was performed before adaptation ( pre ) and after participants had completed the immediate neuropsychological tests ( post ) as in rossetti et al . . the participant was seated blindfolded in front of a horizontal box that allowed for an electronic measurement of the finger movement endpoints with an accuracy of 1 deg . participants were required to point straightahead while their head was kept aligned with the body 's sagittal axis . line cancellation this test consists of an a4 page containing 40 lines arranged in different direction . the page is placed at body midline . the balloon test this test consists of two subtests , carried out on two a3 landscape - orientated stimulus sheets , each containing 202 items ( circles or balloons ) . in the first subtest pop - out , 22 target balloons are interspersed between 180 circles which play the role of distractor . this test is based on the phenomenon of perceptual po - pout , that is , the time taken to detect target of this kind does not increase significantly as the number of distractors increase . in the second subtest search , the number and position of the balloons and circles are exactly the reverse ; thus 22 of the 202 items are circles to be cancelled and the other 180 items are balloons . in this test , subjects were required to cancel out as many circles as they could find . in this test rather they have to be searched , and therefore , this test requires a greater demand on attention . in both subtests , this test consists of two subtests , carried out on two a3 landscape - orientated stimulus sheets , each containing 202 items ( circles or balloons ) . in the first subtest pop - out , 22 target balloons are interspersed between 180 circles which play the role of distractor . this test is based on the phenomenon of perceptual po - pout , that is , the time taken to detect target of this kind does not increase significantly as the number of distractors increase . search , the number and position of the balloons and circles are exactly the reverse ; thus 22 of the 202 items are circles to be cancelled and the other 180 items are balloons . in this test , subjects were required to cancel out as many circles as they could find . in this test , the targets do not pop out . rather they have to be searched , and therefore , this test requires a greater demand on attention . in both subtests , line bisection participants were presented with an a4 page , in front of their body midline , containing twenty lines of different length ranging from 100 mm to 200 mm . participants were instructed to cut each line in half by placing a small pencil mark through each line as close to its center as possible . the score was the mean percentage of deviation from the true center of the line ( the score is positive when the deviation is in the right direction and negative when the deviation is in the left direction ) . participants were presented with an a4 page , in front of their body midline , containing twenty lines of different length ranging from 100 mm to 200 mm . participants were instructed to cut each line in half by placing a small pencil mark through each line as close to its center as possible . the score was the mean percentage of deviation from the true center of the line ( the score is positive when the deviation is in the right direction and negative when the deviation is in the left direction ) . copy of a scene participants were required to reproduce a picture made up of five items ( 4 trees and a house ) in the space bellow it . performances were assessed by two scores : ( i ) the number of items reproduced and ( ii ) the number of items symmetrically depicted . participants were required to reproduce a picture made up of five items ( 4 trees and a house ) in the space bellow it . performances were assessed by two scores : ( i ) the number of items reproduced and ( ii ) the number of items symmetrically depicted . a score of 1 was given when the daisy was highly asymmetrical , 2 when the drawing was moderately asymmetrical , and 3 when the drawing was symmetrical . a score of 1 was given when the daisy was highly asymmetrical , 2 when the drawing was moderately asymmetrical , and 3 when the drawing was symmetrical . the adaptation procedure involved the participants having to wear prismatic goggles that produced a 10 leftward shift of the visual wide - field that is in the opposite direction to rossetti et al . while wearing prisms , the participant was required to make as fast as possible a series of approximately 50 pointing responses , with his / her right hand , to visual targets located to the left and right side of midline . the procedure lasted approximately five minutes . in order to ensure optimal adaptation , visual feedback of the starting point of the hand the first analysis was to test whether healthy subjects and patients correctly adapted to left - deviating prisms . we carried out t - tests to compare the average end - position before ( pre ) and after ( post ) prism adaptation . in order to evaluate the presence of an amelioration of left neglect symptoms after prism adaptation , an analysis of variance with repeated measure ( anova ) was performed on each neuropsychological test , using sessions ( pre , post , late ) as factor . hence , for a specified test , the null hypothesis ( p value > 0.05 ) is the absence of difference between sessions of the mean score across patients . the alternative hypothesis ( p value < 0.05 ) can be written as follow : at least one of the mean score differs between sessions . in this latter case , a post hoc sheff test was carried out in order to compare the mean scores across sessions : pre versus post , pre versus late , and post versus late . for the healthy controls , a significant displacement of the straightahead pointings to the right was observed after exposure to left deviating prisms without significant modification of the performances on the attentional paper and pencil tests . for the neglect patients , no significant effect of prism exposure was observed neither on the straight - ahead pointing task nor on the neuropsychological tests . controls . before left - deviating prism adaptation ( pre ) , the group analysis showed that the mean end - position of 7 straightahead pointing trials was shifted 1.3 degrees to the right of the body midline ( range : 2.3 to 5.1 ) . after prism adaptation ( post ) , the mean deviation was significantly displaced to the right ( mean position after prism adaptation : 5.8 degrees to the right of the body midline ; range : 0.7 to 9.0 ) . comparison between trials performed before and after prism adaptation was significant ( t = 3.15 ; p = 0.026 ) . , the group analysis showed that the mean end - position of 7 straightahead pointing trials was shifted 3.7 degrees to the right of the body midline ( range : 2.0 to 4.9 ) . after prism adaptation ( post ) , the mean end - position was unchanged ( mean : 3.7 degrees to the right ; range : 2.4 to 4 ) . comparison between trials performed before and after prism adaptation was not significant ( t = 0.74 ; p = 0.48 ) . individually , the difference of end - position before and after prism adaptation was always less than 1 degree of angle ( cf . none of the paper and pencil attentional tests have been significantly modified by left - deviating prisms in the control group . the 95% confident interval of healthy subjects ' performances is displayed on figure 3(b ) for each test . for the neglect group , numerical results are reported for each test in the following section and in figure 3(b ) . an average of 36.4 lines were cancelled before prism adaptation , 33.8 immediately after prism adaptation , and 35.6 two hours later ( standard error of mean = 3.3 ) . analysis of variance showed no significant difference between sessions , f ( 2 , 8) = 1.31 ; p = 0.32 . in the pop - out subtest , a mean of 11.2 targets balloons were crossed before prism adaptation , 9.8 immediately after , and 15.2 two hours later ( standard error of mean = 3.0 ) . analysis of variance showed no significant difference between sessions f ( 2 , 8) = 0.86 ; p = 0.46 . in the search subtest , a mean of 8.4 circles were crossed before prism adaptation , 8.0 immediately after prism adaptation , and 8.2 two hours later ( standard error of mean = 2.1 ) . analysis of variance showed no significant difference between sessions f ( 2 , 8) = 0.02 ; p = 0.98 . before prism adaptation , patients bisected lines with a mean deviation calculated at 50.3 percent on the right of the true centre ; this deviation was 35.8 immediately after prism adaptation and 39.5 two hours later ( standard error of mean = 9.0 ) . analysis of variance showed no significant difference between sessions f ( 2 , 8) = 2.27 ; p = 0.17 . before prism adaptation , an average of 3.6 of the five items was copied and an average of 2.2 items was symmetrically copied . immediately after prism adaptation , an average of 4 items was copied and an average of 2.4 items was symmetrically copied . two hours after prism adaptation , an average of 2.4 items was copied and an average of 1.6 items was symmetrically copied . standard error of mean was 0.64 for the total number of items copied and 0.91 for items symmetrically copied . analysis of variance showed no significant difference between sessions both for the total number of items copied f ( 2 , 8) = 3.92 ; p = 0.06 and for the number of items symmetrically copied f ( 2 , 8) = 0.78 ; p = 0.49 . the daisy was moderately asymmetrical before prism adaptation ( mean = 2 ) , immediately after ( mean = 2.2 ) , and two hours later ( mean = 2 ) . analysis of variance showed no significant difference between sessions f ( 2 , 8) = 2.21 ; p = 0.81 . before prism adaptation , an average of 13.5 words were omitted , 8.5 immediately after prism adaptation , and 8.0 two hours later ( standard error of mean = 6.7 ) . analysis of variance showed no significant difference between sessions f ( 2 , 8) = 0.92 ; p = 0.45 . the present study showed that patients with left spatial neglect are not affected by prism adaptation to a leftward optical shift . indeed , neither the rightward deviation of straightahead pointing nor left spatial neglect , as assessed by a battery of classical paper and pencil tests , has been significantly improved or modified after a single session of visuomotor adaptation to left - deviating prisms . not only do these results suggest that there is a directional specificity of the prisms , but they also show that no cognitive effects are found in the absence of adaptation . the present results play against the hypothesis that active exposure to a simple modification of sensori - motor coordinates is sufficient to reduce left spatial neglect . the short duration of the adaptation procedure can not explain independently the absence of sensorimotor after - effects given that healthy controls adapt to prisms with the same procedure and neglect patients show sensori - motor after - effects , even larger than controls , when exposed to right - deviating prisms during the same amount of time . in our experiment , none of the 5 neglect patients showed a consistent sensori - motor adaptation to left - deviating prisms . a similar result was already reported in experiment 1 of the original research performed by rossetti et al . . in this latter study , eight patients with left spatial neglect were randomly assigned to a session of left- or right - deviating prism adaptation . adaptability was assessed by measuring body - midline demonstration ( i.e. straightahead pointing in the dark ) . in contrast to normal subjects , results showed that patients with left neglect adapted only to right - deviating prism and not to left - deviating prism . the effect of left - deviating prism adaptation on left spatial neglect symptoms was not specifically assessed in this latter work . these results suggest that patients with left spatial neglect after right - brain damage are not able to adapt to left - deviating prisms whereas they are able to adapt to right - deviating prisms . this result contrasts with the finding of weiner et al . that the only lesion site that impaired prism adaptation was within the cerebellum ( see ) . although weiner et al . tested groups of patients with left versus right hemisphere lesion , no information is provided concerning the assessment of left spatial neglect . in their study it was stated that only patients with occipital lesion exhibited reduced negative after - effects . however in our group , the lesion overlapped the occipital cortex in only two out of the five patients . one explanation to this intriguing negative result could be related to the absence of detection of the visual errors by left neglect patients in the case of left - deviating prisms . indeed , the first pointing movements with left - deviating prisms are shifted to the left side of the visual target , and considering that patients focus their vision on the target position , the visual error lies in the left visual field . hence , it is not surprising that this visual error , which represents the first necessary signal for prism adaptation , is not even implicitly detected in patients with left spatial neglect . alternatively , it is possible that visual realignment after leftward - deviating prisms critically requires the integrity of the right hemisphere in contrast to visual realignment after rightward deviating prisms . as regard to this hypothesis , it is interesting to consider the directional asymmetry for visual after - effects observed in healthy subjects after a visual adaptation to leftward versus rightward prism displacement . this could explain why right - brain - lesioned patients are only able to adapt to rightward deviating prisms . the larger amplitude of sensori - motor after - effects observed in right brain damaged neglect patients compared to healthy controls is another interesting issue which could be related to the asymmetrical integration of the prism adaptation process . our results showed for the first time that left - deviating prisms had no effect on various symptoms of left spatial neglect . previous studies have reported a similar lateralized specificity of prism adaptation on several neglect - related symptoms . investigated the effect of prism adaptation on postural imbalance in a group of 15 left hemiparetic patients , randomly exposed to right - deviating prism , left - deviating prism , or neutral goggles . the lateral displacement of the centre of pressure observed in the pretest was significantly reduced specifically following right - deviating prisms . finally , for one of the neglect patients ( patient 4 ) included in the experiment performed by maravita et al . , contralesional tactile perception and visual extinction were improved only after adaptation to right - deviating prism and not after adaptation to left - deviating prism . hence , these results favour a specificity of prism adaptation in terms of the direction of optical shift : only adaptation to right - deviating prisms can improve left spatial neglect . the most obvious explanation to account for these results is related to the absence of adaptability to left - deviating prism for patients with left spatial neglect ( cf . these results support the hypothesis that the presence of sensori - motor after - effect is a necessary condition to influence the highest cognitive levels of space and action representation subserving neglect recovery . , several studies have shown that the quantitative relationship between the amplitude of after - effect and neglect amelioration is not obvious ( e.g. , ) . interestingly , the cognitive effects of prism , in non - brain - damaged subjects , are also supported by an asymmetrical pattern of performance . examined the possibility that visuomotor adaptation to left- or right - deviating prisms could generate a bias on a line bisection task . only adaptation to left - deviating prisms induced a rightward bias on the perceptual version of the line bisection task . fourteen participants were either adapted to a leftward or rightward visual shift and it was found again that only adaptation to a leftward visual shift induced significant rightward postural bias . . showed asymmetric effects after manual or locomotor adaptation ( walking along a rectangle drawn on the floor with prismatic google ) to a leftward or ritghtward optical deviation on a goal - oriented locomotor task ( estimation of the spatial location of a visual target with body displacement ) . on the goal - oriented locomotor task which comprises a spatial dimension , the rightward after - effects generated by left - deviating prisms were greater than the leftward after - effects generated by right - deviating prisms . this result suggests that in contrast to rightward - deviating prisms generating only sensori - motor adaptation , leftward - deviating prisms may induce both sensori - motor and an additional cognitive after - effect . investigated whether prism adaptation could influence visual attention , as assessed by a visual attention cueing paradigm . two versions of the task were employed depending on the delay separating cue onset and target onset . in the reflexive version , the delay was short ( 50 to 300 ms ) , whereas it was longer in the voluntary version ( 300 to 500 ms ) . healthy participants were divided in three groups : left - deviating prisms , right - deviating prisms , and neutral goggles . the main result was an increase of voluntary attention efficiency for both left and right visual space after adaptation to left - deviating prisms . in contrast , right - deviating prisms decreased the efficiency of voluntary attention in both left and right space . the experiment performed by morris et al . was less conclusive in the sense that neither adaptation to left - deviating prism nor adaptation to right - deviating prism significantly modified a visual search task . however , results presented in this latter article showed a clear decrease of reaction time and percentage of error in the left visual space ( not present in the right visual space ) after left - deviating prism . altogether , the data available in the literature suggest that the cognitive effects of prism adaptation in healthy subjects depend on the direction of the optical shift . the present results suggest that the same is true for unilateral neglect patients , but in the opposite direction . spatial neglect is improved only by adaptation to rightward optical shifts and spatial cognitive functions tested on healthy subjects are affected mainly after adaptation to a left - ward shift . this coherence allows proposing an integrated model of the effects of prism adaptation on spatial cognition , whereby the lateralized effects of adaptation on the cerebellum would affect the controlateral hemisphere . the neural substrate underlying the therapeutic effect of this method remains to be fully elucidated . our study was not specifically designed to deal with this issue but argues at least for an initial lateralized bottom - up activation implicated in the detection of the right visual error during the first pointing movements through prisms . in a recent functional imaging study performed on healthy subjects , we used event - related fmri to analyze dynamic changes in brain activity during a prolonged exposure to visual prisms . results suggest that during exposure to a leftward prismatic deviation , error - detection was processed in the left intraparietal sulcus , errorcorrection involved the left parietooccipital sulcus , and visuomotor realignment implicated the right cerebellum . furthermore , the activation observed bilaterally in the superior temporal cortex during the late phase of prism exposure was thought to mediate the effects of prism adaptation on cognitive spatial representations . the mechanism by which such lateralized sensori - motor plasticity induced by prism adaptation can improve spatial neglect remains unclear . moreover , the gap might be important between what we know about sensori - motor plasticity in normal subjects and what happens in brain - damaged neglect patients . in a functional imaging pet study , we investigated the anatomical substrates underlying the beneficial effect of prism adaptation in five patients with left spatial neglect following right stroke . we used a covariation analysis to examine linear changes over sessions as a function of neglect improvement . this study confirmed that a low - level sensori - motor adaptation can modulate several cortical areas involved in spatial cognition and gives further support to a bottom - up mechanism . altogether , the following model is proposed : error signals induced by prisms are initially processed by the left occipitoparietal cortex , then forwarded to the right cerebellum where visuomotor realignment takes place . the clinical benefit would result from the modulation of left - hemisphere areas via a bottom - up signal produced by the cerebellum . these areas would partially match those involved in spatial cognition in the right hemisphere , and their modulation would improve interhemispheric rebalancing . the basic idea proposed here is that the activation of the right cerebellum by prism adaptation would play a negative influence on the activation of the left cerebral hemisphere . a recent support for such interaction was provided by pope and miall who explored the effect of cerebellar activity modulation on cognitive tasks . one classic idea about the contribution of the cerebellum to cognitive function has been that the processing capacities developed in the cerebellum for sensorimotor control could also turn out to be useful for cognitive operations . accordingly , the expectation is that reducing cerebellar activity on one side would impair contralateral hemispheric functions . however pope and miall revealed that cathodal tdcs on the right cerebellum resulted in an improvement of several cognitive tasks known to rely on the left cerebral hemisphere functions . the reciprocal arguments that enhancing right cerebellum activity by prism adaptation may inhibit left cerebral cortex function and that downregulating right cerebellum activity by cathodal tdcs may enhance left cerebral cortex function provide a general coherence to the idea that cerebellocortical interactions contribute to the expansion of prism adaptation effects to cognitive functions .

adaptation to right - deviating prisms is a promising intervention for the rehabilitation of patients with left spatial neglect . in order to test the lateral specificity of prism adaptation on left neglect , the present study evaluated the effect of left - deviating prism on straight - ahead pointing movements and on several classical neuropsychological tests in a group of five right brain - damaged patients with left spatial neglect . a group of healthy subjects was also included for comparison purposes . after a single session of exposing simple manual pointing to left - deviating prisms , contrary to healthy controls , none of the patients showed a reliable change of the straight - ahead pointing movement in the dark . no significant modification of attentional paper - and - pencil tasks was either observed immediately or 2 hours after prism adaptation . these results suggest that the therapeutic effect of prism adaptation on left spatial neglect relies on a specific lateralized mechanism . evidence for a directional effect for prism adaptation both in terms of the side of the visuomanual adaptation and therefore possibly in terms of the side of brain affected by the stimulation is discussed .

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

patients with right cerebral hemisphere lesions often show a reduced tendency to respond to stimuli and to search actively for them in the contralateral part of space . this condition described as left spatial neglect is typically demonstrated by clinical observation and simple perceptual motor tests such as a line bisection or cancellation test . left spatial neglect occurs in about 2530% of all stroke patients , and although some degrees of spontaneous recovery occurs , the disorder persists chronically in many cases . frequently associated with contralesional motor or somatosensory deficit , left spatial neglect is recognized as one of the main factors associated with poor functional outcome [ 68 ] . for these reasons , the improvement of left spatial neglect over and above spontaneous recovery represents a challenge in the area of neurological rehabilitation . among these , prism adaptation has been widely used since the end of the nineteenth century as a paradigm to demonstrate visuomotor short - term plasticity . exposure to prisms produces a lateral shift of the visual field so that the visual target appears at a displaced position . while the initial movements tend to approximate to the virtual position of the target , subsequent pointing movements ensure that the pointing error rapidly decreases so that subjects can readily point towards the real target position . to obtain robust compensatory after - effects following removal of prisms , further pointing movements are required . these reinforce the sensory - motor adaptation and are considered characteristic of the real or true after - effects result from a compensatory shift in manual straightahead pointing in a direction opposite to the original visual shift produced by prisms . proposed that the adaptation to right - deviating prisms with leftward compensatory after - effects ( using the intact right hand ) improved left neglect symptoms . from a rehabilitation perspective , the long - term beneficial effect on several functional measures set this intervention apart from the other attempts . found a reduction of spatial dyslexia still present one day after a single session of prism adaptation in a group of 6 patients with left spatial neglect . reported a positive effect of prism adaptation on spatial dysgraphia the improvement concerned the right - page preference and was maintained up to 4 days after a single session of prism adaptation . watanabe and amimoto reported an improvement of wheelchair navigation after a single session of prism adaptation in two single - case studies . this impressive generalization and long - standing effects of prism adaptation have revived interest in the neuro - cognitive mechanisms by which it has been achieved . the most two basic questions about the mechanisms of action of prism adaptation are ( i ) whether adaptation per se is necessary to produce cognitive after - effects or whether simple visuomanual pointing could produce similar effects , and ( ii ) whether this adaptation is specific in terms of its direction . the purpose of the present study was to evaluate the directional specificity of prism adaptation in neglect patients . given that the effect of right - deviating prisms on left spatial neglect is already well documented ( cf . supra ) , this work was designed to evaluate the effect of left - deviating prisms on left spatial neglect . since it has been shown that adaptation to right - deviating prism may affect differently straightahead pointing movements and attentional tasks , the effect of left - deviating prism was measured both on straightahead pointing movements and several attentional tasks classically used to assess left spatial neglect . inclusion criteria were right - handed patients with left spatial neglect after right hemispheric ischemic or hemorrhagic stroke . left spatial neglect was assessed using a battery of six paper and pencil tests : line cancellation , balloon test , line bisection , copy of a scene , drawing from memory , simple text reading ( cf . a cerebral computerized tomography ( ct ) or mri scan was performed for each patient in order to specify the type of lesion ( ischemic or hemorrhagic ) , to rule out any other relevant prestroke lesions and to determine the anatomic location of the lesion . one patient had a hemorrhagic stroke ( patient 2 ) ; the four others had an ischemic stroke : two in the posterior part of the superficial middle cerebral artery territory ( patients 1 and 3 ) , one in the anterior part of the superficial middle cerebral artery territory ( patient 5 ) , and one in the deep part of the middle cerebral artery territory ( patient 4 ) . patients ' performance was investigated in sessions that took place before prism adaptation ( referred to as pre ) , immediately after ( post ) , and 2 hours after ( late ) . healthy subjects performed the same tasks as patients before ( pre ) and immediately after ( post ) prism adaptation . during each session , left spatial neglect was assessed using line cancellation , balloon test , line bisection , copy of a scene , drawing from memory , and a simple text reading ( cf . in order to check whether healthy subjects and patients correctly adapted to prisms , a measure of straightahead pointing was performed before adaptation ( pre ) and after participants had completed the immediate neuropsychological tests ( post ) as in rossetti et al . in the second subtest search , the number and position of the balloons and circles are exactly the reverse ; thus 22 of the 202 items are circles to be cancelled and the other 180 items are balloons . search , the number and position of the balloons and circles are exactly the reverse ; thus 22 of the 202 items are circles to be cancelled and the other 180 items are balloons . the score was the mean percentage of deviation from the true center of the line ( the score is positive when the deviation is in the right direction and negative when the deviation is in the left direction ) . the score was the mean percentage of deviation from the true center of the line ( the score is positive when the deviation is in the right direction and negative when the deviation is in the left direction ) . copy of a scene participants were required to reproduce a picture made up of five items ( 4 trees and a house ) in the space bellow it . participants were required to reproduce a picture made up of five items ( 4 trees and a house ) in the space bellow it . the adaptation procedure involved the participants having to wear prismatic goggles that produced a 10 leftward shift of the visual wide - field that is in the opposite direction to rossetti et al . while wearing prisms , the participant was required to make as fast as possible a series of approximately 50 pointing responses , with his / her right hand , to visual targets located to the left and right side of midline . in order to ensure optimal adaptation , visual feedback of the starting point of the hand the first analysis was to test whether healthy subjects and patients correctly adapted to left - deviating prisms . in order to evaluate the presence of an amelioration of left neglect symptoms after prism adaptation , an analysis of variance with repeated measure ( anova ) was performed on each neuropsychological test , using sessions ( pre , post , late ) as factor . hence , for a specified test , the null hypothesis ( p value > 0.05 ) is the absence of difference between sessions of the mean score across patients . in this latter case , a post hoc sheff test was carried out in order to compare the mean scores across sessions : pre versus post , pre versus late , and post versus late . for the healthy controls , a significant displacement of the straightahead pointings to the right was observed after exposure to left deviating prisms without significant modification of the performances on the attentional paper and pencil tests . for the neglect patients , no significant effect of prism exposure was observed neither on the straight - ahead pointing task nor on the neuropsychological tests . before left - deviating prism adaptation ( pre ) , the group analysis showed that the mean end - position of 7 straightahead pointing trials was shifted 1.3 degrees to the right of the body midline ( range : 2.3 to 5.1 ) . after prism adaptation ( post ) , the mean deviation was significantly displaced to the right ( mean position after prism adaptation : 5.8 degrees to the right of the body midline ; range : 0.7 to 9.0 ) . , the group analysis showed that the mean end - position of 7 straightahead pointing trials was shifted 3.7 degrees to the right of the body midline ( range : 2.0 to 4.9 ) . after prism adaptation ( post ) , the mean end - position was unchanged ( mean : 3.7 degrees to the right ; range : 2.4 to 4 ) . comparison between trials performed before and after prism adaptation was not significant ( t = 0.74 ; p = 0.48 ) . individually , the difference of end - position before and after prism adaptation was always less than 1 degree of angle ( cf . none of the paper and pencil attentional tests have been significantly modified by left - deviating prisms in the control group . the 95% confident interval of healthy subjects ' performances is displayed on figure 3(b ) for each test . an average of 36.4 lines were cancelled before prism adaptation , 33.8 immediately after prism adaptation , and 35.6 two hours later ( standard error of mean = 3.3 ) . in the search subtest , a mean of 8.4 circles were crossed before prism adaptation , 8.0 immediately after prism adaptation , and 8.2 two hours later ( standard error of mean = 2.1 ) . before prism adaptation , patients bisected lines with a mean deviation calculated at 50.3 percent on the right of the true centre ; this deviation was 35.8 immediately after prism adaptation and 39.5 two hours later ( standard error of mean = 9.0 ) . before prism adaptation , an average of 3.6 of the five items was copied and an average of 2.2 items was symmetrically copied . immediately after prism adaptation , an average of 4 items was copied and an average of 2.4 items was symmetrically copied . two hours after prism adaptation , an average of 2.4 items was copied and an average of 1.6 items was symmetrically copied . analysis of variance showed no significant difference between sessions both for the total number of items copied f ( 2 , 8) = 3.92 ; p = 0.06 and for the number of items symmetrically copied f ( 2 , 8) = 0.78 ; p = 0.49 . the present study showed that patients with left spatial neglect are not affected by prism adaptation to a leftward optical shift . indeed , neither the rightward deviation of straightahead pointing nor left spatial neglect , as assessed by a battery of classical paper and pencil tests , has been significantly improved or modified after a single session of visuomotor adaptation to left - deviating prisms . not only do these results suggest that there is a directional specificity of the prisms , but they also show that no cognitive effects are found in the absence of adaptation . the present results play against the hypothesis that active exposure to a simple modification of sensori - motor coordinates is sufficient to reduce left spatial neglect . the short duration of the adaptation procedure can not explain independently the absence of sensorimotor after - effects given that healthy controls adapt to prisms with the same procedure and neglect patients show sensori - motor after - effects , even larger than controls , when exposed to right - deviating prisms during the same amount of time . in our experiment , none of the 5 neglect patients showed a consistent sensori - motor adaptation to left - deviating prisms . in this latter study , eight patients with left spatial neglect were randomly assigned to a session of left- or right - deviating prism adaptation . straightahead pointing in the dark ) . in contrast to normal subjects , results showed that patients with left neglect adapted only to right - deviating prism and not to left - deviating prism . the effect of left - deviating prism adaptation on left spatial neglect symptoms was not specifically assessed in this latter work . these results suggest that patients with left spatial neglect after right - brain damage are not able to adapt to left - deviating prisms whereas they are able to adapt to right - deviating prisms . that the only lesion site that impaired prism adaptation was within the cerebellum ( see ) . tested groups of patients with left versus right hemisphere lesion , no information is provided concerning the assessment of left spatial neglect . however in our group , the lesion overlapped the occipital cortex in only two out of the five patients . one explanation to this intriguing negative result could be related to the absence of detection of the visual errors by left neglect patients in the case of left - deviating prisms . indeed , the first pointing movements with left - deviating prisms are shifted to the left side of the visual target , and considering that patients focus their vision on the target position , the visual error lies in the left visual field . hence , it is not surprising that this visual error , which represents the first necessary signal for prism adaptation , is not even implicitly detected in patients with left spatial neglect . alternatively , it is possible that visual realignment after leftward - deviating prisms critically requires the integrity of the right hemisphere in contrast to visual realignment after rightward deviating prisms . as regard to this hypothesis , it is interesting to consider the directional asymmetry for visual after - effects observed in healthy subjects after a visual adaptation to leftward versus rightward prism displacement . this could explain why right - brain - lesioned patients are only able to adapt to rightward deviating prisms . the larger amplitude of sensori - motor after - effects observed in right brain damaged neglect patients compared to healthy controls is another interesting issue which could be related to the asymmetrical integration of the prism adaptation process . our results showed for the first time that left - deviating prisms had no effect on various symptoms of left spatial neglect . previous studies have reported a similar lateralized specificity of prism adaptation on several neglect - related symptoms . investigated the effect of prism adaptation on postural imbalance in a group of 15 left hemiparetic patients , randomly exposed to right - deviating prism , left - deviating prism , or neutral goggles . the lateral displacement of the centre of pressure observed in the pretest was significantly reduced specifically following right - deviating prisms . finally , for one of the neglect patients ( patient 4 ) included in the experiment performed by maravita et al . , contralesional tactile perception and visual extinction were improved only after adaptation to right - deviating prism and not after adaptation to left - deviating prism . hence , these results favour a specificity of prism adaptation in terms of the direction of optical shift : only adaptation to right - deviating prisms can improve left spatial neglect . the most obvious explanation to account for these results is related to the absence of adaptability to left - deviating prism for patients with left spatial neglect ( cf . these results support the hypothesis that the presence of sensori - motor after - effect is a necessary condition to influence the highest cognitive levels of space and action representation subserving neglect recovery . interestingly , the cognitive effects of prism , in non - brain - damaged subjects , are also supported by an asymmetrical pattern of performance . examined the possibility that visuomotor adaptation to left- or right - deviating prisms could generate a bias on a line bisection task . only adaptation to left - deviating prisms induced a rightward bias on the perceptual version of the line bisection task . showed asymmetric effects after manual or locomotor adaptation ( walking along a rectangle drawn on the floor with prismatic google ) to a leftward or ritghtward optical deviation on a goal - oriented locomotor task ( estimation of the spatial location of a visual target with body displacement ) . on the goal - oriented locomotor task which comprises a spatial dimension , the rightward after - effects generated by left - deviating prisms were greater than the leftward after - effects generated by right - deviating prisms . this result suggests that in contrast to rightward - deviating prisms generating only sensori - motor adaptation , leftward - deviating prisms may induce both sensori - motor and an additional cognitive after - effect . in the reflexive version , the delay was short ( 50 to 300 ms ) , whereas it was longer in the voluntary version ( 300 to 500 ms ) . healthy participants were divided in three groups : left - deviating prisms , right - deviating prisms , and neutral goggles . the main result was an increase of voluntary attention efficiency for both left and right visual space after adaptation to left - deviating prisms . in contrast , right - deviating prisms decreased the efficiency of voluntary attention in both left and right space . was less conclusive in the sense that neither adaptation to left - deviating prism nor adaptation to right - deviating prism significantly modified a visual search task . however , results presented in this latter article showed a clear decrease of reaction time and percentage of error in the left visual space ( not present in the right visual space ) after left - deviating prism . altogether , the data available in the literature suggest that the cognitive effects of prism adaptation in healthy subjects depend on the direction of the optical shift . the present results suggest that the same is true for unilateral neglect patients , but in the opposite direction . spatial neglect is improved only by adaptation to rightward optical shifts and spatial cognitive functions tested on healthy subjects are affected mainly after adaptation to a left - ward shift . this coherence allows proposing an integrated model of the effects of prism adaptation on spatial cognition , whereby the lateralized effects of adaptation on the cerebellum would affect the controlateral hemisphere . the neural substrate underlying the therapeutic effect of this method remains to be fully elucidated . our study was not specifically designed to deal with this issue but argues at least for an initial lateralized bottom - up activation implicated in the detection of the right visual error during the first pointing movements through prisms . results suggest that during exposure to a leftward prismatic deviation , error - detection was processed in the left intraparietal sulcus , errorcorrection involved the left parietooccipital sulcus , and visuomotor realignment implicated the right cerebellum . furthermore , the activation observed bilaterally in the superior temporal cortex during the late phase of prism exposure was thought to mediate the effects of prism adaptation on cognitive spatial representations . the mechanism by which such lateralized sensori - motor plasticity induced by prism adaptation can improve spatial neglect remains unclear . moreover , the gap might be important between what we know about sensori - motor plasticity in normal subjects and what happens in brain - damaged neglect patients . in a functional imaging pet study , we investigated the anatomical substrates underlying the beneficial effect of prism adaptation in five patients with left spatial neglect following right stroke . altogether , the following model is proposed : error signals induced by prisms are initially processed by the left occipitoparietal cortex , then forwarded to the right cerebellum where visuomotor realignment takes place . the clinical benefit would result from the modulation of left - hemisphere areas via a bottom - up signal produced by the cerebellum . the basic idea proposed here is that the activation of the right cerebellum by prism adaptation would play a negative influence on the activation of the left cerebral hemisphere . a recent support for such interaction was provided by pope and miall who explored the effect of cerebellar activity modulation on cognitive tasks . one classic idea about the contribution of the cerebellum to cognitive function has been that the processing capacities developed in the cerebellum for sensorimotor control could also turn out to be useful for cognitive operations . the reciprocal arguments that enhancing right cerebellum activity by prism adaptation may inhibit left cerebral cortex function and that downregulating right cerebellum activity by cathodal tdcs may enhance left cerebral cortex function provide a general coherence to the idea that cerebellocortical interactions contribute to the expansion of prism adaptation effects to cognitive functions .

alzheimer 's disease ( ad ) is the most common form of dementia in adults . pathologically , the hallmarks of ad are amyloid plaques and neurofibrillary tangles , associated with widespread neuronal loss . its fundamental causes and the pathological cascades leading to symptoms , however , remain poorly understood . extensive evidence supports an important role of cholesterol in the development and possibly progression of ad [ 13 ] . the role of other lipids , such as ceramides and related - sphingolipids , ( sphingomyelins , sulfatides , and glycosphingolipids ) is also emerging . they can be produced by hydrolysis of sphingomyelin ( sm ) via sphingomyelinases ( smases ) or synthesized de novo from fatty acyl coa and sphingosine . conversely , in the golgi , ceramides may be transformed into sm by the addition of phosphorylcholine . additionally , glycosyltransferases can attach sugar to ceramide , turning it into glucosylceramide or galactosylceramide ( galcer ) , a key step in the generation of complex glycosphingolipids [ 4 , 5 ] . ceramides are important second messenger molecules that regulate diverse cellular processes including cell growth , differentiation , and apoptosis . ceramide levels also increase in response to aging and various age - related stress factors and are directly involved in apoptotic signaling in various cell types , including neurons [ 68 ] . there is evidence linking sphingolipid abnormalities , app processing , and neuronal death in ad . in vitro , it has been shown that -amyloid added to cultured neurons [ 9 , 10 ] or oligodendrocytes increase smase activity , leading to an increase in ceramide levels . additional reports have found that ceramide levels increase -amyloid synthesis [ 12 , 13 ] and favor gamma secretase processing of app [ 1416 ] so that inhibition of ceramide synthesis confers protection against -amyloid . thus , it has been suggested that ceramides and -amyloid may synergize to induce neuronal death . for example , the total phospholipid and sulfatide content in ad was decreased as compared to normal [ 1719 ] , while the ceramide and galactosylceramide levels were elevated [ 9 , 18 ] . enhanced ceramide levels have been reported in the cerebrospinal fluid ( csf ) of patients with ad and changes in the activity of several key enzymes of ceramide metabolism , in gene expression of pathways associated to sphingolipid metabolism have been found in brains of ad patients [ 21 , 22 ] . during the last years , numerous mouse transgenic lines have been created and have been screened for various aspects of ad pathology [ 23 , 24 ] . unfortunately , very little work has been done on determining if sphingolipid content is likewise perturbed in these rodent models of ad . in one study , long - chain ceramides were shown to be elevated in presenilin 1 ( ps1m146v ) mouse brain and to induce apoptosis in ps1 astrocytes . in a second study , sulfatides , a class of sulfated galactocerebrosides , were found to be decreased in brain tissues from two app transgenic mice ( i.e. , appv717f and appsw ) , whereas no significant changes in the content of other sphingolipid classes including sm , galcer , and ceramides were noted . by contrast , using the new mouse mutant app / ps1knock - in line , we have recently found an early and significant increase of ceramides in the cortex of these mice , without significant changes in other sphingolipid levels . however , the app / ps1ki model is unique for its drastic neuronal loss , not observed in other animal models of ad . the discrepancy in the few data available and the lack of knowledge of sphingolipid levels in the brain of other rodent models of ad prompted us to investigate whether the sphingolipid composition is altered in the brain of two other mouse models of ad : single app and double app / ps1 transgenics . concentrations of ceramides , sm , galcer , and sulfatides were determined in three brain regions : the cortex and the hippocampus , the two brain regions typically associated with the disease , and the cerebellum , a nonvulnerable region with no a plaques . for all analyses , age - matched ps1 ( amyloid free ) mice as well as nontransgenic wild - type mice ( wt ) were used . all organic solvents were of analytical grade and came from vwr international ( strasbourg , france ) . hptlc - plates silicagel 60 , 10 10- or 10 20 cm were purchased from merck ( vwr international ) . lipid standards ( nonhydroxy fatty acid ( nfa ) containing ceramides , ceramides containing 2-hydroxy fatty acids ( 2-hfa ) , sphingomyelins , cerebroside sulfate ( sulfatides ) , and galactosylceramides ( a mixture containing 2-hydroxy fatty acids and nonhydroxy fatty acids ) were purchased from sigma - aldrich ( france ) . aminopropyl - bonded ( lc - nh2 ) silica gel cartridges ( 100 mg matrix ) were from supelco ( saint quentin fallavier , france ) . generation and detailed neuropathological analyses of the app and the app / ps1 transgenic mice have been described earlier [ 29 , 30 ] . in brief , these mice express human app751 with swedish and london mutations ( thy1 promoter ) either alone or in combination with human presenilin-1 ( m146l , hmg - coa promoter ) . in this study , 12-month - old ( n = 5 ) app / ps1 double transgenic , 12-month - old ( n = 5 ) ps1 single - transgenic littermates , and 12-month - old ( n = 6 ) nontransgenic mice as well as 24 month - old ( n = 5 ) app single transgenic and 24-months ( n = 5 ) nontransgenic littermates were used ( generous gift of sanofi - aventis , vitry sur seine , france ) . app mice were analyzed at 24 months of age and app / ps1 mice were assessed at 12 months of age because they revealed comparable levels of amyloid plaques in the brain at these respective ages . all the mice used in this study were female , because a gender effect with female mice displaying more severe pathology than male has been mentioned in several studies [ 27 , 31 , 32 ] . all experiments were performed in compliance and following approval of the sanofi - aventis animal care and use committee and in accordance with standards of the guide for the care and use of laboratory animals ( cnrs ilar ) and with respect to french and european community rules . the mice were anesthetized with pentobarbital ( 40 mg / kg , ip ) and sacrificed . these cerebral regions were homogenized by 10 up - and - down strokes of a prechilled teflon - glass homogenizer in 20 volumes of buffer ( 25 mm tris - hcl , 150 mm nacl , 1 mm edta , ph 7.4 ) and supplemented with 50 mm sodium fluoride , 1 mm phenylmethylsulfonyl fluoride , protease , and phosphatase inhibitor cocktails ( 50 l / g tissue and 10 l / ml lysis buffer , resp . ) . the resulting pellet was resuspended in 3 volumes of ice - cold water , altogether 1.5 ml , homogenized at 4c ( 1015 strokes ) and then sonicated for 20 s using a sonifier ( branson ultrasonics , sonifier 450 , danbury , ct ) . total lipids from brain homogenates were prepared according to previously described procedures [ 27 , 33 ] . the mixture was stirred overnight , and then centrifuged at 1,000 g for 10 min . the pellet was re - extracted with chloroform - methanol - water ( 4 : 8 : 3 , v / v / v ) and the two supernatants combined , evaporated to dryness . partitioning was carried out using diisopropyl ether/1-butanol/50 mm aqueous nacl ( 6 : 4 : 5 , v / v / v ) according to the method of ladish and gillard . the lipids were fractionated using solid - phase extraction on 100 mg lc - nh2 cartridges ( supelco , l'isle d'abeau , france ) as previously described . the eluted fractions containing , respectively , free ceramides , galactosylceramides ( galcer ) , alkali - stable phospholipids ( sm ) , and sulfatides were applied to hptlc plates with a linomat 5 ( camag , switzerland ) . prior to sm analysis , the phospholipid - containing fraction was subjected to mild alkaline methanolysis ( treatment with chloroform : 0.6 n naoh in methanol 1 : 1 ( v / v ) at room temperature for 1 h ) to remove glycerophospholipids . to quantify each lipid species ( ceramides , sm , galcer , and sulfatides ) , calibration curves were obtained by running in parallel known amounts of purified lipid standards . for free ceramides , 1520 mg of brain tissue ( wet weight ) the plates were developed with chloroform - methanol 50 : 3 ( v / v ) and visualized by charring for 10 min at 180c with 3% cupric acetate in 8% phosphoric acid solution . for sm analysis , the plates were developed with chloroform - methanol - water 60 : 35 : 8 ( v / v / v ) . sm was visualized with sulfuric acid - cuso4-ammonium molybdate spray reagent followed by heating at 110c for 15 min . galcer and sulfatides ( about 0.4 mg and 2 mg of tissue per lane , resp . ) were developed in chloroform - methanol - water 65 : 25 : 4 ( v / v / v ) , sprayed with orcinol - h2so4 reagent , and then heated at 150c for 2 min . each sphingolipid species was quantified by scanning densitometry of the plates at 396 nm for ceramides , 540 nm for galcer and sulfatides , and 750 nm for sm with the camag tlc scanner 3/wincats software system . owing to the small number of animals per group , statistical analysis of the data was performed using a nonparametric kruskall - wallis test followed by a posthoc test of dunn for multiple comparisons . for the comparison of two means , all calculations were performed using graphpad prism software 3.02 ( graphpad software , inc . ) . in single app mice ( figure 1(a ) ) , a moderate but not significant elevation of nfa - ceramides was seen in the cortex ( + 22% ) , with no changes of the 2-hfa - ceramide level . conversely , in the hippocampus ( figure 1(b ) ) , the nfa - ceramide level did not differ between wt and app mice , whereas a tendency towards an increase of 2-hfa - ceramides was noted ( + 19% ) , although it was not significant . surprisingly , as shown in figure 1(c ) , the level of 2-hfa - ceramides in the cerebellum of app mice was significantly increased in comparison to the counterpart of their wt littermates ( + 50% , p < .05 ) . conversely , the nfa - ceramides showed a slight but not significant decrease compared to wt control values . however , no difference in the total ceramide content was noticed in the cerebellum of both wt ( 51.35 1.45 nmol / mg tissue ) and app mice ( 50.28 3.31 nmol / mg tissue ) . because the double - transgenic mouse model app / ps1 develops neuropathological features of ad earlier than the single app mice , the sphingolipid analysis was performed in the brain regions of this model in younger animals ( 12 months of age ) . as shown in figures 2(a ) and 2(b ) , concentrations of nfa - ceramides as well as those of 2-hfa - ceramides did not differ between wild - type , ps1 , and app / ps1 mice in the two disease - associated brain regions ( cortex and hippocampus ) . in contrast to the changes of ceramide content in cerebellum of app mice , the nfa - ceramide as well as the 2-hfa - ceramide levels were unchanged in this brain region in app / ps1 mice relative to their wt controls and ps1 littermates ( figure 2(c ) ) . however , nfa - ceramide content showed a tendency towards a decrease , while 2-hfa ceramides tended to slightly increase in the cerebellum of the app / ps1 mice . it should be noted that , although cortex and hippocampus of wt mice displayed almost identical ceramide content ( figures 1(a ) , 1(b ) , 2(a ) , and 2(b ) ) , a relatively lower nfa - ceramide content was manifest in the cerebellum ( figures 1(c ) and 2(c ) ) . in normal human brain , the ceramide levels typical ceramide profiles from either cortex , hippocampus , or cerebellum of the different transgenic mice and wild - type controls were shown in figure 3 . since 2-hfa - ceramides are present in extremely low levels leading to a weak staining on the hptlc plate , densitometric scanning of the plate was shown to visualize the peak corresponding to the 2-hfa - ceramide species ( figure 3 ) . to test whether other related - sphingolipids could be altered in the brain of transgenic mice , the content of sm , galcer , and sulfatides in lipid extracts of the three examined brain regions figures 1(d)1(f ) show that sphingolipids ( sm , galcer , and sulfatides ) did not display significant changes in both cortex , hippocampus , and cerebellum of app mice compared to the wt littermates . similarly , the levels of sm , galcer , and sulfatides did not differ among nontransgenic , ps1 and app / ps1 mice in all brain region examined ( figures 2(d)2(f ) ) . it should be noted that there are differences of sm , galcer , and sulfatide concentrations between cerebral tissues ( cortex and hippocampus ) and cerebellar tissues . in both models , cerebellum displayed higher levels of galcer and sulfatides than cerebral tissues . this is in accordance with cheng et al . who also reported a ~2-fold higher level of sulfatides in the cerebellum compared to the cortex , in two other transgenic mouse models of ad . in contrast , sm levels were almost identical in hippocampus and cerebellum , but higher than those of cerebral cortex ( figures 1(d)1(f ) and 2(d)2(f ) ) . examples of hptlc profiles of sulfatides , galcer , and sm and from either cortex , hippocampus , or cerebellum were represented in figures 4(a ) , 4(b ) , and 4(c ) , respectively . it should be mentioned that the hptlc profiles of each sphingolipid class were similar in all examined brain regions from both mouse models . using the same methodology as in our previous work , we herein analysed the sphingolipid composition of two additional models ( i.e. , single app and double app / ps1 mice ) in which the time - course of pathology is much closer to that seen in the majority of currently available models . the main results of this study are ( 1 ) a moderate but not significant increase of nfa - ceramides and 2-hfa - ceramides in the cortex and the hippocampus respectively , of the app mice , ( 2 ) unaltered ceramide levels in the two disease - associated brain regions from app / ps1 mice , ( 3 ) unexpected alterations of the ceramide profile in the cerebellum of app mice , a region with no a pathology , and ( 4 ) no significant changes in the other related - sphingolipids in all brain structures examined of both transgenic models . based on our results and those from the literature , we will first discuss the possibility of a relationship between neurodegeneration , toxic a accumulation , and ceramide content . for the second time , why an amyloid - free brain region such as cerebellum showed ceramide alterations is discussed . ceramides were shown to accumulate in ad human brain regions and their levels vary by disease severity suggesting that they could be indicators of ad progression [ 9 , 18 , 35 ] . similarly to what happens in human ad , we previously found that ceramides increase very early in the cortex of the app / ps1ki mouse model , preceding the neuronal loss . by contrast , our present results reveal that in single app mice , ceramide levels were not significantly altered in disease - associated brain regions ( e.g. , hippocampus or cortex ) . this is consistent with the findings of cheng et al . who reported no changes in ceramide content in any brain region from appsw and app transgenic mice . an important difference between these single app mouse lines and the app / ps1ki model is that the latter develops an early and massive neuronal loss which correlates with strong accumulation of intracellular a42 , a aggregates , and a42 oligomers [ 28 , 36 ] . although intraneuronal a accumulation has also been documented in various app models [ 29 , 37 , 38 ] , a striking difference between the models used in the present study and the app / ps1ki mice is the nature of the a peptides which accumulate . indeed , in app / ps1ki mouse brain , extremely high levels of n - truncated ax-42 variants and abundant oligomers were detected , which closely resembles that found in ad brain . by contrast , in app mouse brain , with the same total a levels as in app / ps1ki mice , only very limited levels of a42 n - terminal truncated isovariants were detected . in the app / ps1ki mice , ax-42 is the major form accumulated with a ratio ax-42/total a close to 1 . in comparison , this ratio is approximately of only 0.2 - 0.3 in the app mice , and ~0.3 - 0.4 in the double transgenic app / ps1 mice , similar to the range of values reported for a large number of other app - based transgenics [ 28 , 29 ] . n - truncated a peptides are known to aggregate more readily and are considered to be very toxic species . thus they might play a key role in the neurotoxicity observed in the app / ps1ki model . in particular , the pyroglutamate modified n - terminal truncated form of a at position 3 ( a3(pe)-42 ) , which represents a major species in the brain of ad patients , was recently shown to induce a severe neuron loss in the tba2 mouse model , a new model expressing only n - truncated a3(pe)-42 in neurons . interestingly , there is also a coincidence of considerable amounts of a3(pe)-42 and massive neuron loss in the app / ps1ki mouse model . on the basis of these findings , it is attractive to speculate that in app / ps1ki mice , the strong accumulation of intracellular toxic forms of a induces early elevation of ceramides . conversely , other app transgenic mouse models including the app mice have been reported to show no or very low a3(pe)-42 levels . this is due to the lack of posttranslational modifications such as n - terminal degradation and pyroglutamyl formation in these mice . because app mice display neither neuronal loss nor accumulation of highly toxic a 42 isoforms , this may at least in part explain why no significant accumulation of ceramides occurred in the disease - associated brain regions of these mice . thus , despite numerous neuropathological , biochemical , and even behavioral changes representative of ad developed by these app mice [ 23 , 2830 , 33 , 44 ] , they may not constitute a relevant model to further explore the role of ceramides in ad pathology . however , answering the question of the relationship between neurodegeneration , toxic a accumulation , and ceramide elevation could be facilitated using restricted models either lacking ( i.e. , single app mice ) or mimicking only some specific ad - related neuropathological alterations ( i.e. , tba2 mice mentioned above ) . indeed , owing to the simultaneous occurrence of numerous pathological features of ad , the connection between them is often difficult to unravel . we next determined the ceramide content in the double transgenic mouse model app / ps1 , but in younger animals because the app / ps1 mice develop neuropathological features of ad earlier than single app mice . at 12 months of age , these double transgenics revealed comparable levels of amyloid deposits than 24-month - old app mice . our results showed that at 12 months of age , ceramide levels were unaltered in both cortex and hippocampus of app / ps1 mice in comparison to age - matched ps1 mice and wild - type controls . it should be noted that there is no neuronal loss in these mice as old as 17 months , but unfortunately , older app / ps1 mice were not available for this study . however , in our previous work , we demonstrated that elevation of ceramides occurred very early ( 3 months of age ) in the cortex of the app / ps1ki model , preceding by far the neuronal loss detectable at 6 months of age . why the app / ps1ki mice showed an increase of ceramides several months before the appearance of neuronal death while the app / ps1 mice did not is currently unknown . one possible explanation is that the neuronal loss reported in the app / ps1 mice is moderate and more restricted than in the app / ps1ki model . indeed , the loss of neurons in the former involves only the hippocampal pyramidal cell layer ( loss of ~30% in 17-month - old animals ) . this may in some way account for the lack of ceramide accumulation in the cortex of these mice . by comparison , in the app / ps1ki model , the cell loss is greater ( ~50% at 10 months of age ) and has been shown to extend to other brain areas such as frontal cortex and cholinergic system . moreover , as discussed above , accumulation of n - truncated a peptides should be lesser in app / ps1 mice , since the ratio ax-42/total a is of 0.3 only , versus 1 in the app / ps1ki mice . in this context , it would seem likely that the level of highly toxic a3(pe)-42 form , which is thought to be involved in neuronal death , is reduced in the app / ps1 model . it is also possible that , at 12 months of age , it is too early to visualize an elevation of ceramides , owing to the slowest progression of ad lesions in these mice than in the app / ps1ki model . since we did not have older app / ps1 mice , we should therefore be cautious to interpret the results of ceramide composition in these mice , because we can not exclude the possibility that ceramides increase at a later age . the most unexpected finding of the present study was alteration of the ceramide composition in the cerebellum of app mice , a brain region lacking a deposits and regarded as nonvulnerable to the disease . intriguingly , we noted a significant increase of 2-hfa - ceramides ( + 50% ) which was concomitant with a slight decrease in nfa - ceramides . however , considering the total ceramide content , it was almost similar between wild - type and app mice . previously , we found a more dramatic 161% increase of 2-hfa - ceramides in the cortex of app / ps1ki mice but contrary to the present results , it was accompanied by an elevation of nfa - ceramides . as evident from the literature , the current knowledge about the metabolism and physiological function of 2-hfa - ceramides is very limited . in particular in ad studies , no attention was paid to 2-hfa - ceramides , rendering it very difficult to draw conclusions about the role of these ceramide species in ad physiopathology . nevertheless , a couple of interesting facts suggest that these hfa species may participate to ad pathology : ( i ) it was recently found that a selectively bound to sphingolipids that contained a 2-oh group on the ceramide backbone and did not effectively interact with sphingolipids that contained a nonhydroxylated fatty acid , favoring a conformational shift that disrupts membrane stability and promotes peptide - peptide interactions and oligomer formation ; ( ii ) the enzyme udp - galactose : ceramide galactosyltransferase ( cgt ) , which transfers galactose to both nfa- and 2-hfa - ceramides , was found to be upregulated in human ad brain . interestingly , overexpression of cgt in transgenic mice led to a reversal nfa : hfa - galcer ratio which resulted in both decrease in hfa - galcer and increase in nfa - galcer levels . reducing the hfa - galcer level would lead to an accumulation of their immediate precursors , 2-hfa - ceramides ; ( iii ) there is some evidence for enhanced apoptosis - inducing activity of 2-hfa - ceramides compared to nfa - ceramides , and this effect seems to be cell type specific . in this sight , mouse mutants with defective saposin d dramatically accumulate hfa - ceramides in cerebellum , resulting in a selective loss of cerebellar purkinje cells ; ( iv ) we reported a gender - specific expression of hfa- versus nfa - ceramides in the app / ps1ki mouse model of ad , and this biochemical feature could be related to the increase propensity of females to develop earlier neuronal loss . although the degree of 2-hfa - ceramide accumulation in the cerebellum of app mice is much lesser than that seen in the cortex of the app / ps1ki mice , the reasons for these biochemical changes in this amyloid - free brain area are currently unknown . possibly , it might reflect alterations of ceramide metabolism , since there is evidence that other metabolic pathways are perturbed in the cerebellum of these mice . hydroxy fa sphingolipids are synthesized by the same set of enzymes as nonhydroxy sphingolipids , except for fatty acid 2-hydroxylase ( fa2h ) . the expression of fa2h is highly variable among cell types and is inducible by certain stimuli . one may speculate that , upon unknown signal , 2-hfa - ceramides may be preferentially synthesized instead of nfa - species . it has been shown that galactosylceramidase , which forms 2-hfa - ceramides from galcer is up - regulated , whereas acid ceramidase which hydrolyzed 2-hfa - ceramides into hfa and sphingosine is down - regulated in human ad brain . thus , with combinatorial up- and down - regulation of enzymes involved in sphingolipid metabolism , the cell could modify the levels of 2-hfa - ceramides in response to the changing cellular environment . however , this is highly speculative and further investigation is warranted to determine whether these factors or other unknown factors contribute to such changes of the ceramide profile in the app cerebellum . it should be noted that also intriguing is the substantial elevation of ceramide reported by han et al . in the cerebellum of ad patients , we also examined the content of other ceramide - related sphingolipid classes including sm , galcer , and sulfatides . similarly to what we observed in the app / ps1ki model , we did not found any changes of sm and galactolipid levels in all brain regions from the two transgenic lines investigated . similar findings were seen in appsw and appv717f transgenic mice , respectively , except for sulfatides . indeed , by contrast to our results , a loss of sulfatide content was observed in multiple brain regions of these animals . the reasons for such discrepancies are still unclear , but it may be ascribed to the different genetic background of mouse lines and/or the genetic constructs based on different app mutation and different promoters . supporting this , it has been shown for example , that app transgenic and wildtype mice on c57bl/6 background have lower basal cholesterol levels than the ki mouse lines which are on c57bl/6 50%-cba 25%-129sv 25% background . another example is the difference of lipid composition reported by sawamura and coworkers between mouse lines with c57bl/6j and fvb / n backgrounds , respectively . moreover , these authors found that ps2 transgenic mice with c57bl/6j background displayed significant phospholipid alterations , particularly of sm , as compared to their wild - type controls , while ps2 transgenic mice with fvb / n background did not . these few examples point out the difficulties to compare the results from various mouse lines and reinforce the idea that additional studies in this field are required . in summary , this study extends previous observations on sphingolipid alterations in animal models of ad . despite several limitations , in particular the lack of old double transgenics , the present results demonstrated that , in the absence of neurodegeneration , no elevation of ceramides occurred in disease - affected brain regions from single app mice , thus corroborating recent findings in two other single app mice . moreover , since both neuronal loss and accumulation of toxic n - truncated a peptides are lacking in the app model , this might suggest that a-induced ceramide production is an important event leading to neuronal death . in the future , to support this hypothesis , it will be interesting to analyse the sphingolipid composition of the tba2 mice , the new model expressing only n - truncated a3(pe)-42 and which develops a severe neuronal loss , to evaluate whether or not ceramides , especially the 2-oh species , accumulate in the brain of these mice . accumulating and crossing the information obtained from various animal models will help to better understand the exact mechanism by which these sphingolipids contribute to ad pathogenesis .

there is evidence linking sphingolipid abnormalities , app processing , and neuronal death in alzheimer 's disease ( ad ) . we previously reported a strong elevation of ceramide levels in the brain of the appsl / ps1ki mouse model of ad , preceding the neuronal death . to extend these findings , we analyzed ceramide and related - sphingolipid contents in brain from two other mouse models ( i.e. , appsl and appsl / ps1m146l ) in which the time - course of pathology is closer to that seen in most currently available models . conversely to our previous work , ceramides did not accumulate in disease - associated brain regions ( cortex and hippocampus ) from both models . however , the appsl / ps1ki model is unique for its drastic neuronal loss coinciding with strong accumulation of neurotoxic a isoforms , not observed in other animal models of ad . since there are neither neuronal loss nor toxic a species accumulation in appsl mice , we hypothesized that it might explain the lack of ceramide accumulation , at least in this model .

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

alzheimer 's disease ( ad ) is the most common form of dementia in adults . pathologically , the hallmarks of ad are amyloid plaques and neurofibrillary tangles , associated with widespread neuronal loss . the role of other lipids , such as ceramides and related - sphingolipids , ( sphingomyelins , sulfatides , and glycosphingolipids ) is also emerging . conversely , in the golgi , ceramides may be transformed into sm by the addition of phosphorylcholine . there is evidence linking sphingolipid abnormalities , app processing , and neuronal death in ad . additional reports have found that ceramide levels increase -amyloid synthesis [ 12 , 13 ] and favor gamma secretase processing of app [ 1416 ] so that inhibition of ceramide synthesis confers protection against -amyloid . for example , the total phospholipid and sulfatide content in ad was decreased as compared to normal [ 1719 ] , while the ceramide and galactosylceramide levels were elevated [ 9 , 18 ] . enhanced ceramide levels have been reported in the cerebrospinal fluid ( csf ) of patients with ad and changes in the activity of several key enzymes of ceramide metabolism , in gene expression of pathways associated to sphingolipid metabolism have been found in brains of ad patients [ 21 , 22 ] . unfortunately , very little work has been done on determining if sphingolipid content is likewise perturbed in these rodent models of ad . in a second study , sulfatides , a class of sulfated galactocerebrosides , were found to be decreased in brain tissues from two app transgenic mice ( i.e. , appv717f and appsw ) , whereas no significant changes in the content of other sphingolipid classes including sm , galcer , and ceramides were noted . by contrast , using the new mouse mutant app / ps1knock - in line , we have recently found an early and significant increase of ceramides in the cortex of these mice , without significant changes in other sphingolipid levels . however , the app / ps1ki model is unique for its drastic neuronal loss , not observed in other animal models of ad . the discrepancy in the few data available and the lack of knowledge of sphingolipid levels in the brain of other rodent models of ad prompted us to investigate whether the sphingolipid composition is altered in the brain of two other mouse models of ad : single app and double app / ps1 transgenics . concentrations of ceramides , sm , galcer , and sulfatides were determined in three brain regions : the cortex and the hippocampus , the two brain regions typically associated with the disease , and the cerebellum , a nonvulnerable region with no a plaques . lipid standards ( nonhydroxy fatty acid ( nfa ) containing ceramides , ceramides containing 2-hydroxy fatty acids ( 2-hfa ) , sphingomyelins , cerebroside sulfate ( sulfatides ) , and galactosylceramides ( a mixture containing 2-hydroxy fatty acids and nonhydroxy fatty acids ) were purchased from sigma - aldrich ( france ) . in this study , 12-month - old ( n = 5 ) app / ps1 double transgenic , 12-month - old ( n = 5 ) ps1 single - transgenic littermates , and 12-month - old ( n = 6 ) nontransgenic mice as well as 24 month - old ( n = 5 ) app single transgenic and 24-months ( n = 5 ) nontransgenic littermates were used ( generous gift of sanofi - aventis , vitry sur seine , france ) . app mice were analyzed at 24 months of age and app / ps1 mice were assessed at 12 months of age because they revealed comparable levels of amyloid plaques in the brain at these respective ages . each sphingolipid species was quantified by scanning densitometry of the plates at 396 nm for ceramides , 540 nm for galcer and sulfatides , and 750 nm for sm with the camag tlc scanner 3/wincats software system . in single app mice ( figure 1(a ) ) , a moderate but not significant elevation of nfa - ceramides was seen in the cortex ( + 22% ) , with no changes of the 2-hfa - ceramide level . conversely , in the hippocampus ( figure 1(b ) ) , the nfa - ceramide level did not differ between wt and app mice , whereas a tendency towards an increase of 2-hfa - ceramides was noted ( + 19% ) , although it was not significant . surprisingly , as shown in figure 1(c ) , the level of 2-hfa - ceramides in the cerebellum of app mice was significantly increased in comparison to the counterpart of their wt littermates ( + 50% , p < .05 ) . however , no difference in the total ceramide content was noticed in the cerebellum of both wt ( 51.35 1.45 nmol / mg tissue ) and app mice ( 50.28 3.31 nmol / mg tissue ) . because the double - transgenic mouse model app / ps1 develops neuropathological features of ad earlier than the single app mice , the sphingolipid analysis was performed in the brain regions of this model in younger animals ( 12 months of age ) . as shown in figures 2(a ) and 2(b ) , concentrations of nfa - ceramides as well as those of 2-hfa - ceramides did not differ between wild - type , ps1 , and app / ps1 mice in the two disease - associated brain regions ( cortex and hippocampus ) . in contrast to the changes of ceramide content in cerebellum of app mice , the nfa - ceramide as well as the 2-hfa - ceramide levels were unchanged in this brain region in app / ps1 mice relative to their wt controls and ps1 littermates ( figure 2(c ) ) . however , nfa - ceramide content showed a tendency towards a decrease , while 2-hfa ceramides tended to slightly increase in the cerebellum of the app / ps1 mice . it should be noted that , although cortex and hippocampus of wt mice displayed almost identical ceramide content ( figures 1(a ) , 1(b ) , 2(a ) , and 2(b ) ) , a relatively lower nfa - ceramide content was manifest in the cerebellum ( figures 1(c ) and 2(c ) ) . in normal human brain , the ceramide levels typical ceramide profiles from either cortex , hippocampus , or cerebellum of the different transgenic mice and wild - type controls were shown in figure 3 . to test whether other related - sphingolipids could be altered in the brain of transgenic mice , the content of sm , galcer , and sulfatides in lipid extracts of the three examined brain regions figures 1(d)1(f ) show that sphingolipids ( sm , galcer , and sulfatides ) did not display significant changes in both cortex , hippocampus , and cerebellum of app mice compared to the wt littermates . similarly , the levels of sm , galcer , and sulfatides did not differ among nontransgenic , ps1 and app / ps1 mice in all brain region examined ( figures 2(d)2(f ) ) . it should be noted that there are differences of sm , galcer , and sulfatide concentrations between cerebral tissues ( cortex and hippocampus ) and cerebellar tissues . who also reported a ~2-fold higher level of sulfatides in the cerebellum compared to the cortex , in two other transgenic mouse models of ad . it should be mentioned that the hptlc profiles of each sphingolipid class were similar in all examined brain regions from both mouse models . using the same methodology as in our previous work , we herein analysed the sphingolipid composition of two additional models ( i.e. , single app and double app / ps1 mice ) in which the time - course of pathology is much closer to that seen in the majority of currently available models . the main results of this study are ( 1 ) a moderate but not significant increase of nfa - ceramides and 2-hfa - ceramides in the cortex and the hippocampus respectively , of the app mice , ( 2 ) unaltered ceramide levels in the two disease - associated brain regions from app / ps1 mice , ( 3 ) unexpected alterations of the ceramide profile in the cerebellum of app mice , a region with no a pathology , and ( 4 ) no significant changes in the other related - sphingolipids in all brain structures examined of both transgenic models . based on our results and those from the literature , we will first discuss the possibility of a relationship between neurodegeneration , toxic a accumulation , and ceramide content . ceramides were shown to accumulate in ad human brain regions and their levels vary by disease severity suggesting that they could be indicators of ad progression [ 9 , 18 , 35 ] . similarly to what happens in human ad , we previously found that ceramides increase very early in the cortex of the app / ps1ki mouse model , preceding the neuronal loss . by contrast , our present results reveal that in single app mice , ceramide levels were not significantly altered in disease - associated brain regions ( e.g. an important difference between these single app mouse lines and the app / ps1ki model is that the latter develops an early and massive neuronal loss which correlates with strong accumulation of intracellular a42 , a aggregates , and a42 oligomers [ 28 , 36 ] . although intraneuronal a accumulation has also been documented in various app models [ 29 , 37 , 38 ] , a striking difference between the models used in the present study and the app / ps1ki mice is the nature of the a peptides which accumulate . indeed , in app / ps1ki mouse brain , extremely high levels of n - truncated ax-42 variants and abundant oligomers were detected , which closely resembles that found in ad brain . by contrast , in app mouse brain , with the same total a levels as in app / ps1ki mice , only very limited levels of a42 n - terminal truncated isovariants were detected . in the app / ps1ki mice , ax-42 is the major form accumulated with a ratio ax-42/total a close to 1 . in comparison , this ratio is approximately of only 0.2 - 0.3 in the app mice , and ~0.3 - 0.4 in the double transgenic app / ps1 mice , similar to the range of values reported for a large number of other app - based transgenics [ 28 , 29 ] . thus they might play a key role in the neurotoxicity observed in the app / ps1ki model . in particular , the pyroglutamate modified n - terminal truncated form of a at position 3 ( a3(pe)-42 ) , which represents a major species in the brain of ad patients , was recently shown to induce a severe neuron loss in the tba2 mouse model , a new model expressing only n - truncated a3(pe)-42 in neurons . interestingly , there is also a coincidence of considerable amounts of a3(pe)-42 and massive neuron loss in the app / ps1ki mouse model . on the basis of these findings , it is attractive to speculate that in app / ps1ki mice , the strong accumulation of intracellular toxic forms of a induces early elevation of ceramides . this is due to the lack of posttranslational modifications such as n - terminal degradation and pyroglutamyl formation in these mice . because app mice display neither neuronal loss nor accumulation of highly toxic a 42 isoforms , this may at least in part explain why no significant accumulation of ceramides occurred in the disease - associated brain regions of these mice . thus , despite numerous neuropathological , biochemical , and even behavioral changes representative of ad developed by these app mice [ 23 , 2830 , 33 , 44 ] , they may not constitute a relevant model to further explore the role of ceramides in ad pathology . however , answering the question of the relationship between neurodegeneration , toxic a accumulation , and ceramide elevation could be facilitated using restricted models either lacking ( i.e. , single app mice ) or mimicking only some specific ad - related neuropathological alterations ( i.e. indeed , owing to the simultaneous occurrence of numerous pathological features of ad , the connection between them is often difficult to unravel . we next determined the ceramide content in the double transgenic mouse model app / ps1 , but in younger animals because the app / ps1 mice develop neuropathological features of ad earlier than single app mice . our results showed that at 12 months of age , ceramide levels were unaltered in both cortex and hippocampus of app / ps1 mice in comparison to age - matched ps1 mice and wild - type controls . it should be noted that there is no neuronal loss in these mice as old as 17 months , but unfortunately , older app / ps1 mice were not available for this study . however , in our previous work , we demonstrated that elevation of ceramides occurred very early ( 3 months of age ) in the cortex of the app / ps1ki model , preceding by far the neuronal loss detectable at 6 months of age . why the app / ps1ki mice showed an increase of ceramides several months before the appearance of neuronal death while the app / ps1 mice did not is currently unknown . one possible explanation is that the neuronal loss reported in the app / ps1 mice is moderate and more restricted than in the app / ps1ki model . indeed , the loss of neurons in the former involves only the hippocampal pyramidal cell layer ( loss of ~30% in 17-month - old animals ) . this may in some way account for the lack of ceramide accumulation in the cortex of these mice . by comparison , in the app / ps1ki model , the cell loss is greater ( ~50% at 10 months of age ) and has been shown to extend to other brain areas such as frontal cortex and cholinergic system . moreover , as discussed above , accumulation of n - truncated a peptides should be lesser in app / ps1 mice , since the ratio ax-42/total a is of 0.3 only , versus 1 in the app / ps1ki mice . in this context , it would seem likely that the level of highly toxic a3(pe)-42 form , which is thought to be involved in neuronal death , is reduced in the app / ps1 model . it is also possible that , at 12 months of age , it is too early to visualize an elevation of ceramides , owing to the slowest progression of ad lesions in these mice than in the app / ps1ki model . since we did not have older app / ps1 mice , we should therefore be cautious to interpret the results of ceramide composition in these mice , because we can not exclude the possibility that ceramides increase at a later age . the most unexpected finding of the present study was alteration of the ceramide composition in the cerebellum of app mice , a brain region lacking a deposits and regarded as nonvulnerable to the disease . previously , we found a more dramatic 161% increase of 2-hfa - ceramides in the cortex of app / ps1ki mice but contrary to the present results , it was accompanied by an elevation of nfa - ceramides . nevertheless , a couple of interesting facts suggest that these hfa species may participate to ad pathology : ( i ) it was recently found that a selectively bound to sphingolipids that contained a 2-oh group on the ceramide backbone and did not effectively interact with sphingolipids that contained a nonhydroxylated fatty acid , favoring a conformational shift that disrupts membrane stability and promotes peptide - peptide interactions and oligomer formation ; ( ii ) the enzyme udp - galactose : ceramide galactosyltransferase ( cgt ) , which transfers galactose to both nfa- and 2-hfa - ceramides , was found to be upregulated in human ad brain . reducing the hfa - galcer level would lead to an accumulation of their immediate precursors , 2-hfa - ceramides ; ( iii ) there is some evidence for enhanced apoptosis - inducing activity of 2-hfa - ceramides compared to nfa - ceramides , and this effect seems to be cell type specific . in this sight , mouse mutants with defective saposin d dramatically accumulate hfa - ceramides in cerebellum , resulting in a selective loss of cerebellar purkinje cells ; ( iv ) we reported a gender - specific expression of hfa- versus nfa - ceramides in the app / ps1ki mouse model of ad , and this biochemical feature could be related to the increase propensity of females to develop earlier neuronal loss . although the degree of 2-hfa - ceramide accumulation in the cerebellum of app mice is much lesser than that seen in the cortex of the app / ps1ki mice , the reasons for these biochemical changes in this amyloid - free brain area are currently unknown . possibly , it might reflect alterations of ceramide metabolism , since there is evidence that other metabolic pathways are perturbed in the cerebellum of these mice . however , this is highly speculative and further investigation is warranted to determine whether these factors or other unknown factors contribute to such changes of the ceramide profile in the app cerebellum . in the cerebellum of ad patients , we also examined the content of other ceramide - related sphingolipid classes including sm , galcer , and sulfatides . similarly to what we observed in the app / ps1ki model , we did not found any changes of sm and galactolipid levels in all brain regions from the two transgenic lines investigated . similar findings were seen in appsw and appv717f transgenic mice , respectively , except for sulfatides . indeed , by contrast to our results , a loss of sulfatide content was observed in multiple brain regions of these animals . in summary , this study extends previous observations on sphingolipid alterations in animal models of ad . despite several limitations , in particular the lack of old double transgenics , the present results demonstrated that , in the absence of neurodegeneration , no elevation of ceramides occurred in disease - affected brain regions from single app mice , thus corroborating recent findings in two other single app mice . moreover , since both neuronal loss and accumulation of toxic n - truncated a peptides are lacking in the app model , this might suggest that a-induced ceramide production is an important event leading to neuronal death . in the future , to support this hypothesis , it will be interesting to analyse the sphingolipid composition of the tba2 mice , the new model expressing only n - truncated a3(pe)-42 and which develops a severe neuronal loss , to evaluate whether or not ceramides , especially the 2-oh species , accumulate in the brain of these mice .

rheumatoid arthritis ( ra ) is characterized by chronic systemic autoimmune inflammation of the connective tissue and is mostly accompanied by lesions in peripheral joints , erosion and degenerative changes in joints , and joint ankylosis . a broad range of cells , from monocyte / macrophage subsets to t and b cells , cytokines , the main mediators of intercellular communication , play a crucial role at all stages of the development of immune responses in this disease . as a broad - spectrum proinflammatory cytokine , interleukin-1 ( il-1 ) is involved in the development of both local and systemic inflammation in rheumatoid arthritis [ 7 , 8 ] . its hypersecretion destroys the bone tissue at the local level and also results in a significant disruption of hemodynamics at the systemic level . there are two types of membrane - bound il-1 receptors ( il-1r1 and il-1r2 ) ; however , il-1r2 does not contain a full - fledged cytoplasmic domain . hence , il-1r2 can not transmit signals to cells and thus acts as a decoy receptor [ 9 , 10 ] . therefore , il-1 produces its biological activity against cells only through type 1 receptor [ 11 , 12 ] ; however , the signaling pathways can be initiated only if the interleukin-1 receptor accessory protein ( il1racp ) is present in the receptor - cytokine complex . furthermore , cytokine activity is also regulated by the circulating interleukin-1 receptor antagonist ( rail-1 ) that competes for binding to receptors , thus acting as a specific il-1 inhibitor [ 13 , 14 ] . soluble il-1 receptors are the extracellular domains of membrane - bound il-1 receptors [ 15 , 16 ] , which are formed either via proteolytic cleavage catalyzed by specific metalloproteinases [ 17 , 18 ] or by alternative splicing ( so far demonstrated only for type 2 receptors ) . the key function of soluble il-1 receptors is inhibiting the biological effects of cytokines by competing with membrane - bound receptors for binding to the ligand [ 15 , 20 ] . additionally , soluble type 1 receptors were shown to have a buffering function for il-1 ligands . the modulation of cytokine activity depends on a number of parameters , including the levels of soluble mediators , percentage of receptor - carrying cells , ratios between subpopulations by percentage or density of receptor expression , and the ratio between types 1 and 2 receptors per cell . however , simultaneous comprehensive investigation and comparison of the various components of the il-1 receptor apparatus in rheumatoid arthritis using both qualitative ( counting the percentage of il-1r1 and il-1r2 cells ) and accurate quantitative characteristics ( density of receptor expression on cell surface ) have not been performed . hence , our study aimed to investigate the changes in expression of il-1 receptors in rheumatoid arthritis . the group of patients with ra consisted of 40 persons who were hospitalized at the clinic of immunopathology under the federal state budgetary scientific institution research institute of fundamental and clinical immunology , in novosibirsk , russia . diagnosis of ra was verified in accordance with the acr ( american college of rheumatology ) criteria ( 2010 ) . the severity of ra was determined by counting the number of painful and swollen joints among 28 specified joints , determination of the erythrocyte sedimentation rate , assessment of each patient 's general well - being according to the visual analogue scale ( range 0100 mm ) , and subsequent calculation of the das28 index . at the time of their admission to the clinic , all patients had a high disease activity ( das28 > 5.9 ) . blood samples were collected from each patient during the acute stage ( n = 40 , aged 2875 years , six men and 34 women ) and after effective course of treatment ( n = 21 , aged 2875 years , two men and 19 women ) that involved besides basic methotrexate therapy ( 1520 mg in week ) either biological agents ( rituximab , 500 mg intravenous in first and 15th days , 7 women ) or methylprednisolone ( intravenous pulse therapy , 500 mg for three days , two men and 12 women ) one day before discharge from a hospital . none of the 23 investigated parameters was shown to have significant differences between patients who received different types of therapy . additionally , there were no significant differences between men and women and between patients of different age groups , so the data presented were not divided by therapy or age . in all cases , therapeutic efficacy was assessed using the criteria established by the european league against rheumatism and revealed positive clinical or laboratory dynamics : an alteration of das28 by > 1.2 from the initial value was used to classify patients as having responded to therapy . we obtained human peripheral blood mononuclear cells ( pbmc ) from 150 healthy individuals ( aged 1859 years , 67 men and 83 women ) from the novosibirsk blood center . the status of the donors was determined through a questionnaire and a latex test for c - reactive protein ( crp ; llc olvex diagnosticum , russia ) . individuals with serum crp concentration < 6 mg / ml were included as donors . the study was approved by the local ethics committee of the federal state budgetary scientific institution we isolated pbmcs from whole blood by ficoll ( pharmacia fine chemicals , uppsala , sweden ) density centrifugation ( = 1.077 g / l ) as previously described . cells were cultured in 96-well plates in rpmi-1640 medium ( biolot , russia ) supplemented with 10% fetal calf serum , 2 mm l - glutamine , 10 mm hepes buffer , 0.5 mm 2-mercaptoethanol , 80 mg / ml gentamicin , and 100 mg / ml benzylpenicillin for 24 h at 37c with 5% co2 in the presence or absence of lps ( 055:b5 ; sigma - aldrich , st . louis , mo , usa ) at a final concentration of 200 ng / ml . we identified subsets of mononuclear cells by immunophenotyping using human - specific fluorescently labeled mouse monoclonal antibodies : allophycocyanin- ( apc- ) conjugated anti - cd3 ( clone okt3 ) , fluorescein isothiocyanate- ( fitc- ) conjugated anti - cd14 ( clone 61d3 ) and phycoerythrin - cyanine 7 ( pe - cy7 ) conjugated anti - cd19 ( clone hib19 ) ( ebioscience , san diego , ca , usa ) , anti - human il-1r1 , and anti - human il-1r2 pe ( r&d systems , minneapolis , mn , usa ) . bd quantibrite pe beads ( bd biosciences , san jose , ca , usa ) were used to convert cell fluorescence data of positive cells into the absolute number of receptors per cell . we evaluated protein levels in peripheral blood serum using elisa kits for sil-1r1 , sil-1r2 ( raybiotech , norcross , ga , usa ) , il-1 , and rail-1 ( jsc vector - best , novosibirsk , russia ) , as per the manufacturer 's instructions . we analyzed statistical data with statistica 7.0 software ( statsoft , tulsa , ok , usa ) . independent samples were tested for statistical significance using the mann - whitney test and nonparametric kruskal - wallis analysis of variance by rank and median multiple comparisons . we evaluated correlations among parameters with the pearson test ( p < 0.05 ) . a p value of p < 0.05 was considered statistically significant . to study expression of membrane - bound receptors of the proinflammatory cytokine il-1 , which plays a key role in both the development and severity of the pathological process in ra , one needs to assess expression of types 1 and 2 receptors on cells involved in the pathology . the results of previous studies involving healthy individuals demonstrate that there are differences both in the number of cells expressing il-1 receptors types 1 and 2 and in the number of membrane - bound receptors on subpopulations of peripheral blood mononuclear cells ( pbmc ) . however , the high percentage of receptor - expressing cells in subpopulations does not always associate with high receptor level , and vice versa . we selected three main subpopulations of peripheral blood immunocompetent cells : t cells ( cd3 ) , b cells ( cd19 ) , and monocytes ( cd14 ) , because they are most actively involved in the systemic inflammatory response and also take part in local responses . we tested pbmcs derived from ra patients during the acute disease stage and after they had responded to therapy to study the features of the pathogenic process and the reaction of cytokine receptors expression to treatment . the percentage of cells carrying types 1 and 2 receptors was assessed in each subpopulation ( figure 1 ) and the number of membrane - bound receptors on cells was calculated ( figure 2 ) . the distribution of receptors on cell subpopulations was found to differ between ra patients and healthy individuals . whereas in healthy individuals , monocytes had the highest percentage of cells expressing type 1 receptors , the density of expression of these receptors was the lowest . in contrast , in ra patients in the acute phase , the percentage of cd14 cells expressing il-1r was much lower , while the density of expression was significantly higher compared both with healthy volunteers and with the other cell subsets studied . in terms of expression of il receptors types 1 and 2 , consistent differences in percentage of positive cells between t cell , b cell , and monocyte subpopulations were detected neither in healthy individuals nor in ra patients regardless of the disease phase . however , in terms of density of expression of these receptors , t cells were characterized by the lowest number of receptors compared with b cells and monocytes in all the studied groups , while the greatest number of receptors was detected on monocytes in ra patients , both in the acute phase and in patients who showed a clinical response to treatment . it was demonstrated that the percentage of monocytes expressing type 1 receptors was consistently decreased in ra patients who showed a clinical response to treatment , while the density of expression of these receptors on monocytes remained unchanged . thus , it was found that b cells and monocytes showed oppositely directed changes in percentage of il-1r1-positive cells and the number of receptors on these cells in ra patients compared with normal controls , but the change in il-1r1 on b cells in ra patients correlated with the change on monocytes ( as confirmed by its positive correlation with r = 0.66 , p < 0.05 , for both indicators during the acute phase of the disease and r = 0.71 and r = 0.48 for the percentage of positive cells , resp . , in patients who had responded to treatment ) but in an opposite direction . this indicates that there are different possible mechanisms for changing the common pool of membrane - bound receptors in a subpopulation and there is an interrelationship between indicators of receptor expression for different subpopulations of immunocompetent cells . it was found by comparing the expression parameters of different types of receptors that both the percentage of positive cells and the number of receptors per cell in healthy individuals significantly differ for il-1r1 and il-1r2 in each subpopulation studied . ra patients do not exhibit these differences , regardless of the phase of the disease . expression of il-1 receptors on monocytes ( cd14 ) in unstimulated and lps - stimulated human pbmc cultures was assessed in a more detailed study of changes in the system of il-1 receptors under a simulated proinflammatory response . to study the ability of cells to respond to stimulation , we assessed the expression of il-1 receptors types 1 and 2 on monocytes of pbmc cultured for 24 h in the presence and absence of lps ( o55 : b5 ) . we determined the percentage of monocytes expressing types 1 and 2 receptors ( figure 3 ) and calculated the absolute number of receptors per cell ( figure 4 ) . culturing in the presence of lps for 24 h increased the density of il-1 receptor expression in both healthy individuals and ra patients with higher indices in ra patients compared with healthy volunteers . the stimulation coefficient ( quotient of dividing the percentage of cells or the number of receptors in lps - stimulated culture to that in unstimulated one ) for the number of il-1r1 receptors in ra patients in the acute phase and ra patients who had responded to treatment was consistently lower than that in healthy individuals ( 1.29 and 1.49 versus 2.19 , resp . ) , while the stimulation coefficient for the number of il-1r2 receptors was significantly higher ( 1.52 and 1.43 versus 0.91 , resp . ) . however , consistent lps - induced changes in the percentage of receptor - carrying cells were observed only for healthy individuals ( with an increase for type 1 receptor and a decrease for type 2 receptor ) , while no response to stimulation was observed in ra patients according to the percentage of positive cells . when comparing types 1 and type 2 receptors , we found that , unlike intact monocytes ( for which the differences were shown only for healthy individuals ) , the ratio between the number of cells expressing il-1 receptors of different types in unstimulated and lps - stimulated cultures changed significantly . the percentage of il-1r1 monocytes was significantly higher than that of il-1r2 in all the groups under study . however , the densities of expression of types 1 and 2 receptors in monocytes of healthy individuals in unstimulated cultures were almost identical ; stimulation with lps consistently increased the density of type 1 receptors and reduced the density of type 2 receptors . the average number of il-1r1 and il-1r2 receptors on monocytes derived from ra patients was not significantly different . because the effect of il-1 on cells depends significantly on the relative serum concentration of soluble il-1 receptors and cytokine , the serum contents of il-1 ( figure 5 ) and soluble il-1 receptors types 1 and 2 ( figures 6 and 7 ) were studied by elisa in ra patients in the acute phase and ra patients who responded to treatment . according to the peripheral blood serum level of the cytokine , the il-1 level in ra patients was higher than that in healthy individuals and did not decrease after patients had exhibited a clinical response to treatment . ra patients had higher levels of both types of soluble receptors compared with healthy individuals . patients who had exhibited a response to therapy had a lower level of type 1 receptor but not type 2 , which may be due to the fact that type 2 receptor does not function as a buffer for il-1 . furthermore , the system regulating the biological effects of il-1 also includes il-1 receptor antagonist , because it competes for binding to cytokine receptors , thus modulating the effect of il-1 on cells . hence , it was important to assess the serum content of rail-1 in ra patients in the acute phase and those who had responded to treatment compared with healthy individuals ( figure 8) . ra patients in the acute phase had a significantly increased receptor antagonist level compared with those of healthy individuals and ra patients who had responded to treatment . a positive correlation between mediator level in the acute phase and after effective therapy was detected , suggesting that this indicator can be used as a marker of treatment effectiveness . also we found several statistically significant correlations between il-1 receptors parameters and serum contents of soluble mediators . we established a number of correlations between serum levels of tnf and its soluble receptors and parameters of il-1 receptors expression in pbmc culture . particularly content of tnf in ra patients in acute stage negatively correlated ( r = 0.43 , p < 0.05 ) with the percentage of intact il-1r2 monocytes and had no correlations with any parameter of receptor expression in the cultures . however , in ra patients responding to therapy content of tnf positively correlated ( r = + 0.45 , p < 0.05 ) with the percentage of il-1r2 monocytes in lps - stimulated cultures and negatively ( r = 0.48 , p < 0.05 ) with the number of receptors on these cells . the proinflammatory cytokine il-1 has a broad range of effects on the development and course of ra through its receptors , at both the local and systemic levels . however , at the molecular level these effects are regulated by mediator production and changes in the systems of signal transmission to immunocompetent cells or its blockade . the role of il-1 , rail-1 , and soluble il-1 receptors in this pathology however , the changes in the expression of membrane - bound receptors have been insufficiently characterized . il-1 exhibits its proinflammatory effect only after binding to specific membrane - bound type 1 receptors , while type 2 receptors act as decoy receptors . therefore , the balance between different types of soluble and membrane - bound receptors will determine whether a cell responds to il-1 or not and variability in intracellular effects . additionally , the biological effects of cytokines depend on the concentration of their soluble forms both due to binding to the membrane forms of receptors and due to induction of expression and/or shedding of cytokine receptors . hence , the comprehensive assessment and comparison of the levels of soluble factors regulating cytokine activity and expression of membrane - bound forms of receptors enable a richer and more accurate understanding of the system regulating the biological effects of cytokines . one should bear in mind that changes in density of receptor expression on the cell surface are a key mechanism of regulation of the biological properties of cytokines [ 2729 ] . however , cytokine function can be minimal if receptor expression is downregulated . in the case of excessive expression of receptors moreover , it has been demonstrated for some immune mediators that there is a certain threshold level of expression density , which switches the signaling pathways between the fundamentally different functions [ 30 , 31 ] . thus , it is insufficient to only know the percentage of positive cells in a subpopulation when studying the membrane forms of receptors . one also needs to determine the density of receptor expression on the cell surface in standardized units that are independent of the equipment or settings used ( as opposed to the commonly employed direct measures of fluorescence intensity given in terms of arbitrary units ) . hence , we used calibration particles ( bd biosciences ) to convert the units of mean fluorescence intensity ( mfi ) to the number of receptor molecules on the cell surface . tables 1 and 2 summarize the data on differences obtained for ra patients in the acute phase and after they had responded to therapy , as compared with healthy individuals , as well as the differences between the phases of the disease . we demonstrated that intact pbmcs differ both in terms of the relative percentage of cells expressing il-1 receptors types 1 and 2 and in terms of the absolute number of receptors on them . the high density of receptor expression on a cell does not always imply a high percentage of positive cells , and vice versa . moreover , increased or reduced density of receptor expression on cells in pathology compared with normal does not always correlate with the percentage of cells carrying these receptors . it was demonstrated for the expression of il-1 receptors type 1 that the percentage of il-1r1 cells increases while the density of expression of these receptors on these cells decreases simultaneously in b cells of patients who had responded to treatment . an opposite ratio is observed in monocytes of ra patients regardless of the stage of the disease ( i.e. , reduced percentage of il-1r1 cells with a simultaneous increase in density ) . thus , the opposing changes in the percentage of cells and density of expression of receptor in these subpopulations of immunocompetent cells can act as a mechanism for cytokine binding to target receptors on specific subpopulations . the changes in the percentage of receptor - expressing cells in subpopulations or the changes in density of receptor expression in pathology compared with health are caused by different mechanisms . it was demonstrated for il-1 that upregulated expression of il-1r2 on cells may either cause the absence of cellular response to il-1 [ 32 , 33 ] or reduce the cytokine effect on cells . the number of type 2 decoy receptors on b lymphocytes is reduced in ra patients in the acute phase , but it is compensated for by the higher percentage of il-1r2 cells in patients who had a clinical response to treatment as compared to with healthy individuals . furthermore , the percentage of il-1r2 t cells increased significantly in patients who had responded to therapy , while upregulation of expression of these receptors was observed in monocytes in ra patients regardless of the phase of the disease . thus , these changes demonstrate that il-1r2 plays a crucial role in cellular response to treatment in ra patients . a hypothesis can be put forward that this type of membrane - bound receptor could be targeted in future therapeutic approaches in rheumatoid arthritis . according to the changes in function of membrane - bound receptors and their competition for binding to a corresponding cytokine , the balance between the two receptor types is a key factor showing the status of the system regulating the biological effects of cytokines . the changes in the percentage of receptor - carrying cells in a subpopulation , the changes in mean density of expression of types 1 and 2 receptors , and the changes in this balance in pathology compared with health provide the two alternative mechanisms of modulation of cytokine effect to increase its chances for binding to a certain receptor type on cells of a certain subpopulation . we demonstrated that the percentage of cells expressing il-1r1 is higher than the percentage of il-1r2 cells among t and b cells and monocytes only in healthy donors ; after the cells are cultured , this percentage is higher among monocytes in all the groups under study . in terms of the number of receptors on cells of intact subpopulations in healthy individuals , t and b cells are characterized by upregulated expression of type 1 receptors , while monocytes are characterized by upregulated expression of type 2 receptors . however , this balance is disturbed in ra patients and there are changes between the mean number of types 1 and 2 receptors in neither subpopulation . thus , the balance between different types of cytokine receptors having different functions is changed in pathology , which explains the disturbance in regulation of effects of proinflammatory mediators in rheumatoid arthritis . in addition , for a more detailed consideration and establishment of causes and effects of these differences also the relationship with other components of cytokine network and regulatory proteins ( such other proinflammatory cytokines and il1racp ) need to be considered . the detected differences in intact cells and in the monocyte response to activation by lps demonstrate that monocytes can regulate the activity of il-1 both by changing the percentage of positive cells or the number of receptors per cell and by simultaneously changing these two indicators in opposite directions . soluble receptors are powerful regulators of cytokine activity as they can neutralize il-1 in the systemic circulation [ 15 , 20 ] and cytokine complexed with soluble receptors can have a buffering function . however , certain differences in implementation of their functions have been demonstrated for different types of soluble receptors . soluble il-1r2 is characterized by higher binding affinity compared with the corresponding type 1 receptors ( i.e. , they are major contributors to inhibition of cytokine activity ) . sil-1r1 has a better buffering function and is simultaneously characterized by higher affinity to the il-1 receptor antagonist and il-1 , rather than il-1. furthermore , it was shown for il-1 that there exists a specific receptor antagonist competing with cytokine for binding to receptors . we demonstrated that the serum levels of il-1r2 in all the groups under study were significantly higher than those of type 1 receptors . however , ra patients in the acute phase had higher levels of type 1 receptors compared with healthy individuals . we revealed the correlations between parameters of soluble mediators in ra patients who had responded to treatment and those in the same patients in the acute phase ( reduced serum levels of il-1r1 and rail-1 ) , which can be used to attest to treatment effectiveness . the resulting data are indicative of differences in expression of il-1 receptors in various subpopulations of immunocompetent cells in normal cells and in pathology . additionally , they show changes both in indicators of mediator production accompanying inflammatory pathologies and in the system of receptor regulation on the cell surface . therefore , it is important to determine both the relative percentage of cells expressing receptors to immunomodulatory cytokines and the levels of membrane - bound receptors , because the density of expression is characterized by disease - induced changes that can not be detected when assessing the percentage of positive cells . it was found that oppositely directed changes in the density of expression of il-1 receptors type 1 and in the percentage of il-1r1 cells in b cells and monocytes take place , thus resulting in different mechanisms of regulation of cellular response to cytokines via changing the parameters of expression of different types of receptors .

il-1 is involved in the induction and maintenance of chronic inflammation in rheumatoid arthritis ( ra ) . its activity is regulated and induced by soluble and membrane - bound receptors , respectively . the effectiveness of the cytokine depends not only on the percentage of receptor - positive cells in an immunocompetent subset but also on the density of receptor expression . the objective of this study was to investigate the expression of il-1 membrane - bound receptors ( il-1r1 and il-1r2 ) in terms of the percentage of receptor - positive cells and the number of receptors per cell in different subsets of immune cells in ra patients before and after a course of basic ( excluding anticytokine ) therapy and in healthy individuals . the resulting data indicate differences in the expression of il-1 receptors among t cells , b cells , and monocytes in healthy volunteers and in rheumatoid arthritis patients . the importance of determining both the relative percentage of cells expressing receptors to immunomodulatory cytokines and the number of membrane - bound receptors per cell is highlighted by evidence of unidirectional or multidirectional changing of these parameters according to cell subset and health status .

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

rheumatoid arthritis ( ra ) is characterized by chronic systemic autoimmune inflammation of the connective tissue and is mostly accompanied by lesions in peripheral joints , erosion and degenerative changes in joints , and joint ankylosis . a broad range of cells , from monocyte / macrophage subsets to t and b cells , cytokines , the main mediators of intercellular communication , play a crucial role at all stages of the development of immune responses in this disease . as a broad - spectrum proinflammatory cytokine , interleukin-1 ( il-1 ) is involved in the development of both local and systemic inflammation in rheumatoid arthritis [ 7 , 8 ] . there are two types of membrane - bound il-1 receptors ( il-1r1 and il-1r2 ) ; however , il-1r2 does not contain a full - fledged cytoplasmic domain . therefore , il-1 produces its biological activity against cells only through type 1 receptor [ 11 , 12 ] ; however , the signaling pathways can be initiated only if the interleukin-1 receptor accessory protein ( il1racp ) is present in the receptor - cytokine complex . furthermore , cytokine activity is also regulated by the circulating interleukin-1 receptor antagonist ( rail-1 ) that competes for binding to receptors , thus acting as a specific il-1 inhibitor [ 13 , 14 ] . soluble il-1 receptors are the extracellular domains of membrane - bound il-1 receptors [ 15 , 16 ] , which are formed either via proteolytic cleavage catalyzed by specific metalloproteinases [ 17 , 18 ] or by alternative splicing ( so far demonstrated only for type 2 receptors ) . the key function of soluble il-1 receptors is inhibiting the biological effects of cytokines by competing with membrane - bound receptors for binding to the ligand [ 15 , 20 ] . the modulation of cytokine activity depends on a number of parameters , including the levels of soluble mediators , percentage of receptor - carrying cells , ratios between subpopulations by percentage or density of receptor expression , and the ratio between types 1 and 2 receptors per cell . however , simultaneous comprehensive investigation and comparison of the various components of the il-1 receptor apparatus in rheumatoid arthritis using both qualitative ( counting the percentage of il-1r1 and il-1r2 cells ) and accurate quantitative characteristics ( density of receptor expression on cell surface ) have not been performed . hence , our study aimed to investigate the changes in expression of il-1 receptors in rheumatoid arthritis . the severity of ra was determined by counting the number of painful and swollen joints among 28 specified joints , determination of the erythrocyte sedimentation rate , assessment of each patient 's general well - being according to the visual analogue scale ( range 0100 mm ) , and subsequent calculation of the das28 index . blood samples were collected from each patient during the acute stage ( n = 40 , aged 2875 years , six men and 34 women ) and after effective course of treatment ( n = 21 , aged 2875 years , two men and 19 women ) that involved besides basic methotrexate therapy ( 1520 mg in week ) either biological agents ( rituximab , 500 mg intravenous in first and 15th days , 7 women ) or methylprednisolone ( intravenous pulse therapy , 500 mg for three days , two men and 12 women ) one day before discharge from a hospital . the study was approved by the local ethics committee of the federal state budgetary scientific institution we isolated pbmcs from whole blood by ficoll ( pharmacia fine chemicals , uppsala , sweden ) density centrifugation ( = 1.077 g / l ) as previously described . cells were cultured in 96-well plates in rpmi-1640 medium ( biolot , russia ) supplemented with 10% fetal calf serum , 2 mm l - glutamine , 10 mm hepes buffer , 0.5 mm 2-mercaptoethanol , 80 mg / ml gentamicin , and 100 mg / ml benzylpenicillin for 24 h at 37c with 5% co2 in the presence or absence of lps ( 055:b5 ; sigma - aldrich , st . we identified subsets of mononuclear cells by immunophenotyping using human - specific fluorescently labeled mouse monoclonal antibodies : allophycocyanin- ( apc- ) conjugated anti - cd3 ( clone okt3 ) , fluorescein isothiocyanate- ( fitc- ) conjugated anti - cd14 ( clone 61d3 ) and phycoerythrin - cyanine 7 ( pe - cy7 ) conjugated anti - cd19 ( clone hib19 ) ( ebioscience , san diego , ca , usa ) , anti - human il-1r1 , and anti - human il-1r2 pe ( r&d systems , minneapolis , mn , usa ) . bd quantibrite pe beads ( bd biosciences , san jose , ca , usa ) were used to convert cell fluorescence data of positive cells into the absolute number of receptors per cell . to study expression of membrane - bound receptors of the proinflammatory cytokine il-1 , which plays a key role in both the development and severity of the pathological process in ra , one needs to assess expression of types 1 and 2 receptors on cells involved in the pathology . the results of previous studies involving healthy individuals demonstrate that there are differences both in the number of cells expressing il-1 receptors types 1 and 2 and in the number of membrane - bound receptors on subpopulations of peripheral blood mononuclear cells ( pbmc ) . however , the high percentage of receptor - expressing cells in subpopulations does not always associate with high receptor level , and vice versa . we selected three main subpopulations of peripheral blood immunocompetent cells : t cells ( cd3 ) , b cells ( cd19 ) , and monocytes ( cd14 ) , because they are most actively involved in the systemic inflammatory response and also take part in local responses . we tested pbmcs derived from ra patients during the acute disease stage and after they had responded to therapy to study the features of the pathogenic process and the reaction of cytokine receptors expression to treatment . the percentage of cells carrying types 1 and 2 receptors was assessed in each subpopulation ( figure 1 ) and the number of membrane - bound receptors on cells was calculated ( figure 2 ) . the distribution of receptors on cell subpopulations was found to differ between ra patients and healthy individuals . whereas in healthy individuals , monocytes had the highest percentage of cells expressing type 1 receptors , the density of expression of these receptors was the lowest . in contrast , in ra patients in the acute phase , the percentage of cd14 cells expressing il-1r was much lower , while the density of expression was significantly higher compared both with healthy volunteers and with the other cell subsets studied . in terms of expression of il receptors types 1 and 2 , consistent differences in percentage of positive cells between t cell , b cell , and monocyte subpopulations were detected neither in healthy individuals nor in ra patients regardless of the disease phase . however , in terms of density of expression of these receptors , t cells were characterized by the lowest number of receptors compared with b cells and monocytes in all the studied groups , while the greatest number of receptors was detected on monocytes in ra patients , both in the acute phase and in patients who showed a clinical response to treatment . it was demonstrated that the percentage of monocytes expressing type 1 receptors was consistently decreased in ra patients who showed a clinical response to treatment , while the density of expression of these receptors on monocytes remained unchanged . thus , it was found that b cells and monocytes showed oppositely directed changes in percentage of il-1r1-positive cells and the number of receptors on these cells in ra patients compared with normal controls , but the change in il-1r1 on b cells in ra patients correlated with the change on monocytes ( as confirmed by its positive correlation with r = 0.66 , p < 0.05 , for both indicators during the acute phase of the disease and r = 0.71 and r = 0.48 for the percentage of positive cells , resp . this indicates that there are different possible mechanisms for changing the common pool of membrane - bound receptors in a subpopulation and there is an interrelationship between indicators of receptor expression for different subpopulations of immunocompetent cells . it was found by comparing the expression parameters of different types of receptors that both the percentage of positive cells and the number of receptors per cell in healthy individuals significantly differ for il-1r1 and il-1r2 in each subpopulation studied . expression of il-1 receptors on monocytes ( cd14 ) in unstimulated and lps - stimulated human pbmc cultures was assessed in a more detailed study of changes in the system of il-1 receptors under a simulated proinflammatory response . to study the ability of cells to respond to stimulation , we assessed the expression of il-1 receptors types 1 and 2 on monocytes of pbmc cultured for 24 h in the presence and absence of lps ( o55 : b5 ) . we determined the percentage of monocytes expressing types 1 and 2 receptors ( figure 3 ) and calculated the absolute number of receptors per cell ( figure 4 ) . culturing in the presence of lps for 24 h increased the density of il-1 receptor expression in both healthy individuals and ra patients with higher indices in ra patients compared with healthy volunteers . the stimulation coefficient ( quotient of dividing the percentage of cells or the number of receptors in lps - stimulated culture to that in unstimulated one ) for the number of il-1r1 receptors in ra patients in the acute phase and ra patients who had responded to treatment was consistently lower than that in healthy individuals ( 1.29 and 1.49 versus 2.19 , resp . ) , while the stimulation coefficient for the number of il-1r2 receptors was significantly higher ( 1.52 and 1.43 versus 0.91 , resp . ) however , consistent lps - induced changes in the percentage of receptor - carrying cells were observed only for healthy individuals ( with an increase for type 1 receptor and a decrease for type 2 receptor ) , while no response to stimulation was observed in ra patients according to the percentage of positive cells . when comparing types 1 and type 2 receptors , we found that , unlike intact monocytes ( for which the differences were shown only for healthy individuals ) , the ratio between the number of cells expressing il-1 receptors of different types in unstimulated and lps - stimulated cultures changed significantly . the percentage of il-1r1 monocytes was significantly higher than that of il-1r2 in all the groups under study . however , the densities of expression of types 1 and 2 receptors in monocytes of healthy individuals in unstimulated cultures were almost identical ; stimulation with lps consistently increased the density of type 1 receptors and reduced the density of type 2 receptors . the average number of il-1r1 and il-1r2 receptors on monocytes derived from ra patients was not significantly different . because the effect of il-1 on cells depends significantly on the relative serum concentration of soluble il-1 receptors and cytokine , the serum contents of il-1 ( figure 5 ) and soluble il-1 receptors types 1 and 2 ( figures 6 and 7 ) were studied by elisa in ra patients in the acute phase and ra patients who responded to treatment . according to the peripheral blood serum level of the cytokine , the il-1 level in ra patients was higher than that in healthy individuals and did not decrease after patients had exhibited a clinical response to treatment . ra patients had higher levels of both types of soluble receptors compared with healthy individuals . furthermore , the system regulating the biological effects of il-1 also includes il-1 receptor antagonist , because it competes for binding to cytokine receptors , thus modulating the effect of il-1 on cells . hence , it was important to assess the serum content of rail-1 in ra patients in the acute phase and those who had responded to treatment compared with healthy individuals ( figure 8) . ra patients in the acute phase had a significantly increased receptor antagonist level compared with those of healthy individuals and ra patients who had responded to treatment . a positive correlation between mediator level in the acute phase and after effective therapy was detected , suggesting that this indicator can be used as a marker of treatment effectiveness . we established a number of correlations between serum levels of tnf and its soluble receptors and parameters of il-1 receptors expression in pbmc culture . particularly content of tnf in ra patients in acute stage negatively correlated ( r = 0.43 , p < 0.05 ) with the percentage of intact il-1r2 monocytes and had no correlations with any parameter of receptor expression in the cultures . however , in ra patients responding to therapy content of tnf positively correlated ( r = + 0.45 , p < 0.05 ) with the percentage of il-1r2 monocytes in lps - stimulated cultures and negatively ( r = 0.48 , p < 0.05 ) with the number of receptors on these cells . the proinflammatory cytokine il-1 has a broad range of effects on the development and course of ra through its receptors , at both the local and systemic levels . the role of il-1 , rail-1 , and soluble il-1 receptors in this pathology however , the changes in the expression of membrane - bound receptors have been insufficiently characterized . il-1 exhibits its proinflammatory effect only after binding to specific membrane - bound type 1 receptors , while type 2 receptors act as decoy receptors . therefore , the balance between different types of soluble and membrane - bound receptors will determine whether a cell responds to il-1 or not and variability in intracellular effects . additionally , the biological effects of cytokines depend on the concentration of their soluble forms both due to binding to the membrane forms of receptors and due to induction of expression and/or shedding of cytokine receptors . hence , the comprehensive assessment and comparison of the levels of soluble factors regulating cytokine activity and expression of membrane - bound forms of receptors enable a richer and more accurate understanding of the system regulating the biological effects of cytokines . one should bear in mind that changes in density of receptor expression on the cell surface are a key mechanism of regulation of the biological properties of cytokines [ 2729 ] . in the case of excessive expression of receptors moreover , it has been demonstrated for some immune mediators that there is a certain threshold level of expression density , which switches the signaling pathways between the fundamentally different functions [ 30 , 31 ] . thus , it is insufficient to only know the percentage of positive cells in a subpopulation when studying the membrane forms of receptors . one also needs to determine the density of receptor expression on the cell surface in standardized units that are independent of the equipment or settings used ( as opposed to the commonly employed direct measures of fluorescence intensity given in terms of arbitrary units ) . hence , we used calibration particles ( bd biosciences ) to convert the units of mean fluorescence intensity ( mfi ) to the number of receptor molecules on the cell surface . tables 1 and 2 summarize the data on differences obtained for ra patients in the acute phase and after they had responded to therapy , as compared with healthy individuals , as well as the differences between the phases of the disease . we demonstrated that intact pbmcs differ both in terms of the relative percentage of cells expressing il-1 receptors types 1 and 2 and in terms of the absolute number of receptors on them . the high density of receptor expression on a cell does not always imply a high percentage of positive cells , and vice versa . moreover , increased or reduced density of receptor expression on cells in pathology compared with normal does not always correlate with the percentage of cells carrying these receptors . it was demonstrated for the expression of il-1 receptors type 1 that the percentage of il-1r1 cells increases while the density of expression of these receptors on these cells decreases simultaneously in b cells of patients who had responded to treatment . an opposite ratio is observed in monocytes of ra patients regardless of the stage of the disease ( i.e. thus , the opposing changes in the percentage of cells and density of expression of receptor in these subpopulations of immunocompetent cells can act as a mechanism for cytokine binding to target receptors on specific subpopulations . the changes in the percentage of receptor - expressing cells in subpopulations or the changes in density of receptor expression in pathology compared with health are caused by different mechanisms . the number of type 2 decoy receptors on b lymphocytes is reduced in ra patients in the acute phase , but it is compensated for by the higher percentage of il-1r2 cells in patients who had a clinical response to treatment as compared to with healthy individuals . furthermore , the percentage of il-1r2 t cells increased significantly in patients who had responded to therapy , while upregulation of expression of these receptors was observed in monocytes in ra patients regardless of the phase of the disease . thus , these changes demonstrate that il-1r2 plays a crucial role in cellular response to treatment in ra patients . a hypothesis can be put forward that this type of membrane - bound receptor could be targeted in future therapeutic approaches in rheumatoid arthritis . according to the changes in function of membrane - bound receptors and their competition for binding to a corresponding cytokine , the balance between the two receptor types is a key factor showing the status of the system regulating the biological effects of cytokines . the changes in the percentage of receptor - carrying cells in a subpopulation , the changes in mean density of expression of types 1 and 2 receptors , and the changes in this balance in pathology compared with health provide the two alternative mechanisms of modulation of cytokine effect to increase its chances for binding to a certain receptor type on cells of a certain subpopulation . we demonstrated that the percentage of cells expressing il-1r1 is higher than the percentage of il-1r2 cells among t and b cells and monocytes only in healthy donors ; after the cells are cultured , this percentage is higher among monocytes in all the groups under study . in terms of the number of receptors on cells of intact subpopulations in healthy individuals , t and b cells are characterized by upregulated expression of type 1 receptors , while monocytes are characterized by upregulated expression of type 2 receptors . however , this balance is disturbed in ra patients and there are changes between the mean number of types 1 and 2 receptors in neither subpopulation . the detected differences in intact cells and in the monocyte response to activation by lps demonstrate that monocytes can regulate the activity of il-1 both by changing the percentage of positive cells or the number of receptors per cell and by simultaneously changing these two indicators in opposite directions . however , ra patients in the acute phase had higher levels of type 1 receptors compared with healthy individuals . we revealed the correlations between parameters of soluble mediators in ra patients who had responded to treatment and those in the same patients in the acute phase ( reduced serum levels of il-1r1 and rail-1 ) , which can be used to attest to treatment effectiveness . the resulting data are indicative of differences in expression of il-1 receptors in various subpopulations of immunocompetent cells in normal cells and in pathology . additionally , they show changes both in indicators of mediator production accompanying inflammatory pathologies and in the system of receptor regulation on the cell surface . therefore , it is important to determine both the relative percentage of cells expressing receptors to immunomodulatory cytokines and the levels of membrane - bound receptors , because the density of expression is characterized by disease - induced changes that can not be detected when assessing the percentage of positive cells . it was found that oppositely directed changes in the density of expression of il-1 receptors type 1 and in the percentage of il-1r1 cells in b cells and monocytes take place , thus resulting in different mechanisms of regulation of cellular response to cytokines via changing the parameters of expression of different types of receptors .

exclusive breastfeeding is the practice of feeding infants only on breast milk without any additional food or drink , even water , for the first six months of life . exclusive breastfeeding enhances the overall benefits of breast milk , which is widely recognized as the ideal form of nutrition for infants . to achieve optimal growth , development , and health of infants , major national , global health , and professional organizations recommend exclusive breastfeeding of infants for the first six months of life , and continued breastfeeding along with appropriate complementary foods for up to two years of age or beyond . in addition to exclusive breastfeeding , experts recommend that breastfeeding be initiated within the first hour of an infant s life , it should be on demand that is as often as the child wants and without the use of bottles , teats , or pacifiers . the short and long - term health benefits of exclusive breastfeeding for infants and mothers have been well - reported . these benefits include protection from infectious diseases , reduction in the risks of chronic diseases , diabetes , asthma , lymphoma , leukemia , hodgkin s disease , gastrointestinal tract infection , atopic eczema , and improvement in cognitive functioning.[4 - 6 ] breast milk contains and provides all the nutrients an infant needs for healthy development ; it is safe and rich in antibodies that protect infants from the two leading causes of child mortality namely pneumonia and diarrhea worldwide ; and breast milk is readily available and affordable . optimal breastfeeding of all children aged 0 - 23 months could lead to an annual savings of about 800,000 under - five children s lives around the world . exclusive breastfeeding has also been associated with natural birth control for the first six months after birth , reduction in the risks of breast and ovarian cancers among mothers later in life , reduction in obesity rates , and faster return to their pre - pregnancy weights . national and global health efforts aimed at promoting exclusive breastfeeding have primarily focused on increasing and tracking the number of mothers around the world who exclusively breastfeed their infants and on individual - level determinants and effects of exclusive breastfeeding on specific health outcomes.[7,9 - 12 ] given this interest , many prior studies have focused on the benefits of exclusive breastfeeding in mitigating health care risks and preventing chronic and infectious diseases such as gastrointestinal problems , asthma , diabetes , obesity , leukemia , lymphoma , and reproductive - aged cancers . there is a paucity of recent studies examining the long - term , cross - national , and population - level impacts of exclusive breastfeeding on major global child health indicators such as under - five mortality rates . to our knowledge , the prior studies that examined the protective effects of exclusive breastfeeding on under - five mortality were based in single sub - saharan african countries and involved a sample of women.[13 - 16 ] investing all research examining increase in breastfeeding rates alone could be counterproductive given that , despite concerted efforts , only modest improvements have been achieved in increasing exclusive breastfeeding rates in several decades globally . in this study , we investigate the association between exclusive breastfeeding and under - five mortality , a leading child health indicator , using data from 57 low- and middle - income countries . first , we investigated whether the rates of exclusive breastfeeding were independently associated with under - five mortality . secondly , we investigated whether the associations , if any , identified in the above were attenuated or otherwise in the presence of other health systems - level factors in 57 countries in our study . in other words , we expected that under - five mortality rates would be lower in countries with higher rates of exclusive breastfeeding . understanding how exclusive breastfeeding rates and under - five mortality rates are associated in the presence of other population - level and health - systems factors in countries that share similar socioeconomic , demographic , and health characteristics is important . this information would be useful for policy and program planners in identifying areas of potential intervention that will make a difference in the lives of infants and mothers in their respective countries , and ultimately lead to increase in the uptake of exclusive breastfeeding . to quantify the association between exclusive breastfeeding and under - five mortality , we obtained data on indicators from multiple global health data sources for the 57- low and middle - income countries included in our analyses spanning 2006 to 2014 . the data on exclusive breastfeeding were from the global databases on infant and young child feeding . data on under - five mortality were from the world health statistics 2015 published by the world health organization ( who ) . data on the human development index ( hdi ) and gender inequality index ( gii ) were from the united nations 2014 human development report , while data on health - related indicators namely improved drinking water usage , sanitation usage , physician density , antenatal coverage , births attended , and healthcare expenditure were from the who s 2015 world health statistics . where necessary , we augmented missing data on few countries with data from comparable countries as determined by their gross national income ( gni ) . countries and study covariates were selected based on prior research , field experience , and the need to examine countries with similar socioeconomic and global health systems indicators . our dependent variable was under - five mortality which is defined as the probability of dying by age 5 per 1,000 live births . our independent variable was exclusive breastfeeding greater than or equal to 6 months which is defined as the percentage of infants 0 to 5 months of age who are fed exclusively with breast milk . our study covariates were eight national / health systems - level sociodemographic indicators : hdi , gii , improved drinking water usage , sanitation usage , physician density , antenatal coverage , births attended by trained health personnel , and health care expenditure . hdi is a composite measure of three basic human development indices , which includes a life expectancy index , an education index , and an income index . values for hdi vary between 0 and 1 , with 0 representing the lowest level of development and 1 representing the highest level . gii is an aggregate measure that depicts gender - based disadvantages based on three dimensions : reproductive health , empowerment , and the labor market . gii values vary between 0 and 1 , with 0 indicating equal faring between men and women . healthcare expenditure is the per capita total expenditure on health expressed in purchasing power parity ( ppp ) and is internationally - denominated to the united states dollar . improved drinking water usage is measured as the percentage of the population using an improved drinking water source and improved sanitation usage is measured as the percentage of the population using an improved sanitation facility . physician density is measured as the number of medical doctors per 10,000 population in a particular area . antenatal care coverage is the percentage of women between the ages of 15 and 49 with a live birth within a given time period that received four or more antenatal care visits during pregnancy . pearson correlation coefficients were computed to examine the bivariate associations between under - five mortality , exclusive breastfeeding , and other covariates . multivariate ordinary least squares ( ols ) regression models were used to determine the independent effects of exclusive breastfeeding and other covariates on under - five mortality rates . unstandardized regression coefficients ( b s ) were used to estimate the effect on child mortality associated with per unit change in exclusive breastfeeding and other independent variables . standardized regression coefficients ( s ) were used to estimate the relative impacts of the predictors on under - five mortality rates . percentage variance explained ( r ) determined the goodness of fit of the multivariate models . our dependent variable was under - five mortality which is defined as the probability of dying by age 5 per 1,000 live births . our independent variable was exclusive breastfeeding greater than or equal to 6 months which is defined as the percentage of infants 0 to 5 months of age who are fed exclusively with breast milk . our study covariates were eight national / health systems - level sociodemographic indicators : hdi , gii , improved drinking water usage , sanitation usage , physician density , antenatal coverage , births attended by trained health personnel , and health care expenditure . hdi is a composite measure of three basic human development indices , which includes a life expectancy index , an education index , and an income index . values for hdi vary between 0 and 1 , with 0 representing the lowest level of development and 1 representing the highest level . gii is an aggregate measure that depicts gender - based disadvantages based on three dimensions : reproductive health , empowerment , and the labor market . gii values vary between 0 and 1 , with 0 indicating equal faring between men and women . healthcare expenditure is the per capita total expenditure on health expressed in purchasing power parity ( ppp ) and is internationally - denominated to the united states dollar . improved drinking water usage is measured as the percentage of the population using an improved drinking water source and improved sanitation usage is measured as the percentage of the population using an improved sanitation facility . physician density is measured as the number of medical doctors per 10,000 population in a particular area . antenatal care coverage is the percentage of women between the ages of 15 and 49 with a live birth within a given time period that received four or more antenatal care visits during pregnancy . pearson correlation coefficients were computed to examine the bivariate associations between under - five mortality , exclusive breastfeeding , and other covariates . multivariate ordinary least squares ( ols ) regression models were used to determine the independent effects of exclusive breastfeeding and other covariates on under - five mortality rates . unstandardized regression coefficients ( b s ) were used to estimate the effect on child mortality associated with per unit change in exclusive breastfeeding and other independent variables . standardized regression coefficients ( s ) were used to estimate the relative impacts of the predictors on under - five mortality rates . percentage variance explained ( r ) determined the goodness of fit of the multivariate models . table 1 presents the 57 countries included in the study , while figure 1 presents the prevalence of exclusive breastfeeding and under - five mortality rates across the countries . the average under - five mortality rate across the 57 countries was 69.0 child deaths under age 5 per 1,000 live births . singapore had the lowest under - five mortality rate ( 2.8 deaths per 1,000 live births ) , while angola had the highest under - five mortality rate ( 167.4 ) . majority of sub - saharan african countries had higher under - five mortality rates than their asian counterparts . djibouti had the lowest exclusive breastfeeding rate of 1% while rwanda had the highest rate of exclusive breastfeeding ( 85% ) followed by cambodia ( 74% ) and malawi ( 71% ) . although not shown here , data for the study covariates varied across the countries . in general , asian countries had an hdi greater than 0.5 , with singapore having the highest hdi of 0.90 among all 57 countries in our analysis . sub - saharan african countries of niger republic , the democratic republic of congo , and central african republic had the lowest hdi at 0.34 . nigeria and singapore had very low gii values of 0.07 and 0.09 , respectively , while niger and chad had the highest gii value of 0.71 . singapore and malaysia both had 100% of their populations using an improved drinking water source , while the democratic republic of congo and mozambique had the lowest improved drinking water usage at 46% and 49% , respectively . very low levels of sanitation usage were observed among many of the sub - saharan african countries ; niger republic had the lowest percentage at 9% followed by malawi at 10% . all african countries except algeria , tunisia , libya , and egypt had physician densities of less than 10 , meaning that , for every 10,000 persons in these countries , there were fewer than 10 available physicians . many of the sub - saharan african countries had an even lower number of less than one available physician per 10,000 persons . egypt had the highest physician density at 28.3 among all 57 countries , followed by singapore at 19.5 . antenatal care coverage was highest for thailand and brunei ( 93% ) and lowest for djibouti ( 7% ) . the percentage of births attended by skilled health personnel was 100% for 5 of the 57 countries : libya , singapore , china , thailand , and brunei . countries included in the analysis , according to world geographical region ( n = 57 ) notes : n = number of countries . based on the united nations geographical regions exclusive breastfeeding and under - five mortality in 57 low - and - middle - income countries . sources : global databases on infant and young child feeding , 2015 ; world health statistics , 2015 . descriptive statistics of the study variables of all countries ( n = 57 ) note : n = number of countries , se mean = standard error of mean , sd = standard deviation table 3 presents the results of our bivariate correlation analysis . there was a significant positive relationship between under - five mortality rate and gii ( r = 0.41 , p < 0.01 ) . this indicated that a higher under - five mortality rate was associated with increased gender inequality . there was a significant inverse relationship between under - five mortality rate and improved drinking water usage ( r = -0.57 , p<0.01 ) , sanitation usage ( r = -0.62 , p<0.01 ) , physician density ( r = -0.65 , p<0.01 ) , antenatal coverage ( r = -0.51 , p<0.01 ) , and births attended ( r = -0.61 , p<0.01 ) . a lower under - five mortality rate was associated with higher improved drinking water usage , sanitation usage , physician density , and births attended . in addition , the correlation between hdi and the under - five mortality rate was very strong , as indicated by the correlation coefficient of -0.81 . there was no significant linear relationship between under - five mortality rate and exclusive breastfeeding ( r = 0.05 , p > 0.05 ) . bivariate correlations showing the relationship between exclusive breastfeeding , human development , health systems , and socioeconomic risk factors and under - five mortality in selected developing countries , 2006 - 2014 ( n=57 countries ) note : statistically significant at p<0.05 level table 4 presents the results of our multivariate regression analysis . in multivariate model 1 , one unit increase ( i.e. , one percentage point increase ) in exclusive breastfeeding was associated with 0.5 units decrease in under - five mortality rate . equivalently , a 10 percentage - points increase in exclusive breastfeeding was associated with a reduction of 5 child deaths per 1,000 live births . a one unit increase in hdi was associated with a decrease of 231 under - five deaths per 1,000 live births . one percentage point increase in improved drinking water source was associated with 0.47 units decrease in under - five mortality . in multivariate model 1 , a comparison of b s ( standardized regression coefficients ) indicated that hdi had the strongest effect on under - five mortality , followed by exclusive breastfeeding and improved drinking water . in multivariate model 2 , a $ 100 us ppp increase in per capita health care expenditure was associated with a decrease of 2 under - five child deaths per 1,000 live births . in multivariate model 2 , physician density had the largest impact on child mortality , followed by improved drinking water source , exclusive breastfeeding , and healthcare expenditure . covariates in the multivariate models 1 and 2 explained 72% and 54% of the cross - national variance in under - five mortality , respectively . multivariate regression models showing the effects of exclusive breastfeeding , health systems and socioeconomic risk factors on under - five mortality in selected low - and - middle - income countries , 2006 - 2014 ( n=57 countries ) notes : b = unstandardized regression coefficient ; = standardized regression coefficient ; r = percentage variance explained ; vif = variance inflation factor ; p0.05 using one - tailed t - test table 1 presents the 57 countries included in the study , while figure 1 presents the prevalence of exclusive breastfeeding and under - five mortality rates across the countries . the average under - five mortality rate across the 57 countries was 69.0 child deaths under age 5 per 1,000 live births . singapore had the lowest under - five mortality rate ( 2.8 deaths per 1,000 live births ) , while angola had the highest under - five mortality rate ( 167.4 ) . majority of sub - saharan african countries had higher under - five mortality rates than their asian counterparts . djibouti had the lowest exclusive breastfeeding rate of 1% while rwanda had the highest rate of exclusive breastfeeding ( 85% ) followed by cambodia ( 74% ) and malawi ( 71% ) . although not shown here , data for the study covariates varied across the countries . in general , asian countries had an hdi greater than 0.5 , with singapore having the highest hdi of 0.90 among all 57 countries in our analysis . sub - saharan african countries of niger republic , the democratic republic of congo , and central african republic had the lowest hdi at 0.34 . nigeria and singapore had very low gii values of 0.07 and 0.09 , respectively , while niger and chad had the highest gii value of 0.71 . singapore and malaysia both had 100% of their populations using an improved drinking water source , while the democratic republic of congo and mozambique had the lowest improved drinking water usage at 46% and 49% , respectively . very low levels of sanitation usage were observed among many of the sub - saharan african countries ; niger republic had the lowest percentage at 9% followed by malawi at 10% . all african countries except algeria , tunisia , libya , and egypt had physician densities of less than 10 , meaning that , for every 10,000 persons in these countries , there were fewer than 10 available physicians . many of the sub - saharan african countries had an even lower number of less than one available physician per 10,000 persons . egypt had the highest physician density at 28.3 among all 57 countries , followed by singapore at 19.5 . antenatal care coverage was highest for thailand and brunei ( 93% ) and lowest for djibouti ( 7% ) . the percentage of births attended by skilled health personnel was 100% for 5 of the 57 countries : libya , singapore , china , thailand , and brunei . countries included in the analysis , according to world geographical region ( n = 57 ) notes : n = number of countries . based on the united nations geographical regions exclusive breastfeeding and under - five mortality in 57 low - and - middle - income countries . sources : global databases on infant and young child feeding , 2015 ; world health statistics , 2015 . descriptive statistics of the study variables of all countries ( n = 57 ) note : n = number of countries , se mean = standard error of mean , sd = standard deviation table 3 presents the results of our bivariate correlation analysis . there was a significant positive relationship between under - five mortality rate and gii ( r = 0.41 , p < 0.01 ) . this indicated that a higher under - five mortality rate was associated with increased gender inequality . there was a significant inverse relationship between under - five mortality rate and improved drinking water usage ( r = -0.57 , p<0.01 ) , sanitation usage ( r = -0.62 , p<0.01 ) , physician density ( r = -0.65 , p<0.01 ) , antenatal coverage ( r = -0.51 , p<0.01 ) , and births attended ( r = -0.61 , p<0.01 ) . a lower under - five mortality rate was associated with higher improved drinking water usage , sanitation usage , physician density , and births attended . in addition , the correlation between hdi and the under - five mortality rate was very strong , as indicated by the correlation coefficient of -0.81 . there was no significant linear relationship between under - five mortality rate and exclusive breastfeeding ( r = 0.05 , p > 0.05 ) . bivariate correlations showing the relationship between exclusive breastfeeding , human development , health systems , and socioeconomic risk factors and under - five mortality in selected developing countries , 2006 - 2014 ( n=57 countries ) note : statistically significant at p<0.05 level table 4 presents the results of our multivariate regression analysis . in multivariate model 1 , one unit increase ( i.e. , one percentage point increase ) in exclusive breastfeeding was associated with 0.5 units decrease in under - five mortality rate . equivalently , a 10 percentage - points increase in exclusive breastfeeding was associated with a reduction of 5 child deaths per 1,000 live births . a one unit increase in hdi was associated with a decrease of 231 under - five deaths per 1,000 live births . one percentage point increase in improved drinking water source was associated with 0.47 units decrease in under - five mortality . in multivariate model 1 , a comparison of b s ( standardized regression coefficients ) indicated that hdi had the strongest effect on under - five mortality , followed by exclusive breastfeeding and improved drinking water . in multivariate model 2 , a $ 100 us ppp increase in per capita health care expenditure was associated with a decrease of 2 under - five child deaths per 1,000 live births . in multivariate model 2 , physician density had the largest impact on child mortality , followed by improved drinking water source , exclusive breastfeeding , and healthcare expenditure . covariates in the multivariate models 1 and 2 explained 72% and 54% of the cross - national variance in under - five mortality , respectively . multivariate regression models showing the effects of exclusive breastfeeding , health systems and socioeconomic risk factors on under - five mortality in selected low - and - middle - income countries , 2006 - 2014 ( n=57 countries ) notes : b = unstandardized regression coefficient ; = standardized regression coefficient ; r = percentage variance explained ; vif = variance inflation factor ; p0.05 using one - tailed t - test using multiple global health databases , we have quantified the associations between under - five mortality and exclusive breastfeeding in the presence of key socioeconomic and policy indicators in developing countries . our findings support prior country - based studies in ghana , india , and tanzania that reported the protective effects of exclusive breastfeeding on under - five mortality.[13 - 15,22 ] however , our study adds upon these studies by providing quantifiable evidence across multiple countries using the latest cross - national sociodemographic , human development , and health care data . our findings on the overwhelming effect of hdi on population - level health indicators in general and under - five mortality in particular are consistent with earlier findings . prior studies have identified some of the social and economic barriers to exclusive breastfeeding in developing countries at the individual level including unemployment , low income , lack of breastfeeding friendly workplaces , and traditional practices . our study goes beyond the individual - level barriers and demonstrates the diversity of mediating macro - socioeconomic policy factors that affect the ultimate benefit of exclusive breastfeeding in reducing under - five mortality . to the best of our knowledge , this is one of the first studies to empirically document the direct impact of macro socioeconomic factors and exclusive breastfeeding on under - five mortality across various developing countries . under - five mortality rate is a key indicator of infant and child health as well as reflection of the socioeconomic and surrounding health and healthcare conditions of a child s environment . as exclusive breastfeeding is thought to directly impact a child s nutrition and subsequent survival , we performed a study to examine the possible effect of exclusive breastfeeding on the under - five mortality rate . the most recent global health statistics were used to assess the relationship between exclusive breastfeeding and under - five mortality rates in the presence of several sociodemographic and health indicators in 57 low- and middle - income countries . this is an ecologic cross - sectional study ; thus , the associations we observed in the study may not be causally - related . some of the observed associations at the cross - national level may not hold at the individual level . however , given that a health indicator such as under - five mortality can not be subjected to a randomized controlled trial , due to ethical concerns , ecological studies remain one of the most - robust methodologies for investigating critical issue such as national variations in under - five mortality rates . further studies are needed to investigate whether the associations observed in this study hold for specific countries and for more advanced , industrialized countries . subsequent research should also explore the quantifiable link between exclusive breastfeeding and other child health indicators such as infant mortality , stunting , and educational achievement . in conclusion , this study found that in the presence of enabling health systems and sociodemographic indicators such as hdi , physician density , and healthcare expenditure , the association between exclusive breastfeeding and the under - five mortality rate in the 57 countries investigated becomes substantial and statistically significant . these findings suggest that the ability of exclusive breastfeeding to impact the under - five mortality rate is determined , in part , upon other sociodemographic and health factors and , in the presence of these other factors , the effect that exclusive breastfeeding has on the under - five mortality rate is increased . our findings support the importance of a health system as the overall hub upon which health governance , financing , service delivery , health workforce , information and medicines and vaccines and other technologies work together in improving health outcomes for all citizens . the results of this study indicate that while exclusive breastfeeding is critical in improving child survival , its benefits to the overall improvement of child health , as measured by under - five mortality , are maximized in the presence of a robust environment of functional health systems and capacities . there also needs to be a supportive environment and overall health system of improved sociodemographic and health care factors that will help to drive the benefits of exclusive breastfeeding for the first 6 months of an infant s life . from a public health viewpoint , increasing the awareness of the importance of having supporting environment to further enhance the positive effects of exclusive breastfeeding may help policymakers and other healthcare decision makers improve the policy and programs that deal with infant and child health outcomes . our study findings are essential to better inform programs and policies aimed at not only increasing exclusive breastfeeding and decreasing under - five mortality , but also improving the lives of children and mothers in these developing countries . under - five mortality rate is a key indicator of infant and child health as well as reflection of the socioeconomic and surrounding health and healthcare conditions of a child s environment . human development index , physician density , healthcare expenditure , and exclusive breastfeeding are significantly associated with under - five mortality.a one unit increase in hdi was associated with a reduction of 231 under - five deaths per 1,000 live births . one unit increase in the number of physicians per 10,000 population was associated with a 2.8 units decrease in under - five mortality.a $ 100 increase in the per capita total expenditure on health was associated with a decrease of 2 child deaths per 1,000 live births , while a 10 percentage - points increase in exclusive breastfeeding was associated with a reduction of 5 child deaths per 1,000 live births . under - five mortality rate is a key indicator of infant and child health as well as reflection of the socioeconomic and surrounding health and healthcare conditions of a child s environment . human development index , physician density , healthcare expenditure , and exclusive breastfeeding are significantly associated with under - five mortality . a one unit increase in hdi was associated with a reduction of 231 under - five deaths per 1,000 live births . one unit increase in the number of physicians per 10,000 population was associated with a 2.8 units decrease in under - five mortality . a $ 100 increase in the per capita total expenditure on health was associated with a decrease of 2 child deaths per 1,000 live births , while a 10 percentage - points increase in exclusive breastfeeding was associated with a reduction of 5 child deaths per 1,000 live births .

background : few studies have examined the long - term , cross - national , and population - level impacts of exclusive breastfeeding on major global child health indicators . we investigated the overall and independent associations between exclusive breastfeeding and under - five mortality in 57 low- and - middle - income countries.methods:data were obtained from the latest world health organization , united nations , and united nations children s fund databases for 57 low- and middle - income countries covering the periods 2006 - 2014 . multivariate linear regression was used to estimate the effects of exclusive breastfeeding on under - five mortality after adjusting for differences in socioeconomic , demographic , and health - related factors.results:in multivariate models , exclusive breastfeeding was independently associated with under - five mortality after adjusting for sociodemographic and health systems - related factors . a 10 percentage - points increase in exclusive breastfeeding was associated with a reduction of 5 child deaths per 1,000 live births . a one - unit increase in human development index was associated with a decrease of 231 under - five child deaths per 1,000 live births . a $ 100 increase in per capita health care expenditure was associated with a decrease of 2 child deaths per 1,000 live births . one unit increase in physician density was associated with 2.8 units decrease in the under - five mortality rate.conclusions and global health implications : population - level health system and socioeconomic factors exert considerable effect on the association between exclusive breastfeeding and under - five mortality . given that the health policy and socioeconomic indicators shown to influence exclusive breastfeeding and under - five mortality are modifiable , policy makers could potentially target specific policies and programs to address national - level deficiencies in these sectors to reduce under - five mortality in their countries .

Introduction Methods Study variables Statistical analysis Results Descriptive statistics Bivariate analysis Multivariate regression analysis Discussion Conclusions and Global Health Implications

exclusive breastfeeding enhances the overall benefits of breast milk , which is widely recognized as the ideal form of nutrition for infants . to achieve optimal growth , development , and health of infants , major national , global health , and professional organizations recommend exclusive breastfeeding of infants for the first six months of life , and continued breastfeeding along with appropriate complementary foods for up to two years of age or beyond . the short and long - term health benefits of exclusive breastfeeding for infants and mothers have been well - reported . these benefits include protection from infectious diseases , reduction in the risks of chronic diseases , diabetes , asthma , lymphoma , leukemia , hodgkin s disease , gastrointestinal tract infection , atopic eczema , and improvement in cognitive functioning. optimal breastfeeding of all children aged 0 - 23 months could lead to an annual savings of about 800,000 under - five children s lives around the world . exclusive breastfeeding has also been associated with natural birth control for the first six months after birth , reduction in the risks of breast and ovarian cancers among mothers later in life , reduction in obesity rates , and faster return to their pre - pregnancy weights . national and global health efforts aimed at promoting exclusive breastfeeding have primarily focused on increasing and tracking the number of mothers around the world who exclusively breastfeed their infants and on individual - level determinants and effects of exclusive breastfeeding on specific health outcomes. [7,9 - 12 ] given this interest , many prior studies have focused on the benefits of exclusive breastfeeding in mitigating health care risks and preventing chronic and infectious diseases such as gastrointestinal problems , asthma , diabetes , obesity , leukemia , lymphoma , and reproductive - aged cancers . there is a paucity of recent studies examining the long - term , cross - national , and population - level impacts of exclusive breastfeeding on major global child health indicators such as under - five mortality rates . to our knowledge , the prior studies that examined the protective effects of exclusive breastfeeding on under - five mortality were based in single sub - saharan african countries and involved a sample of women. [13 - 16 ] investing all research examining increase in breastfeeding rates alone could be counterproductive given that , despite concerted efforts , only modest improvements have been achieved in increasing exclusive breastfeeding rates in several decades globally . in this study , we investigate the association between exclusive breastfeeding and under - five mortality , a leading child health indicator , using data from 57 low- and middle - income countries . first , we investigated whether the rates of exclusive breastfeeding were independently associated with under - five mortality . secondly , we investigated whether the associations , if any , identified in the above were attenuated or otherwise in the presence of other health systems - level factors in 57 countries in our study . in other words , we expected that under - five mortality rates would be lower in countries with higher rates of exclusive breastfeeding . understanding how exclusive breastfeeding rates and under - five mortality rates are associated in the presence of other population - level and health - systems factors in countries that share similar socioeconomic , demographic , and health characteristics is important . this information would be useful for policy and program planners in identifying areas of potential intervention that will make a difference in the lives of infants and mothers in their respective countries , and ultimately lead to increase in the uptake of exclusive breastfeeding . to quantify the association between exclusive breastfeeding and under - five mortality , we obtained data on indicators from multiple global health data sources for the 57- low and middle - income countries included in our analyses spanning 2006 to 2014 . the data on exclusive breastfeeding were from the global databases on infant and young child feeding . data on under - five mortality were from the world health statistics 2015 published by the world health organization ( who ) . data on the human development index ( hdi ) and gender inequality index ( gii ) were from the united nations 2014 human development report , while data on health - related indicators namely improved drinking water usage , sanitation usage , physician density , antenatal coverage , births attended , and healthcare expenditure were from the who s 2015 world health statistics . countries and study covariates were selected based on prior research , field experience , and the need to examine countries with similar socioeconomic and global health systems indicators . our dependent variable was under - five mortality which is defined as the probability of dying by age 5 per 1,000 live births . our study covariates were eight national / health systems - level sociodemographic indicators : hdi , gii , improved drinking water usage , sanitation usage , physician density , antenatal coverage , births attended by trained health personnel , and health care expenditure . hdi is a composite measure of three basic human development indices , which includes a life expectancy index , an education index , and an income index . pearson correlation coefficients were computed to examine the bivariate associations between under - five mortality , exclusive breastfeeding , and other covariates . multivariate ordinary least squares ( ols ) regression models were used to determine the independent effects of exclusive breastfeeding and other covariates on under - five mortality rates . unstandardized regression coefficients ( b s ) were used to estimate the effect on child mortality associated with per unit change in exclusive breastfeeding and other independent variables . standardized regression coefficients ( s ) were used to estimate the relative impacts of the predictors on under - five mortality rates . our dependent variable was under - five mortality which is defined as the probability of dying by age 5 per 1,000 live births . our study covariates were eight national / health systems - level sociodemographic indicators : hdi , gii , improved drinking water usage , sanitation usage , physician density , antenatal coverage , births attended by trained health personnel , and health care expenditure . hdi is a composite measure of three basic human development indices , which includes a life expectancy index , an education index , and an income index . pearson correlation coefficients were computed to examine the bivariate associations between under - five mortality , exclusive breastfeeding , and other covariates . multivariate ordinary least squares ( ols ) regression models were used to determine the independent effects of exclusive breastfeeding and other covariates on under - five mortality rates . unstandardized regression coefficients ( b s ) were used to estimate the effect on child mortality associated with per unit change in exclusive breastfeeding and other independent variables . standardized regression coefficients ( s ) were used to estimate the relative impacts of the predictors on under - five mortality rates . table 1 presents the 57 countries included in the study , while figure 1 presents the prevalence of exclusive breastfeeding and under - five mortality rates across the countries . the average under - five mortality rate across the 57 countries was 69.0 child deaths under age 5 per 1,000 live births . singapore had the lowest under - five mortality rate ( 2.8 deaths per 1,000 live births ) , while angola had the highest under - five mortality rate ( 167.4 ) . majority of sub - saharan african countries had higher under - five mortality rates than their asian counterparts . djibouti had the lowest exclusive breastfeeding rate of 1% while rwanda had the highest rate of exclusive breastfeeding ( 85% ) followed by cambodia ( 74% ) and malawi ( 71% ) . all african countries except algeria , tunisia , libya , and egypt had physician densities of less than 10 , meaning that , for every 10,000 persons in these countries , there were fewer than 10 available physicians . based on the united nations geographical regions exclusive breastfeeding and under - five mortality in 57 low - and - middle - income countries . there was a significant positive relationship between under - five mortality rate and gii ( r = 0.41 , p < 0.01 ) . this indicated that a higher under - five mortality rate was associated with increased gender inequality . there was a significant inverse relationship between under - five mortality rate and improved drinking water usage ( r = -0.57 , p<0.01 ) , sanitation usage ( r = -0.62 , p<0.01 ) , physician density ( r = -0.65 , p<0.01 ) , antenatal coverage ( r = -0.51 , p<0.01 ) , and births attended ( r = -0.61 , p<0.01 ) . a lower under - five mortality rate was associated with higher improved drinking water usage , sanitation usage , physician density , and births attended . in addition , the correlation between hdi and the under - five mortality rate was very strong , as indicated by the correlation coefficient of -0.81 . there was no significant linear relationship between under - five mortality rate and exclusive breastfeeding ( r = 0.05 , p > 0.05 ) . bivariate correlations showing the relationship between exclusive breastfeeding , human development , health systems , and socioeconomic risk factors and under - five mortality in selected developing countries , 2006 - 2014 ( n=57 countries ) note : statistically significant at p<0.05 level table 4 presents the results of our multivariate regression analysis . in multivariate model 1 , one unit increase ( i.e. , one percentage point increase ) in exclusive breastfeeding was associated with 0.5 units decrease in under - five mortality rate . equivalently , a 10 percentage - points increase in exclusive breastfeeding was associated with a reduction of 5 child deaths per 1,000 live births . a one unit increase in hdi was associated with a decrease of 231 under - five deaths per 1,000 live births . one percentage point increase in improved drinking water source was associated with 0.47 units decrease in under - five mortality . in multivariate model 1 , a comparison of b s ( standardized regression coefficients ) indicated that hdi had the strongest effect on under - five mortality , followed by exclusive breastfeeding and improved drinking water . in multivariate model 2 , a $ 100 us ppp increase in per capita health care expenditure was associated with a decrease of 2 under - five child deaths per 1,000 live births . in multivariate model 2 , physician density had the largest impact on child mortality , followed by improved drinking water source , exclusive breastfeeding , and healthcare expenditure . covariates in the multivariate models 1 and 2 explained 72% and 54% of the cross - national variance in under - five mortality , respectively . multivariate regression models showing the effects of exclusive breastfeeding , health systems and socioeconomic risk factors on under - five mortality in selected low - and - middle - income countries , 2006 - 2014 ( n=57 countries ) notes : b = unstandardized regression coefficient ; = standardized regression coefficient ; r = percentage variance explained ; vif = variance inflation factor ; p0.05 using one - tailed t - test table 1 presents the 57 countries included in the study , while figure 1 presents the prevalence of exclusive breastfeeding and under - five mortality rates across the countries . the average under - five mortality rate across the 57 countries was 69.0 child deaths under age 5 per 1,000 live births . singapore had the lowest under - five mortality rate ( 2.8 deaths per 1,000 live births ) , while angola had the highest under - five mortality rate ( 167.4 ) . majority of sub - saharan african countries had higher under - five mortality rates than their asian counterparts . all african countries except algeria , tunisia , libya , and egypt had physician densities of less than 10 , meaning that , for every 10,000 persons in these countries , there were fewer than 10 available physicians . based on the united nations geographical regions exclusive breastfeeding and under - five mortality in 57 low - and - middle - income countries . there was a significant positive relationship between under - five mortality rate and gii ( r = 0.41 , p < 0.01 ) . this indicated that a higher under - five mortality rate was associated with increased gender inequality . there was a significant inverse relationship between under - five mortality rate and improved drinking water usage ( r = -0.57 , p<0.01 ) , sanitation usage ( r = -0.62 , p<0.01 ) , physician density ( r = -0.65 , p<0.01 ) , antenatal coverage ( r = -0.51 , p<0.01 ) , and births attended ( r = -0.61 , p<0.01 ) . a lower under - five mortality rate was associated with higher improved drinking water usage , sanitation usage , physician density , and births attended . in addition , the correlation between hdi and the under - five mortality rate was very strong , as indicated by the correlation coefficient of -0.81 . there was no significant linear relationship between under - five mortality rate and exclusive breastfeeding ( r = 0.05 , p > 0.05 ) . bivariate correlations showing the relationship between exclusive breastfeeding , human development , health systems , and socioeconomic risk factors and under - five mortality in selected developing countries , 2006 - 2014 ( n=57 countries ) note : statistically significant at p<0.05 level table 4 presents the results of our multivariate regression analysis . , one percentage point increase ) in exclusive breastfeeding was associated with 0.5 units decrease in under - five mortality rate . equivalently , a 10 percentage - points increase in exclusive breastfeeding was associated with a reduction of 5 child deaths per 1,000 live births . a one unit increase in hdi was associated with a decrease of 231 under - five deaths per 1,000 live births . one percentage point increase in improved drinking water source was associated with 0.47 units decrease in under - five mortality . in multivariate model 1 , a comparison of b s ( standardized regression coefficients ) indicated that hdi had the strongest effect on under - five mortality , followed by exclusive breastfeeding and improved drinking water . in multivariate model 2 , a $ 100 us ppp increase in per capita health care expenditure was associated with a decrease of 2 under - five child deaths per 1,000 live births . in multivariate model 2 , physician density had the largest impact on child mortality , followed by improved drinking water source , exclusive breastfeeding , and healthcare expenditure . covariates in the multivariate models 1 and 2 explained 72% and 54% of the cross - national variance in under - five mortality , respectively . multivariate regression models showing the effects of exclusive breastfeeding , health systems and socioeconomic risk factors on under - five mortality in selected low - and - middle - income countries , 2006 - 2014 ( n=57 countries ) notes : b = unstandardized regression coefficient ; = standardized regression coefficient ; r = percentage variance explained ; vif = variance inflation factor ; p0.05 using one - tailed t - test using multiple global health databases , we have quantified the associations between under - five mortality and exclusive breastfeeding in the presence of key socioeconomic and policy indicators in developing countries . our findings support prior country - based studies in ghana , india , and tanzania that reported the protective effects of exclusive breastfeeding on under - five mortality. [13 - 15,22 ] however , our study adds upon these studies by providing quantifiable evidence across multiple countries using the latest cross - national sociodemographic , human development , and health care data . our findings on the overwhelming effect of hdi on population - level health indicators in general and under - five mortality in particular are consistent with earlier findings . prior studies have identified some of the social and economic barriers to exclusive breastfeeding in developing countries at the individual level including unemployment , low income , lack of breastfeeding friendly workplaces , and traditional practices . our study goes beyond the individual - level barriers and demonstrates the diversity of mediating macro - socioeconomic policy factors that affect the ultimate benefit of exclusive breastfeeding in reducing under - five mortality . to the best of our knowledge , this is one of the first studies to empirically document the direct impact of macro socioeconomic factors and exclusive breastfeeding on under - five mortality across various developing countries . under - five mortality rate is a key indicator of infant and child health as well as reflection of the socioeconomic and surrounding health and healthcare conditions of a child s environment . as exclusive breastfeeding is thought to directly impact a child s nutrition and subsequent survival , we performed a study to examine the possible effect of exclusive breastfeeding on the under - five mortality rate . the most recent global health statistics were used to assess the relationship between exclusive breastfeeding and under - five mortality rates in the presence of several sociodemographic and health indicators in 57 low- and middle - income countries . this is an ecologic cross - sectional study ; thus , the associations we observed in the study may not be causally - related . however , given that a health indicator such as under - five mortality can not be subjected to a randomized controlled trial , due to ethical concerns , ecological studies remain one of the most - robust methodologies for investigating critical issue such as national variations in under - five mortality rates . subsequent research should also explore the quantifiable link between exclusive breastfeeding and other child health indicators such as infant mortality , stunting , and educational achievement . in conclusion , this study found that in the presence of enabling health systems and sociodemographic indicators such as hdi , physician density , and healthcare expenditure , the association between exclusive breastfeeding and the under - five mortality rate in the 57 countries investigated becomes substantial and statistically significant . these findings suggest that the ability of exclusive breastfeeding to impact the under - five mortality rate is determined , in part , upon other sociodemographic and health factors and , in the presence of these other factors , the effect that exclusive breastfeeding has on the under - five mortality rate is increased . our findings support the importance of a health system as the overall hub upon which health governance , financing , service delivery , health workforce , information and medicines and vaccines and other technologies work together in improving health outcomes for all citizens . the results of this study indicate that while exclusive breastfeeding is critical in improving child survival , its benefits to the overall improvement of child health , as measured by under - five mortality , are maximized in the presence of a robust environment of functional health systems and capacities . there also needs to be a supportive environment and overall health system of improved sociodemographic and health care factors that will help to drive the benefits of exclusive breastfeeding for the first 6 months of an infant s life . from a public health viewpoint , increasing the awareness of the importance of having supporting environment to further enhance the positive effects of exclusive breastfeeding may help policymakers and other healthcare decision makers improve the policy and programs that deal with infant and child health outcomes . our study findings are essential to better inform programs and policies aimed at not only increasing exclusive breastfeeding and decreasing under - five mortality , but also improving the lives of children and mothers in these developing countries . under - five mortality rate is a key indicator of infant and child health as well as reflection of the socioeconomic and surrounding health and healthcare conditions of a child s environment . human development index , physician density , healthcare expenditure , and exclusive breastfeeding are significantly associated with under - five mortality.a one unit increase in hdi was associated with a reduction of 231 under - five deaths per 1,000 live births . one unit increase in the number of physicians per 10,000 population was associated with a 2.8 units decrease in under - five mortality.a $ 100 increase in the per capita total expenditure on health was associated with a decrease of 2 child deaths per 1,000 live births , while a 10 percentage - points increase in exclusive breastfeeding was associated with a reduction of 5 child deaths per 1,000 live births . under - five mortality rate is a key indicator of infant and child health as well as reflection of the socioeconomic and surrounding health and healthcare conditions of a child s environment . human development index , physician density , healthcare expenditure , and exclusive breastfeeding are significantly associated with under - five mortality . a one unit increase in hdi was associated with a reduction of 231 under - five deaths per 1,000 live births . one unit increase in the number of physicians per 10,000 population was associated with a 2.8 units decrease in under - five mortality . a $ 100 increase in the per capita total expenditure on health was associated with a decrease of 2 child deaths per 1,000 live births , while a 10 percentage - points increase in exclusive breastfeeding was associated with a reduction of 5 child deaths per 1,000 live births .

the hippocampal place cells are thought to collectively form a representation of space , known as a cognitive map , because of their spatially localized firing , which occurs in patches known as place fields ( figure 1(a ) ) . one source of spatial inputs to place cells is the entorhinal grid cells , one synapse upstream , whose activity forms a regular array of firing fields suggestive of an intrinsic odometric ( distance - measuring ) process , which may convey metric information to place cells and allow them to position their place fields accurately in space . the place and grid cells are an excellent model system with which to study the formation and architecture of cognitive knowledge structures . place and grid cells use external environmental cues to anchor their activity to the real world , as evidenced by the fact that their activity appears bound to the local environmental walls [ 2 , 4 , 5 ] and reacts to changes in the environment . however , firing patterns are then stabilized and maintained by internal network dynamics so that activity can be self - sustaining and coherent across the network . these internal dynamics are often considered to arise from the operation of attractor processes [ 79 ] , which are processes that arise from mutually interconnected neurons that collectively have a tendency to find stable states . two kinds of attractors have been proposed to explain place cell behavior : discrete and continuous . the purpose of this paper is to review the evidence for these two attractor types in the hippocampal network and then to explore a phenomenon that can not be easily accounted for by attractors , known as partial remapping . finally , a model will be described that may be able to explain how both attractor dynamics and partial remapping can co - exist in the same network . one of the earliest and most striking observations concerning the place cell representation was the way that the cells can suddenly and collectively alter their activity from one pattern to another , a process known as remapping ( figure 1(b ) ) . this phenomenon led to proposals that the pattern of activity arises from cooperative activity among all involved place neurons , perhaps exerted via the recurrent synapses in the highly interconnected ca3 network . the attractor hypothesis built upon earlier ideas that the hippocampal ca3 network functions as an autoassociative memory [ 1012 ] . attractor networks are a special case of autoassociative memory , and an attractor 's defining characteristic is the existence of stable states , caused by the mutual excitation of neurons within the network , towards which the system gravitates when it is sufficiently close . the process of moving towards and settling into a stable state is what is meant by anatomical and physiological observations of place cells suggest the operation of two kinds of attractor dynamics : discrete and continuous . discrete attractor dynamics enable the system to resist small changes in sensory input but respond collectively and coherently to large ones , while continuous dynamics enable the system to move smoothly from one state to the next as the animal moves through space . these two attractor systems clearly must either be colocalized on the same neurons or else be separate but interacting , since one accounts for the population of place cells active at a given moment and the other for the progression of activity from one set to the next as the animal moves . one possibility , discussed later , is that the source of the discrete attractor dynamics may lie in the place cell network itself [ 79 , 14 ] , and the continuous dynamics may originate upstream in the entorhinal grid cell network . in a discrete attractor network , the possible states are clearly separable , and when the system moves from one state to another , it seems to do so abruptly . the separate states of a discrete attractor are often conceptualized as hollows in an undulating energy landscape ( figure 1(d ) ) into which the system ( represented as a ball ) tends to gravitate ( i.e. , to be attracted to ) . the hollows , also called basins , are low - energy states , but to move from one hollow to the next , the ball requires a substantial perturbation : a small push will not cause it to change basins / states . the states are imprinted onto the network by appropriate modulation of the connection strengths between the neurons in the network . place cell remapping was initially conceptualized as being a sudden transition of the place cell network from one state to the next following the large perturbation arising from environmental change , an idea that has been very influential . experimental evidence for attractor dynamics in the place cell network was initially provided by observations of remapping , but stronger evidence came from a study by wills et al . , who showed that incremental changes in the squareness or circularity of an enclosure would produce no change in place cell activity until the cumulative changes became sufficiently great , at which point the whole system would suddenly switch to the other pattern ( figure 1(c ) ) . interestingly , attractor dynamics do not seem to be invariable observations : for example , they were not seen by leutgeb et al . , who found , by contrast , that gradual transformation of environment shape induced gradual transition of firing patterns from one shape to the other . such differences in network behavior may be accounted for by the attractor landscapethe exact distribution of the imaginary hills and valleys in the attractor state - space which may have been sculpted by the past experience of the animal , via processes described below . attractor properties derive from the pattern of recurrent synaptic connections , and attractor networks can learn new information , presumably by hebbian synaptic plasticity occurring in these connections . in support of this notion , the place cell network is highly plastic , as evidenced by the propensity for its synapses to display changes in strength , either upwards ( long - term potentiation ; ltp ) or downwards ( long - term depression ; ltd ) , in response to activity patterns in its afferents . exactly what this plasticity is for is still unresolved , but one hypothesis has been that one function is for the system to discover for itself the different states the environment can take and to represent this by different states ( also called place cells do indeed have the capacity to acquire different representations of an environment that was previously represented by a single state [ 20 , 21 ] , an observation which is consistent with this idea . the attractor architecture allows for comparison of incoming sensory information with stored information in the network . the incoming information effectively places the attractor network in a state that is nearer to or further from an attractor basin . when presented with an altered state of an environment ( e.g. , an office with the furniture rearranged or a familiar field covered in snow ) , the system compares its stored representations with the current observed state and makes a decision about whether the current state is the same environment after minor changes ( pattern completion ) , or a different environment requiring a new representation ( pattern separation ) . whether or not the system opts to separate ( move to an adjacent basin ) or complete ( return to the previous basin ) the pattern , the cells should all act coherently by virtue of the attractor architecture . experimental evidence for pattern completion in the ca3 hippocampal network was provided by nakazawa et al . who found that partial presentation of a set of environmental cues ( analogous to placing a ball at the edge rather than the centre of an attractor basin ) triggered spontaneous retrieval of the full activation pattern . the other property characteristic of place cells is the way that a given activity pattern can smoothly move from one to the next as an animal moves through space . this smooth movement has also been ascribed to attractor dynamics in a recurrent network , but instead of a discrete attractor in which the state jumps from one pattern to the next , it is thought instead to comprise a continuous attractor around which the activity moves smoothly [ 7 , 17 ] . a continuous attractor can be conceptualized as movement of the imaginary ball across a smooth surface rather than a hilly landscape ( figure 1(e ) ) . the attractors in this network are no longer the possible states of the network in the whole environment , but rather the activity patterns that pertain across the active cells when the animal is at one single place in that environment any neuron that is supposed to be part of this state , at that place , will tend to be pulled into it and held there by the activity of the others to which it is connected . continuous attractors were originally postulated in order to explain the dynamics of the upstream head direction cells . for place cells , the one - dimensional ring attractor of the head direction cell model has been extended to two dimensions . one problem with the notion of attractors in the hippocampal network is that under some situations , place cells fail to act coherently . this phenomenon , which is known as partial remapping , occurs when an environmental change causes some cells to remap and others to maintain their firing patterns unaltered . it is frequently observed when partial changes are made to an environment such that some cues change while others do not [ 13 , 2426 ] . partial remapping is rarely addressed in models of remapping , perhaps because it is not seen in the more typical experimental paradigms involving large environmental changes and also perhaps because of the theoretical difficulties it introduces . partial remapping is difficult for an attractor model to explain , because the defining feature of attractors is the coherence of the network behavior and partial remapping represents a degree of incoherence . it is possible to circumvent this problem by supposing that under some situations attractors can fragment into submaps ( or maplets ) so that some of the neurons ( the ones that did not remap , say ) belong to one attractor system and the others ( the ones that did remap ) to a second one . however , in a study of bidimensional contextual remapping , we found that the attractor could break down even further . here , contextual stimuli were varied across two stimulus dimensions , color and odor , and cells were found to respond essentially arbitrarily to the different combinations . this is shown in the examples in figure 2 , in which the recording environment , a square box , could be varied in either color ( black or white ) and odor ( lemon or vanilla ) . the cells in this example have clearly responded as individuals to these changes . even with these limited environmental changes , the five cells shown here would require five attractor maplets , since no two cells have remapped in quite the same way . once it becomes necessary to propose as many attractors subsystems as there are neurons , the attractor concept starts to lose its explanatory power . one way to rescue the attractor hypothesis is to suppose that perhaps attractors are normally created in the place cell system , but our experiment created a pathological situation in which the ability of the network to discover attractor states was thwarted by the way in which context elements were explicitly decorrelated so that no two always occurred together . perhaps , partial remapping is a reflection of a broken attractor system one in which the neurons act independently because their ability to act cohesively was disrupted by the fragmented nature of the environments we created . an alternative possibility is that partial remapping is a normal reflection of simultaneous encoding of both similarities and differences in two contexts . however , regardless of whether partial remapping reflects a pathology in the discrete attractor network in the hippocampus or is a normal reflection of configural encoding , its existence poses an interesting conundrum , because although the discrete attractors appear to have fragmented , the continuous attractor dynamics seem intact : activity can still move smoothly from one set of neurons to the next even though some of the neurons seem to belong to one subnetwork and some to another , and partial remapping to a new environmental configuration may have thrown up a combination of place fields that has never occurred before . it seems that fragmentation of the discrete attractor networks has not interrupted the continuous attractor dynamics . the discussion below reviews evidence that this may be because the continuous and discrete attractors are in different networks , with the continuous attractor dynamics residing in the network in entorhinal cortex and the discrete attractor landscape resulting from plasticity processes in the dentate - ca3 network in hippocampus . the discrete dynamics the shift of a given place cell from one pattern to another will be explained as the result of contextually modulated switching in the entorhinal - hippocampal connections . the entorhinal grid cells , upstream of the place cells in dorsomedial entorhinal cortex , were discovered by hafting et al . in the moser lab . the firing of grid cells is spatially localized , but firing fields from a given cell are multiple and occur in evenly spaced arrays of circular fields arranged in a hexagonal close - packed configuration ( which happens to be the most efficient way of tiling a plane with circles ) . grid cells have a number of interesting features which make them plausible candidates for the long - postulated continuous attractor system that underlies place cell activity and the ability of place cells to update their activity in response to movement . to begin with , they are always active in any environment , like head direction cells but unlike place cells . also , as far as we know , closely colocalized ( and possibly also more distant ) cells having the same grid scale maintain the same relative firing field locations , regardless of the absolute location with respect to the world outside . this suggests the operation of intrinsic network dynamics , in which activity is modulated by movement of the animal in any direction but reinforced by intrinsic connections in the grid cell network itself . how is this network activity conveyed to place cells ? the spatial nature of grid cell firing makes these cells natural candidates to underlie place field formation , but the issue of how the grid cell activity patterns are converted into place fields is not yet resolved . evidence suggests that there is a high convergence from grid cells to dentate granule cells , ca3 cells , and ca1 cells : for example , de almeida et al . estimated that each granule cell receives around 1200 synapses from grid cells . importantly , grid fields occur at different spacings at different dorsoventral levels , and a place cell receives inputs from a variety of different scales . however , modeling shows that even when multiple grid scales converge , this number of grids impinging on a single neuron results in a cell being activated uniformly across the whole environment . even when the gain of the inputs is turned down so that the place cell only becomes active at the peaks of this drive , the resulting activity hotspots occur in a highly regular , multipeaked symmetrical array that does not resemble real place fields . one way around this problem is to produce some kind of inhomogeneity or chunking of the grid cell inputs so that the resulting pattern becomes lumpy and more likely to produce a small number of fields . this can be done by increasing the sparseness of the network , varying orientation and spatial phase , adding phase precession , grouping the inputs according to spatial phase , or adding feedback inhibition and varying synaptic strengths . the resulting patterns have something of a resemblance to dentate granule cell place fields , which are multipeaked , like grid fields , but irregular like place fields , and intermediate in sparsity between grid and place fields . such schemes do not , however , account for some features of place cell activity , such as why fields tend to be elongated near walls , and so clearly , some additional factor is needed to fully account for place field morphology . having established a basic possible connectivity between grid and place cells and also that the grid cells are the likeliest source of the continuous attractor dynamics in the system , the question now is how the continuous attractor dynamics of the place cells can be explained as the animal moves around . this is relatively straightforward : because the activity of place cells derives from the grids , then as the grid cell activity rises and falls with locomotion , so too should the drive onto the place cells , with the irregularity resulting from a combination of the multiple scales and ( perhaps ) the chunked inputs . because the place cells inherit the attractor dynamics from the grid cells and not from each other , it therefore does not matter if the place cell network is disrupted by partial remapping . like place cells , grid cells change their firing patterns following environmental change [ 2 , 30 ] , but the exact nature of the remapping is different . as mentioned above , grid cells are always active , so they do not switch fields on and off as place cells do . nor do they , as far as is known , modulate their rates in a rate - remapping fashion , like place cells do in some situations [ 21 , 40 ] . rather , their remapping consists of translation and/or rotation of fields ( figure 3 ) . in the first systematic study of grid cell remapping by fyhn et al . , large changes to the environment , such as moving the animal to a new room , caused both translation and rotation , while small changes ( changing the enclosure but not the room ) did not cause remapping at all . interestingly , the large changes were accompanied by place cell global remapping ( reshuffling of the map ) , while small changes were associated only with rate remapping . rate remapping occurs when the response of a place cell to a change in context is to increase or decrease the intensity of the place field without switching on or off completely [ 21 , 40 ] . following environmental change , the grid cells seem to act coherently , inasmuch as any sufficiently large change seems to cause the firing fields to shift en masse [ 2 , 30 ] . this poses some problems for models of place field generation : if the grid cells are , as popularly supposed , the basis for place field generation , then how can the heterogeneity of place field remapping be accounted for by the apparent homogeneity of grid cell remapping ? the first is that during complete remapping , some cells switch their fields on or off and some cells shift their fields ( figure 3(b ) ) . the resulting pattern has , as far as we can determine , no relationship to the original and the map looks to have been randomly regenerated . thus , the fields alter their position , or their very existence , with respect to each other . since attractor dynamics arise from the connections between the neurons in the map , this means the original attractor , if it were located in the intrahippocampal connections , must have been disrupted . as discussed above , the solution to this problem may be that the continuous attractor dynamics reside in the grid cell network and not the place cell network . while this could explain the continuity of continuous attractor dynamics across a change in environment , it still does not explain the place field response of rearrangement of fields , because mere rotation and translation of a grid array should cause simple rotation and translation of the place field array , which clearly does not happen . suggested two ways around this problem : first , perhaps there is modularity in the grid cell population such that the whole population does shift , or second , perhaps the grid pattern shifts to a point far away on an imaginary , infinite grid field array , which would result in the place fields effectively shifting and rotating by such a large amount that the resulting place field pattern is one that did not previously occur in the enclosure . the other kind of heterogeneity is the partial remapping discussed above , where some cells respond to a particular environmental change , while others do not ( figure 2 ) . this seems harder to explain by grids , because if the grid cells are the drivers of place cell activity , then how can partial remapping occur ? there are no published data to support this yet , but evidence so far suggests that the grid cells mostly tend to act coherently [ 2 , 30 ] . it may be , however , that grids are not really as homogeneous as has been assumed and that they can in fact remap independently , and thus become dislocated with respect to each other . , perhaps more plausible possibility is that the mapping between the grid cells and place cells can be dynamically modulated so that following an environmental change , the population of grid cells that drives a particular place cell becomes altered . we have previously proposed that such modulation could occur by means of concurrently active contextual inputs - the inputs that tell the system that the environment has changed [ 13 , 37 , 41 , 42 ] . this model is described , below , and an outline provided of how it may explain how the homogeneous and always - present pattern of grid cell activity can translate to the heterogeneous and sometimes - present activity of place cells . in 2008 we presented a contextual gating model of grid - cell / place - cell connectivity that may explain how the heterogeneous and seemingly individualistic behavior of place cells might arise from the relatively homogeneous and coherent activity of grid cells . figure 4 illustrates the basic model , comprising a set of grid cells projecting to place cells in hippocampus in order to drive the formation of place fields . converging onto these same cells are a set of inputs conveying information about context , such as whether the box is black / white or lemon / vanilla as in the example given earlier . the function of these context inputs is to interact with the spatial inputs from the grid cells and decide which of the spatial inputs figure 4(a ) shows a schematic of the synaptic matrix arising from the intersection of the context inputs and spatial inputs . this matrix transforms the uniform and coherent pattern of grid cell activity into the discrete and cell - specific patterns seen in dentate gyrus . thus , the contextual inputs have gated the spatial inputs . figure 4(b ) shows the model at the single neuron level . the figure shows a hypothetical hippocampal neuron , in this case a dentate granule cell , which integrates convergent spatial and contextual information in its dendrites . the schematic illustrates how inputs from medial and lateral entorhinal cortex converge , in the dentate gyrus , onto the same set of granule cells , with the grid cell inputs arriving from medial entorhinal cortex ( mec ) and terminating on the middle portion of the dendritic tree , and inputs carrying contextual information arriving from the lateral entorhinal cortex ( lec ) and terminating in the outer part of the dendritic tree . ( there are also feedback inputs from the dentate - ca3 network arriving via the commissural - associational network , which terminate in the proximal dendrites , as discussed later . ) in our model , the basic unit of computation is a branch of the dendritic tree , which receives both spatial and contextual inputs and is able to integrate these . interestingly , the idea that a dendritic branch could be a unit of processing is one that is gaining increasing support in the experimental literature . the figure illustrates how , using this model , complete remapping can be explained as follows : when the context changes , now a different set of context inputs is active and these gate a different set of spatial inputs , driving a different branch of the granule cell dendritic tree , and thus generating a different spatial pattern of activity . if the change to the environment is large , then there is a massive change in the pattern of context inputs which would affect all the grid cell inputs to all the place cells , causing a complete remapping . for smaller changes , some of the inputs to the place cell would alter , and some would stay the same depending on how big these changes were the cell might retain its original place field . similarly , rate remapping can be explained by a change of the facilitation level of the context inputs to the grid cell ones . importantly , different cells are able to be modulated independently , also consistent with real data . the most important feature of the contextual gating model is that it can explain partial remapping . even if activity in the grid cell sheet continues at the same level ( with or without translation / rotation remapping ) , and with the same spatial relationship between the field arrays of individual cells , the model allows for independent tuning of individual cells , meaning that an environmental change is able to affect some cells but not others . we have modeled this contextual gating proposal by simulating networks of grids of varying scales which project to granule cells , and then altering the connection to each granule cell in a context - dependent manner . first , in order to generate realistic - looking dentate granule fields , which could , in turn , produce realistic ca3 fields , we needed to introduce some kind of chunking of the inputs , for reasons discussed earlier . we accomplished this by grouping the inputs according to spatial phase ( offset ) so that grid cells having overlapping grid fields would be more likely to coterminate on a particular branch of a granule cell dendrite , in each dendritic branch , possessing grid cell inputs with an above - average tendency to overlap in a region of the environment , formed the computational unit of the network . the clustering of the grid cell inputs in this way helped to avoid uniform activation across the environment , and with appropriate choice of grouping parameter , we were able to produce granule cell activation that occurred in only a few places in the environment , consistent with the multipeaked irregular place fields in the data from leutgeb et al . . next , we introduced contextual modulation of the kind discussed above so that a given cluster of entorhinal inputs that is , those terminating on one branch of the cell 's dendritic tree could only drive the granule cell if the appropriate contextual inputs were also active and terminating on that same dendritic branch . by switching context inputs ( and hence dendritic branches and their associated inputs ) on and off , we mimicked the effect of placing a rat in different environments . by steadily ( rather than abruptly ) varying the degree of contextual variation , we were able to cause the simulated granule cells to progressively change their firing patterns ( figure 5 ) . interestingly , both the model data from our simulation and the real data from leutgeb et al . show the same effect , which is that rather than producing on - off remapping as is typical with place cells , we saw a gradual refocusing of the subfields of a set of granule cells . thus , as the context was progressively altered ( by varying the contextual drive in the simulation , or by slowly morphing the environment in the real experiment ) , it was possible to slowly shift activity from one subfield to another within the granule cell field cluster . a similar effect has subsequently also been reported by renn - costa et al . who explored the effect quantitatively . these gradual changes translated into place field remapping in a simulated downstream ca3 network ( figure 5(c ) ) . one example is rate remapping , which could be accounted for in the contextual gating model by supposing that rather than altering the contextual inputs completely , and switching in a new set of grids , the environmental change merely decreases the intensity of the inputs coming in , and thus weakens the synergistic interaction between the contextual and spatial inputs . the model can also explain partial remapping in the place cells , downstream from the granule cells . by generating simulated ca3 fields from the granule cells , we found a predominance of unitary fields , which closely resemble real place fields ( although , as mentioned earlier , they lack some notable features such as conformation to wall contours ) . by changing the contextual inputs , we showed that some ca3 fields did not change while others remapped ( figure 5(c ) ) . there is one other phenomenon which the contextual gating model can potentially explain , although we have yet to model it with our simulated grid cells , and that is conditional remapping , which is remapping to changes in one contextual stimulus that depends on what other(s ) may also be present . an example of conditional remapping was given earlier in the discussion of the two - element context experiments ( and figure 2 ) , in which the response to a change in ( say ) color depended on the odor that was present , and vice versa . in this experiment , not only did the place cells act independently , they also acted as though they knew which color and odor belonged together in order to produce a given field . theoretical considerations show that this could not occur if the context elements terminated independently on the place cell : they must be integrated somewhere upstream of the cell , possibly in its dendrites . the contextual gating model that we originally formulated suggested a convergence , upstream of the place cells , between spatial inputs and contextual inputs , which is where the integration could occur , forming a combined associative input that could be thought of as configural ( i.e. , combining stimulus elements together ) . with the subsequent discovery of grid cells , this model was able to be given more specific detail : in the updated version , the integration can occur at the place where the context elements converge , which in our model is in the dendritic tree of a dentate granule cell . by this view , context elements converging in entorhinal cortex from different sensory modalities come to coterminate on the same part of the dendritic tree ( figure 5(a ) ) and are able to act together to gate the same set of grid cell inputs . an illustration of how this might occur is shown in figure 6 . from the foregoing , we can see how it is that the continuous attractor dynamics in the place cell population can survive partial remapping the attractor dynamics are actually generated in the upstream grid cells , which do not fragment when contexts are fragmented . thus , although new place fields occur in the hybrid context states , the underlying generators the grids are unchanged . it is worth noting that although this scheme does not require grid cells to have remapped , they likely do also remap to even nonspatial contextual changes but whether or not they remap , the attractor hypothesis supposes that grids having the same scale will maintain their relative firing locations , thus preserving the continuous attractor dynamics . we have proposed that the discrete attractor states arise as a result of switching in and out of combinations of grids , but we have also noted that evidence suggests that these attractor states are learned by the dentate - ca3 network , and indeed that one function of the hippocampus might be to discover the set of attractor states that best corresponds to the states the environment can exist in . how can attractor states , discovered by the dentate - ca3 autoassociative network as a result of experience , act to shape the switching profiles upstream in the entorhinal - hippocampal connections ? the implication is that there must be some kind of back - propagation from the dentate - ca3 system to the dendritic tree in which the proposed contextual gating occurs . it is suggested here that this back - propagation could occur within the granule cells themselves , since depolarization can travel retrogradely into the dendrites [ 43 , 46 ] . dentate gyrus neurons receive substantial back - projections from the ca3 autoassociative network via a rich set of commissural associational inputs which terminate on the proximal branches of the granule cell dendrites ( , figure 4(a ) ) . also , spikes that have been generated in granule cells are able to back - propagate into the dendrites and thus alter both the degree of depolarization and also the likelihood of synaptic plasticity there [ 43 , 46 ] . an illustration of how this back - propagation might occur at the single - cell level is shown in figure 6 , in which retrograde depolarization from the commissural associational network facilitates ltp of a weak context input onto a branch of the dendritic tree , and compensating homeostatic ltd in other synapses so as to keep drive onto the cell constant . the injection of network information , via the commissural - associational pathway , means that attractor states that have been formed and learned by the dentate - ca3 network can feed back into the entorhinal hippocampal connections , where the switch between patterns occurs . thus , the attractor basins that have been formed by the dentate - ca3 network can act , in principle , to shape themselves by enhancing the connections from active incoming context inputs back in the dendrites of those same cells . this paper has described continuous and discrete attractor dynamics in the hippocampal formation and proposed a mechanism for the interaction between the two attractor systems . the continuous attractor may be located in the entorhinal grid cell network , and it allows the smooth transition from one set of active place cells to the next as the animal moves around . the location of this attractor outside of the place cell network itself allows an explanation for why the continuous dynamics are preserved even when the place cells partially remap . the discrete attractor landscape arises initially from the mapping between entorhinal cortex and hippocampus , by virtue of context - mediated selection of a unique subset of grid cell afferents onto each place cell . contextual gating can explain a number of phenomena such as rate remapping , partial remapping of place cells even when grid cells do not remap , and also conditional remapping in which the response to one contextual stimulus depends on the presence of another . by this view , the discrete attractor landscape is sculpted within the hippocampal place cell network , but the jump in state that occurs following large environmental changes arises upstream of the place cells , in the entorhinal hippocampal connections and ( in situations where grid cells themselves remap ) beyond . new basins in the place cell attractor landscape feed into this connectivity matrix via back - propagation of depolarization into the dendritic tree . in this way , incoming contextual inputs help the place cell network discover and learn the appropriate attractor landscape , and the resultant plasticity , in turn , shapes how the contextual inputs modulate the grid - cell / place - cell connections , and thus the place field remapping dynamics .

place and grid cells are thought to use a mixture of external sensory information and internal attractor dynamics to organize their activity . attractor dynamics may explain both why neurons react coherently following sufficiently large changes to the environment ( discrete attractors ) and how firing patterns move smoothly from one representation to the next as an animal moves through space ( continuous attractors ) . however , some features of place cell behavior , such as the sometimes independent responsiveness of place cells to environmental change ( called remapping ) , seem hard to reconcile with attractor dynamics . this paper suggests that the explanation may be found in an anatomical separation of the two attractor systems coupled with a dynamic contextual modulation of the connection matrix between the two systems , with new learning being back - propagated into the matrix . such a scheme could explain how place cells sometimes behave coherently and sometimes independently .

1. Introduction 2. The Contextual Gating Model 3. Conclusion

the hippocampal place cells are thought to collectively form a representation of space , known as a cognitive map , because of their spatially localized firing , which occurs in patches known as place fields ( figure 1(a ) ) . the place and grid cells are an excellent model system with which to study the formation and architecture of cognitive knowledge structures . place and grid cells use external environmental cues to anchor their activity to the real world , as evidenced by the fact that their activity appears bound to the local environmental walls [ 2 , 4 , 5 ] and reacts to changes in the environment . however , firing patterns are then stabilized and maintained by internal network dynamics so that activity can be self - sustaining and coherent across the network . the purpose of this paper is to review the evidence for these two attractor types in the hippocampal network and then to explore a phenomenon that can not be easily accounted for by attractors , known as partial remapping . finally , a model will be described that may be able to explain how both attractor dynamics and partial remapping can co - exist in the same network . one of the earliest and most striking observations concerning the place cell representation was the way that the cells can suddenly and collectively alter their activity from one pattern to another , a process known as remapping ( figure 1(b ) ) . the attractor hypothesis built upon earlier ideas that the hippocampal ca3 network functions as an autoassociative memory [ 1012 ] . the process of moving towards and settling into a stable state is what is meant by anatomical and physiological observations of place cells suggest the operation of two kinds of attractor dynamics : discrete and continuous . discrete attractor dynamics enable the system to resist small changes in sensory input but respond collectively and coherently to large ones , while continuous dynamics enable the system to move smoothly from one state to the next as the animal moves through space . these two attractor systems clearly must either be colocalized on the same neurons or else be separate but interacting , since one accounts for the population of place cells active at a given moment and the other for the progression of activity from one set to the next as the animal moves . one possibility , discussed later , is that the source of the discrete attractor dynamics may lie in the place cell network itself [ 79 , 14 ] , and the continuous dynamics may originate upstream in the entorhinal grid cell network . the hollows , also called basins , are low - energy states , but to move from one hollow to the next , the ball requires a substantial perturbation : a small push will not cause it to change basins / states . the states are imprinted onto the network by appropriate modulation of the connection strengths between the neurons in the network . place cell remapping was initially conceptualized as being a sudden transition of the place cell network from one state to the next following the large perturbation arising from environmental change , an idea that has been very influential . experimental evidence for attractor dynamics in the place cell network was initially provided by observations of remapping , but stronger evidence came from a study by wills et al . , who showed that incremental changes in the squareness or circularity of an enclosure would produce no change in place cell activity until the cumulative changes became sufficiently great , at which point the whole system would suddenly switch to the other pattern ( figure 1(c ) ) . , who found , by contrast , that gradual transformation of environment shape induced gradual transition of firing patterns from one shape to the other . such differences in network behavior may be accounted for by the attractor landscapethe exact distribution of the imaginary hills and valleys in the attractor state - space which may have been sculpted by the past experience of the animal , via processes described below . in support of this notion , the place cell network is highly plastic , as evidenced by the propensity for its synapses to display changes in strength , either upwards ( long - term potentiation ; ltp ) or downwards ( long - term depression ; ltd ) , in response to activity patterns in its afferents . whether or not the system opts to separate ( move to an adjacent basin ) or complete ( return to the previous basin ) the pattern , the cells should all act coherently by virtue of the attractor architecture . the other property characteristic of place cells is the way that a given activity pattern can smoothly move from one to the next as an animal moves through space . this smooth movement has also been ascribed to attractor dynamics in a recurrent network , but instead of a discrete attractor in which the state jumps from one pattern to the next , it is thought instead to comprise a continuous attractor around which the activity moves smoothly [ 7 , 17 ] . continuous attractors were originally postulated in order to explain the dynamics of the upstream head direction cells . for place cells , the one - dimensional ring attractor of the head direction cell model has been extended to two dimensions . this phenomenon , which is known as partial remapping , occurs when an environmental change causes some cells to remap and others to maintain their firing patterns unaltered . however , in a study of bidimensional contextual remapping , we found that the attractor could break down even further . one way to rescue the attractor hypothesis is to suppose that perhaps attractors are normally created in the place cell system , but our experiment created a pathological situation in which the ability of the network to discover attractor states was thwarted by the way in which context elements were explicitly decorrelated so that no two always occurred together . however , regardless of whether partial remapping reflects a pathology in the discrete attractor network in the hippocampus or is a normal reflection of configural encoding , its existence poses an interesting conundrum , because although the discrete attractors appear to have fragmented , the continuous attractor dynamics seem intact : activity can still move smoothly from one set of neurons to the next even though some of the neurons seem to belong to one subnetwork and some to another , and partial remapping to a new environmental configuration may have thrown up a combination of place fields that has never occurred before . it seems that fragmentation of the discrete attractor networks has not interrupted the continuous attractor dynamics . the discussion below reviews evidence that this may be because the continuous and discrete attractors are in different networks , with the continuous attractor dynamics residing in the network in entorhinal cortex and the discrete attractor landscape resulting from plasticity processes in the dentate - ca3 network in hippocampus . the discrete dynamics the shift of a given place cell from one pattern to another will be explained as the result of contextually modulated switching in the entorhinal - hippocampal connections . the entorhinal grid cells , upstream of the place cells in dorsomedial entorhinal cortex , were discovered by hafting et al . grid cells have a number of interesting features which make them plausible candidates for the long - postulated continuous attractor system that underlies place cell activity and the ability of place cells to update their activity in response to movement . also , as far as we know , closely colocalized ( and possibly also more distant ) cells having the same grid scale maintain the same relative firing field locations , regardless of the absolute location with respect to the world outside . evidence suggests that there is a high convergence from grid cells to dentate granule cells , ca3 cells , and ca1 cells : for example , de almeida et al . even when the gain of the inputs is turned down so that the place cell only becomes active at the peaks of this drive , the resulting activity hotspots occur in a highly regular , multipeaked symmetrical array that does not resemble real place fields . one way around this problem is to produce some kind of inhomogeneity or chunking of the grid cell inputs so that the resulting pattern becomes lumpy and more likely to produce a small number of fields . such schemes do not , however , account for some features of place cell activity , such as why fields tend to be elongated near walls , and so clearly , some additional factor is needed to fully account for place field morphology . having established a basic possible connectivity between grid and place cells and also that the grid cells are the likeliest source of the continuous attractor dynamics in the system , the question now is how the continuous attractor dynamics of the place cells can be explained as the animal moves around . this is relatively straightforward : because the activity of place cells derives from the grids , then as the grid cell activity rises and falls with locomotion , so too should the drive onto the place cells , with the irregularity resulting from a combination of the multiple scales and ( perhaps ) the chunked inputs . because the place cells inherit the attractor dynamics from the grid cells and not from each other , it therefore does not matter if the place cell network is disrupted by partial remapping . like place cells , grid cells change their firing patterns following environmental change [ 2 , 30 ] , but the exact nature of the remapping is different . as mentioned above , grid cells are always active , so they do not switch fields on and off as place cells do . , large changes to the environment , such as moving the animal to a new room , caused both translation and rotation , while small changes ( changing the enclosure but not the room ) did not cause remapping at all . interestingly , the large changes were accompanied by place cell global remapping ( reshuffling of the map ) , while small changes were associated only with rate remapping . rate remapping occurs when the response of a place cell to a change in context is to increase or decrease the intensity of the place field without switching on or off completely [ 21 , 40 ] . following environmental change , the grid cells seem to act coherently , inasmuch as any sufficiently large change seems to cause the firing fields to shift en masse [ 2 , 30 ] . this poses some problems for models of place field generation : if the grid cells are , as popularly supposed , the basis for place field generation , then how can the heterogeneity of place field remapping be accounted for by the apparent homogeneity of grid cell remapping ? since attractor dynamics arise from the connections between the neurons in the map , this means the original attractor , if it were located in the intrahippocampal connections , must have been disrupted . as discussed above , the solution to this problem may be that the continuous attractor dynamics reside in the grid cell network and not the place cell network . while this could explain the continuity of continuous attractor dynamics across a change in environment , it still does not explain the place field response of rearrangement of fields , because mere rotation and translation of a grid array should cause simple rotation and translation of the place field array , which clearly does not happen . suggested two ways around this problem : first , perhaps there is modularity in the grid cell population such that the whole population does shift , or second , perhaps the grid pattern shifts to a point far away on an imaginary , infinite grid field array , which would result in the place fields effectively shifting and rotating by such a large amount that the resulting place field pattern is one that did not previously occur in the enclosure . this seems harder to explain by grids , because if the grid cells are the drivers of place cell activity , then how can partial remapping occur ? there are no published data to support this yet , but evidence so far suggests that the grid cells mostly tend to act coherently [ 2 , 30 ] . , perhaps more plausible possibility is that the mapping between the grid cells and place cells can be dynamically modulated so that following an environmental change , the population of grid cells that drives a particular place cell becomes altered . we have previously proposed that such modulation could occur by means of concurrently active contextual inputs - the inputs that tell the system that the environment has changed [ 13 , 37 , 41 , 42 ] . this model is described , below , and an outline provided of how it may explain how the homogeneous and always - present pattern of grid cell activity can translate to the heterogeneous and sometimes - present activity of place cells . in 2008 we presented a contextual gating model of grid - cell / place - cell connectivity that may explain how the heterogeneous and seemingly individualistic behavior of place cells might arise from the relatively homogeneous and coherent activity of grid cells . figure 4 illustrates the basic model , comprising a set of grid cells projecting to place cells in hippocampus in order to drive the formation of place fields . converging onto these same cells are a set of inputs conveying information about context , such as whether the box is black / white or lemon / vanilla as in the example given earlier . the function of these context inputs is to interact with the spatial inputs from the grid cells and decide which of the spatial inputs figure 4(a ) shows a schematic of the synaptic matrix arising from the intersection of the context inputs and spatial inputs . the schematic illustrates how inputs from medial and lateral entorhinal cortex converge , in the dentate gyrus , onto the same set of granule cells , with the grid cell inputs arriving from medial entorhinal cortex ( mec ) and terminating on the middle portion of the dendritic tree , and inputs carrying contextual information arriving from the lateral entorhinal cortex ( lec ) and terminating in the outer part of the dendritic tree . if the change to the environment is large , then there is a massive change in the pattern of context inputs which would affect all the grid cell inputs to all the place cells , causing a complete remapping . for smaller changes , some of the inputs to the place cell would alter , and some would stay the same depending on how big these changes were the cell might retain its original place field . similarly , rate remapping can be explained by a change of the facilitation level of the context inputs to the grid cell ones . even if activity in the grid cell sheet continues at the same level ( with or without translation / rotation remapping ) , and with the same spatial relationship between the field arrays of individual cells , the model allows for independent tuning of individual cells , meaning that an environmental change is able to affect some cells but not others . we accomplished this by grouping the inputs according to spatial phase ( offset ) so that grid cells having overlapping grid fields would be more likely to coterminate on a particular branch of a granule cell dendrite , in each dendritic branch , possessing grid cell inputs with an above - average tendency to overlap in a region of the environment , formed the computational unit of the network . the clustering of the grid cell inputs in this way helped to avoid uniform activation across the environment , and with appropriate choice of grouping parameter , we were able to produce granule cell activation that occurred in only a few places in the environment , consistent with the multipeaked irregular place fields in the data from leutgeb et al . next , we introduced contextual modulation of the kind discussed above so that a given cluster of entorhinal inputs that is , those terminating on one branch of the cell 's dendritic tree could only drive the granule cell if the appropriate contextual inputs were also active and terminating on that same dendritic branch . show the same effect , which is that rather than producing on - off remapping as is typical with place cells , we saw a gradual refocusing of the subfields of a set of granule cells . thus , as the context was progressively altered ( by varying the contextual drive in the simulation , or by slowly morphing the environment in the real experiment ) , it was possible to slowly shift activity from one subfield to another within the granule cell field cluster . one example is rate remapping , which could be accounted for in the contextual gating model by supposing that rather than altering the contextual inputs completely , and switching in a new set of grids , the environmental change merely decreases the intensity of the inputs coming in , and thus weakens the synergistic interaction between the contextual and spatial inputs . an example of conditional remapping was given earlier in the discussion of the two - element context experiments ( and figure 2 ) , in which the response to a change in ( say ) color depended on the odor that was present , and vice versa . the contextual gating model that we originally formulated suggested a convergence , upstream of the place cells , between spatial inputs and contextual inputs , which is where the integration could occur , forming a combined associative input that could be thought of as configural ( i.e. by this view , context elements converging in entorhinal cortex from different sensory modalities come to coterminate on the same part of the dendritic tree ( figure 5(a ) ) and are able to act together to gate the same set of grid cell inputs . from the foregoing , we can see how it is that the continuous attractor dynamics in the place cell population can survive partial remapping the attractor dynamics are actually generated in the upstream grid cells , which do not fragment when contexts are fragmented . it is worth noting that although this scheme does not require grid cells to have remapped , they likely do also remap to even nonspatial contextual changes but whether or not they remap , the attractor hypothesis supposes that grids having the same scale will maintain their relative firing locations , thus preserving the continuous attractor dynamics . we have proposed that the discrete attractor states arise as a result of switching in and out of combinations of grids , but we have also noted that evidence suggests that these attractor states are learned by the dentate - ca3 network , and indeed that one function of the hippocampus might be to discover the set of attractor states that best corresponds to the states the environment can exist in . the implication is that there must be some kind of back - propagation from the dentate - ca3 system to the dendritic tree in which the proposed contextual gating occurs . dentate gyrus neurons receive substantial back - projections from the ca3 autoassociative network via a rich set of commissural associational inputs which terminate on the proximal branches of the granule cell dendrites ( , figure 4(a ) ) . also , spikes that have been generated in granule cells are able to back - propagate into the dendrites and thus alter both the degree of depolarization and also the likelihood of synaptic plasticity there [ 43 , 46 ] . an illustration of how this back - propagation might occur at the single - cell level is shown in figure 6 , in which retrograde depolarization from the commissural associational network facilitates ltp of a weak context input onto a branch of the dendritic tree , and compensating homeostatic ltd in other synapses so as to keep drive onto the cell constant . this paper has described continuous and discrete attractor dynamics in the hippocampal formation and proposed a mechanism for the interaction between the two attractor systems . the continuous attractor may be located in the entorhinal grid cell network , and it allows the smooth transition from one set of active place cells to the next as the animal moves around . the location of this attractor outside of the place cell network itself allows an explanation for why the continuous dynamics are preserved even when the place cells partially remap . contextual gating can explain a number of phenomena such as rate remapping , partial remapping of place cells even when grid cells do not remap , and also conditional remapping in which the response to one contextual stimulus depends on the presence of another . by this view , the discrete attractor landscape is sculpted within the hippocampal place cell network , but the jump in state that occurs following large environmental changes arises upstream of the place cells , in the entorhinal hippocampal connections and ( in situations where grid cells themselves remap ) beyond . new basins in the place cell attractor landscape feed into this connectivity matrix via back - propagation of depolarization into the dendritic tree .

historian edward shorter viewed the introduction of the tranquiliser largactil in 1953 as a pivotal moment in psychiatric practice , initiating a revolution in psychiatry that transformed psychiatry from a branch of social work to a field that called for the most precise knowledge of pharmacology.1 in his 1967 autobiography , psychiatrist william sargant expressed similar sentiments , recalling how the introduction of largactil helped realise his dream of transforming psychiatric practice into a branch of general medicine . wonder drug , as he dubbed it , enabled him to treat patients diagnosed with schizophrenia within a general hospital . administer enough largactil to keep even the acutest schizophrenic tranquilised while electric shock treatment and other methods speeded their recovery.2 like shorter , sargant reinforced his point that psychiatry had progressed by drawing a comparison with earlier social work approaches to mental illness , inferring that psychiatric social work was obsolete . at the maudsley hospital in the 1930s , he explained , tactful women interrogators called psychiatric social workers had compiled case histories detailing the family and home circumstances of each patient admitted . these case histories would now be laughed at and were often a waste of time , but what else could one do ? nowadays we may only need to prescribe four or five electric shock treatments , or a new course of some antidepressant drug.3 largactil belonged to a group of psychiatric therapies grouped together under the label of physical treatments . these treatment methods , which included insulin treatment , convulsion , malarial therapy , prefrontal leucotomy , sedatives and stimulants , were designed to alleviate psychological symptoms by inducing physiological change or by altering the structure of the brain.4 if we turn to accounts produced by practitioners of the relatively new profession of psychiatric social work disparaged in sargant s autobiography , we gain a different perspective on the impact of physical treatments on patients . based within a psychiatric hospital , one such psychiatric social worker ( hereafter psw ) madelene crump , found her work expanding when the new sedatives and tranquillisers of the 1950s , such as largactil , produced an increased number of cases deemed by psychiatrists to have recovered sufficiently to be discharged . however , she expressed reservations about the effect of the drugs on patients she described as formerly : bizarre and sometimes spiteful and vindictive ... they seemed to have shown considerable vitality and individuality . one wonders where the vitality has gone now , what is turning over in their minds as they sit there calm and a little rigid in their chairs , giving the polite answer like children anxious to please , when approached by a member of staff.5 bizarre and sometimes spiteful and vindictive ... they seemed to have shown considerable vitality and individuality . one wonders where the vitality has gone now , what is turning over in their minds as they sit there calm and a little rigid in their chairs , giving the polite answer like children anxious to please , when approached by a member of staff.5 this article explores the rise and dominance of physical treatments from the perspective of psws . commencing with an account of the development of psychiatric social work , the article will contend that psws , by virtue of their training and function , were uniquely placed to trace the repercussions of physical therapies through their work with discharged patients in the community . it explores how psws shared their concerns with colleagues via the british journal of psychiatric social work ( hereafter bjpsw ) , which was established in 1947 by the association of psychiatric social workers ( hereafter apsw ) publications sub - committee with the intention of providing a vehicle for the exchange of ideas regarding the methods of psychiatric social work.6 the bjpsw offered psws a space in which to forge a critical analysis of physical treatment methods . it provides historians with an insight into the professionalising aspirations of a nascent occupation ; fragmentary perspectives from disparate locations , provided by authors for whom it is often impossible to ascertain even a first name . the article will then explore how some psws strove to assert their professional status and transcend their auxiliary function by arguing that the care and support they provided to patients and their families could fulfil needs unmet by physical treatments , drew upon a distinctive type of expertise , and could most accurately be conceptualised as psychiatric social treatment. this detailed analysis of specific cases prefaces a consideration of how the objectives of social and the article concludes by stressing the fluidity of the concepts of care , treatment , recovery and cure , tracing how the terms were mobilised in support of professional ideologies and aspirations . the roots of psychiatric social work can be traced back to the work of earlier charitable organisations that worked with those who experienced mental distress and their families within community settings.7 the growing acceptance of psychological explanations for people s behaviour and capabilities and the emergence of the mental hygiene movement also proved influential.8 social worker mary jarrett first developed professional training for social workers in psychiatric fields in 1914 at the boston psychopathic hospital , and psws became part of the team in the newly established child guidance clinics that aimed to prevent juvenile delinquency.9 these american developments dovetailed with the growing interest expressed in the problem child in britain.10 when the commonwealth fund agreed to finance the establishment of child guidance clinics in britain , it stressed the need to train social workers in a university setting . thus in 1929 , the london school of economics established the first course to train social science graduates with some experience of social work as psws . in the same year , the association of psychiatric social work ( hereafter apsw ) was inaugurated with the dual objectives of promoting mental hygiene , and raising the professional status of psychiatric social work.11 edinburgh , manchester and liverpool universities established courses to train psws in 1944 , 1946 and 1954 respectively . by 1944 , 257 people in britain had qualified as psws although not all of these people were using their qualification to work as a psw . those who chose to do so worked either in child guidance clinics , where they undertook casework with mothers , or within psychiatric hospitals , where they compiled a social history of cases admitted and helped patients readjust following discharge from hospital.12 a report undertaken in 1951 found that only eight of the 331 psws working in britain were employed within the mental health departments of local authorities , where they provided support to people living in the community who experienced mental health problems , while 239 were employed in psychiatric social work in mental hospitals , general hospitals or child guidance clinics.13 eight years later , the younghusband report suggested that 325 psws would provide adequate coverage to local authority health services but noted that this field only employed 26 full - time psws.14 it was only by 1969 that the balance was redressed , with 257 psws engaged in community care , 264 in mental hospitals , and 259 working in child guidance clinics.15 with a membership dispersed geographically and occupationally , the apsw endeavoured to facilitate discussion amongst practitioners . general meetings , which over time revolved increasingly around specific issues facing psws , provided one place in which members could exchange views . the development of local branches expanded this forum outside its original london setting , enabling members throughout the country to debate professional issues . from 1950 , the apsw distributed copies of the bjpsw to all subscribing members , facilitating the aspiration expressed by editor margaret ashdown that meetings of the association would discuss the content of the journal , stimulating the interest and initiative of the members.16 ashdown also felt the bjpsw might help publicise the achievements of the apsw to related professions , serving as a whispering gallery , by means of which our voices , which some of us feel to be so feeble , can be made to carry to our professional neighbours , without fear or strain.17 given the circulation of the initial edition of the bjpsw , these hopes appear misplaced : the initial print run of 1,000 copies in 1947 and 1948 had to be reduced to 600 copies by 1949 when it became clear that the apsw had over 200 copies left of each of the previous journals.18 membership of the apsw stood at only 398 by the end of 1948 . proponents of physical therapies , such as william sargant , claimed that mental illnesses had a somatic aetiology and wanted to bridge the gap between psychiatry and general medicine . in an introduction to physical methods of treatment , co - authored with fellow psychiatrist eliot slater nearly a decade before the introduction of largactil , sargant claimed that new treatment methods had transformed mental hospitals beyond recognition within a decade.19 physical treatments , asserted sargant and slater , produce their beneficial effects with greater speed and greater certainty than the older and more well - established psychotherapeutic methods.20 psychiatrists could now interpret the emotional distress and social problems experienced by patients as a product of underlying biological malfunction rather than a cause of their disorder . correspondingly , therapeutic practices designed to unpick the social roots of mental illness by delving into a patient s history or to alleviate the social consequences of mental distress could at best play a supporting role . in the late 1940s and 1950s , two factors converged to create an atmosphere amenable to the dissemination of physical therapies within psychiatric practice . although the board of control continued to regulate the mental health services until the passing of the 1959 mental health act , the nominal incorporation of psychiatric hospitals within the new national health service in 1948 presented an opportunity for psychiatrists to realign their professional activities with general medicine.21 this was also an era of rising patient numbers . overcrowding in mental hospitals was estimated nationally at fourteen per cent in 1950 ; at st andrew s hospital in norfolk , overcrowding had reached twenty - five and fifty per cent on men s and women s wards respectively between 1951 and 1957.22 as the resident population in mental hospitals peaked at 151,400 in 1954,23 many psychiatrists may well have been inclined to agree with sargant and slater regarding the incapacity of highly individual and time - consuming methods to deal with a large - scale problem , viewing physical treatments as more efficacious in terms of speed , convenience and certainty.24 other major psychiatric textbooks , such as clinical psychiatry , co - authored by william - mayer - gross , eliot slater and martin roth in 1954 , also emphasised the organic aetiology of mental illness and the value of physical therapies , largely dismissing the influence of social and psychological factors in mental distress.25 psw cyril greenland , who worked for mayer - gross between 1948 and 1955 , recalled his superior s conviction that since the causes of metal illness would sooner or later be revealed by the biological sciences , sociology and social work had a very limited role to play in psychiatry.26 positioned at the boundary between the hospital and the community , psws were less sanguine in their assessments of the efficacy of physical treatments . in articles contributed to the bjpsw , some stressed that what psychiatrists might define as a medical recovery did not necessarily constitute a social recovery , and suggested that physical therapies could damage patients personalities and consequently destroy families . analysing the social consequences of physical treatments , madelene crump described cases where mrs o , who was brought into hospital in 1944 , began to improve in 1956 after a year s treatment on largactil . she discovered that her husband was living with another woman by whom he had had several children , and that her own children , who lived with him , had been told that she was dead . mrs l was brought into hospital in 1943 and was placed on largactil in 1955 . as her memory started to return , searches were made for her husband and children without any success , and her brother was discovered to have died two months previously . in this case , crump believed that part of her task was to help mrs l accept that for the present time at least , she has no family. mrs p , a widow admitted to hospital in 1948 , started to improve while taking largactil from 1957 . she was discharged and managed to gain work . however , the children s department refused to allow mrs p any contact with her children for fear of disturbing their home environment . crump believed that despite mrs p s longing to have her children back she was never likely to be stable enough to establish and maintain a normal home for them.27 in her analysis of how different psychiatric professions understood mental illness , shulamit ramon used crump s article to evidence her assertion that amongst psws there was no serious debate on the psychiatric means of intervention , which she attributed to the unquestioning acceptance of psychiatric authority by the psw.28 crump , ramon claimed , expressed her enthusiasm at the impact of the new drugs. read closely , the article provides a more unsettling perspective than ramon suggested , offering an insight into the palpable distress experienced by erstwhile long - stay patients and demonstrating the inability of the newly developed physical treatments , in isolation , to remedy interpersonal problems . other contributions to the bjpsw went further , suggesting that the damage caused by physical therapies could outweigh the therapeutic benefits . following women patients who had received leucotomy operations after they left hospital , psw mary lane found that many cases psychiatrists would describe as a clinical success had led to a collapse of the family because of changes in personality or behaviour caused by the operation.29 the husband in case d , for example , had became much more hostile after the operation , telling his wife that she was like an animal. before the leucotomy operation in case e , the husband had been devoted to his wife . after the operation , when the patient became gross in appearance , slack in personal habits and cleanliness but casually cheerful , the husband s hostility was kindled . i can not stand the smiling stranger in my house.30 in case g , the husband had insisted on the leucotomy operation for his wife . her obsessional phobic symptoms disappeared but she became lethargic and exhibited an inappropriate emotional response to situations. this infuriated her husband who turned her out of the house and refused to let her see their son.31 through these accumulated cases of marital breakdown , lane was able to demonstrate an overlooked consequence of the leucotomy operations . most of the husbands , she suggested , seemed better able to deal with the consequences to themselves of prolonged or recurrent illness in the spouse than with the indifference and disturbance which her post operative behaviour showed. other examples illustrated the destabilising impact of a leucotomy upon family relationship more broadly : lane cited these cases to support her suggestion that psychiatrists should inform families about the likely effects of the operation on the behaviour of the patient . in one such case , after the leucotomy , the sister became frustrated by the widow s complacency and casual attitude and complained that she was a different sister. the sister allied herself with a third sister and violent rows ensued . the therapeutic benefits of the treatment were brought further into question in the case of a widow who had been sent to live with her married daughter after her operation . the daughter found the changes in her mother so distressing that the widow had to be transferred back to the hospital . you ca nt converse with her anymore.32 edward shorter has depicted psychosurgery as an anomalous deviation from the progressive path of physical therapies.33 conversely , in his detailed study of psychosurgery , jack pressman demonstrated that leucotomies were by no means an aberration from the logic underpinning physical treatments . psychiatrists , he argued , viewed psychosurgery as therapeutically beneficial because it transformed demanding and troublesome patients into placid , manageable patients who would conform more readily to the regime of the hospital : such considerations would have been particularly compelling in an era of hospital overcrowding . pressman concluded that tranquilisers such as largactil , which slowly displaced psychosurgery , were widely adopted precisely because they produced very similar effects.34 pressman s observations are supported by anecdotal evidence gathered by diana gittins . michael wilson , who had worked at severalls hospital as a nurse , commented that patients after leucotomies were more tranquil , but once you d been attila the hun , with the leucotomies , you could nt put it right again. he recalled that leucotomies had fallen out of favour following the introduction of largactil , what we call the chemical leucotomies.35 unlike psychiatrists , psws were primarily concerned not with how patients behaved in the hospital but how they functioned socially outside of the hospital . by examining the impact on relationships between family members , psws such as lane and crump were able to question , not just the treatment method , but also the criteria by which psychiatrists measured recovery , suggesting that physical treatments could have a detrimental impact on the lives of patients and their families . psws working in mental hospitals attempted to alleviate the social problems arising in the lives of patients who had undergone physical treatments . those who worked for local authorities found that many of their cases had been discharged into their care because hospitals had deemed them to be beyond the help of psychiatric medicine . the psw working in the field of local authority community care , claimed one bjpsw article from 1960 , was likely to find that a high proportion of hopeless cases will come her way semi - stabilised psychotics , chronics of all descriptions , psychopaths , epileptics , dullards until the local authority office may even be regarded as some sort of benevolent dustbin.36 michael power , who worked for a local authority , complained that it was difficult for relatives to understand intellectually and accept emotionally the limitations of psychiatry , because of a natural tendency to regard specialists and hospitals as omnipotent and refuse to accept knowledge as limited and incomplete. nevertheless , he believed that there was often much that could be done to help such people , but socially rather than medically.37 power , and other psws working in this field , developed a model of psychiatric social treatment that aimed to build on what was healthy within their clients by adjusting their social surroundings and interpersonal relations . they described how such an approach enabled them to enhance the lives of people with enduring mental health problems , many of whom had been discarded by psychiatrists as beyond the help of medical treatment , and to challenge the efficacy of a medical approach that did not consider people s social needs and their lives within a community . describing her work with clients diagnosed with paranoid schizophrenia in the community , margaret ferard , who was employed by a psychiatric hospital , coined the phrase psychiatric social treatment , to distinguish her work from psychiatric treatment carried out primarily from a medical standpoint.38 she argued that if a psw was in possession of a professional skill that she consciously employs with a therapeutic aim , it must logically follow that she is in fact carrying out treatment. in contrast to the rationale underpinning physical treatments which aimed to cure incipient cases , ferard defined the objective of such treatment as less ambitious , frankly palliative , designed to help the patient to fit into the community as well as possible in spite of his symptoms.39 ferard focused on assessing and optimising her clients degree of mental health , instead of concentrating on their illness so that she might enable her clients to adjust to society . to prevent the family s anxieties from damaging the social adjustment of her cases , ferard found herself acting as a safety valve for both client and family.40 by focusing on capabilities rather than symptoms , ferard was able to assist her clients to gain employment , and consequently , more independence.41 psw eugene heimler developed ferard s emphasis on maximising the healthy aspects of her cases and restoring their social functioning further . witnessing at first hand how a sense of futility and purposeless had destroyed people s mental health while a prisoner at auschwitz , buchenwald and troglitz , heimler wondered if mental distress could be alleviated if people were given a sense of purpose.42 he sought to adjust the environment to suit individuals , arguing that even people with apparently crippling delusions could lead normal lives if given conditions that suited them.43 heimler drew on psychoanalytic theory to argue that the present could be utilised as a therapeutic tool to induce people to adopt a new pattern of functioning which would assist them to feel differently about the past , explaining satisfaction can alleviate past frustrations.44 heimler extended his analysis to a study of the relationship between an individual s satisfaction levels and their ability to function socially . he recognised that social isolation , prevalent amongst the elderly , might increase an individual s sense of purposelessness and induce mental distress . heimler discussed the case of mrs smith , a widow who had derived satisfaction from bringing up her children and caring for her husband , until her children had left home and her husband died : mrs smith now in her seventies had nothing to do but sit by her window and wait for her children to visit her . as she had lost all sense of purpose , her routine broke down she neglected herself and was generally careless ... mrs smith was admitted to an old people s home where her condition deteriorated . away from her familiar way of life and with no interests to occupy her time , her imagination soon got the better of her . gradually , she became more and more depressed and finally had to be admitted to a mental hospital , where she died.45 mrs smith now in her seventies had nothing to do but sit by her window and wait for her children to visit her . as she had lost all sense of purpose , her routine broke down she neglected herself and was generally careless ... mrs smith was admitted to an old people s home where her condition deteriorated . away from her familiar way of life and with no interests to occupy her time , her imagination soon got the better of her . gradually , she became more and more depressed and finally had to be admitted to a mental hospital , where she died.45 convinced that a sense of futility caused people to breakdown unless counterbalanced by their satisfactions in life , heimler created a social function scale test . this measured levels of satisfaction in the fields of family relationships , friendships , work and hobbies , sexual satisfaction and financial security . low satisfaction level scores indicated an individual s inability to function adequately in a social setting.46 by embracing the ability of the present to change the way a person may feel about negative events in the past , heimler adopted a more therapeutically optimistic attitude than ferard s palliative approach , attempting to sever the link between mental illness and the ability of an individual to function adequately in their community . as heimler explained to journalist christopher driver , people who were revealed to be hopelessly neurotic when scored by doctor eysenck s maudsley personality inventory could score correspondingly high on social functioning . the physical and social treatment approaches advocated by psychiatrists and psws were premised on apparently antithetical conceptualisations of disease causation . many psychiatrists in the 1940s and 1950s believed that mental illness had a biological aetiology and deployed physical treatments which targeted the individual patient as a clinical entity abstracted from his or her social environment . by contrast , most psws believed that mental illness could only be alleviated if social and environmental circumstances were addressed . thus , the psw studied the individual patient in the context of their family and social environment and advocated social psychiatric treatment . in some respects , psychiatrists and psychoanalysts seeking to rehabilitate and re - socialise soldiers suffering from neurosis at mill hill and northfield military hospitals experimented with group treatment methods , utilising interpersonal relations and the social environment of the hospital as a therapeutic tool . after the cessation of the second world war , some hospitals adopted this therapeutic approach , leading to the development of the concept of the therapeutic community.48 in practice , social and physical approaches to treatment frequently co - existed within hospitals , and the desirable clinical outcomes of physical and social treatments were often similar . describing how he had transformed claybury from a traditional authoritarian hospital into a therapeutic community , dennis martin explained that he was opposed not to physical treatments per se but to their deployment for controlling disturbed patients . he felt that physical and psychological treatments could profitably be combined and went on to suggest that many hospitals had neglected psychological or social treatments simply because they lacked sufficient doctors to carry out such treatment on an individual basis . for martin , the therapeutic community was the perfect mechanism to provide psychotherapeutic treatment to the entire patient population without necessitating a rise in staff numbers , offering a form of mass psychotherapy . 49 moreover , the first psws to qualify in the 1930s and 1940s aimed not to fulfil the individual needs of their clients , but to manage and reshape individuals so that they contributed to society : understanding the individual client meant understanding their individualised failings . in this sense , their work reflected a broader willingness to utilise the human sciences to understand , manage and enhance the behaviour of individuals and society.50 the american pioneer of psychiatric social work , mary jarrett , believed that psws were aptly trained to help maladjusted individuals adapt to the workplace . mental hygiene in industry , she wrote , attempted to attain the scientific large - scale production of individualisation.51 ultimately , the emerging profession of scientific management curbed this initiative and consequently psws were never employed in british industry;52 nevertheless , early british psws attempted to instil normative behaviour , seeing it as their responsibility to adjust individuals and families believed to be deviant to social norms . in 1932 , for example , one such case , r d , was described as essentially capable of contributing , as an intelligent and responsible citizen , to the community on which he has so long parasitically depended.53 throughout the 1940s and early 1950s , many psws had been careful to balance the needs of their clients with the perceived interests of society . e. l. thomas warned fellow psws in 1950 that concern for a patient s optimal readjustment can not be pursued to the point of jeopardising the welfare of others.54 molly harrington , who worked in a borstal institution , believed that psws should adopt the stance of a caseworker , not a reformer and accept the present stage of social opinion and , above all , of the work of the people operating the system.55 this authoritarian stance was reflected in the younghusband report , which claimed that the psw understood deviations from the normal and used a professional relationship in a disciplined way to carry out treatment.56 while psws recognised the individuality of their cases , they nevertheless considered it their role to adjust their clients to society . indeed , eliot slater neatly turned the table on critics of physical therapies to suggest that psychosocial approaches to mental illness failed to individualise the patient . many such practitioners , he argued are totally misled by bogus ideas getting their information from social knowledge which is knowledge about societies and groups , not about individuals.57 attempting to adjust the individual suffering from mental illness so that he or she conformed to their social environment was a long - standing objective in mental healthcare and transcended the perceived boundaries of physical and social treatment approaches.58 as pressman argued , many psychiatrists who advocated physical treatments also sought to re - socialise patients they viewed as maladjusted or maladapted , measuring success in terms of a patient s conformity or adjustment to hospital and ward routines.59 the therapeutic approaches may have been at opposite poles , but the objective was virtually identical . the social approaches advocated by psws were thus not unique within the field of psychiatric practice but were out of step with the dominant therapeutic trends of the 1940s and 1950 : the effort to assimilate mental illness with physical illness and to transform asylums into hospitals through the use of physical treatments . by the late 1950s and early 1960s , psws began to focus on the societal problems that might block an individual s adjustment and integration into society . in 1960 , the same year that the younghusband report was published , the apsw described psychiatric social work in a career pamphlet as a branch of social casework which is concerned with helping disturbed people and society adapt themselves to one another.60 as social therapist , the psw s role was now to mediate between the interests of the private individual and the wider public . in 1963 , the apsw launched a sustained attack on the idea that the individual experiencing mental distress should strive to adapt him or herself to society . urging her colleagues to adopt the role of reformer , apsw chairman irene spackman asserted that casework is not a panacea for all social ills.61 a report of the apsw s conference in new society explained : social workers are being asked to help people adjust to society in cases where society should be doing a better job for the individual .... when the welfare services fail , social workers are expected to make life bearable , but it is housing , national assistance and other national needs which are often unfulfilled . it can be said that adjustment is necessary because reality has to be accepted , but the social workers would like to do a little adjustment of reality and society for a change.62 social workers are being asked to help people adjust to society in cases where society should be doing a better job for the individual .... when the welfare services fail , social workers are expected to make life bearable , but it is housing , national assistance and other national needs which are often unfulfilled . it can be said that adjustment is necessary because reality has to be accepted , but the social workers would like to do a little adjustment of reality and society for a change.62 while william sargant complacently relegated psychiatric social work to the dustbin of history in his 1967 memoir , changes in mental healthcare policy and broader cultural developments had led to a reassessment of the place of social therapies in psychiatric practice . in 1961 , the government announced proposals for a reduction in the number of psychiatric hospital beds . as the government implemented a policy of hospital closure and sought to transfer services into the community , attention began to focus on the difficulties posed by long - stay hospital patients . physical treatments , which aimed to facilitate the management of patients in overcrowded hospitals by inducing calmer behaviour , were of limited use for practitioners seeking to deinstitutionalise long - stay patients . social psychiatrists , such as john wing , argued that such patients could only be socially reintegrated through a programme of rehabilitation which focused on employment , family and social functioning.63 the medical psychiatric approach also came under attack from the anti - psychiatry movement , informed by broader social and cultural trends that favoured a social approach to mental disorder.64 joshua bierer , who founded the marlborough day hospital , argued that many mental disorders involved the breakdown of an individual s socialisation skills within the family , the workplace or general interpersonal relationships.65 he believed that mental hospitals contributed towards the de - socialisation of those who experienced mental distress and criticised the increasing specialisation within medicine and psychiatry : we know more and more about less and less ! there is , however , a counter - movement towards specialisation , one which attempts to look at the total patient ; but even this is insufficient in our estimation . considering the total individual when working with patients , all factors are important cultural factors , constitutional factors , everything which has a bearing on interpersonal relations.66 we know more and more about less and less ! there is , however , a counter - movement towards specialisation , one which attempts to look at the total patient ; but even this is insufficient in our estimation . considering the total individual is not enough . when working with patients , all factors are important cultural factors , constitutional factors , everything which has a bearing on interpersonal relations.66 however , the growing appeal of social psychiatry and the shift in government policy was not matched by a commensurate investment in training specialised personnel or providing community - based services . in 1972 , the certificate of qualification in social work , introduced to train a new breed of generic social worker , replaced prior specialist training schemes for different branches of social work including psychiatric social work . drawing on interviews with health service users , peter barham and robert hayward demonstrated how the shortfall of specialised personnel and services affected the lives of people who experienced severe mental illness in the 1980s and 1990s.67 vaughan , for example , had lost possession of his flat during the four months he had been in hospital , but his doctor appeared unable or unwilling to recognise how vaughan s medical and social problems interacted . i said i had nowhere to go , vaughan recollected , to which his doctor responded well , there s nothing i can do , you re better now and you can go home.68 another interviewee , henry , was unemployed and lived in a council flat at the margins of a town . henry described how he believed that his diagnosis of schizophrenia would always make me a second - class citizen , despite the fact that his symptoms were largely under control.69 henry s account , barham and hayward asserted , illustrates not the natural consequences of mental illness , but the social consequences of becoming mentally ill the social pressures and constraints that have turned him into a person devoid of purpose and worth.70 addressing a meeting of the apsw in 1959 , child psychiatrist tom ratcliffe admonished his listeners for transcending their auxiliary role to provide interpretative analytical casework.71 there was a danger , he claimed , that the therapeutic approaches adopted by psws could become governed more by the training level and dare we say the professional ambitions of the therapist or caseworker , than by the level of therapy which is most appropriate to the client s needs and capacity.72 psws , ratcliffe implied , were medical auxiliaries responsible for providing care , not therapists who provided treatment . assertions that psws were practising psychiatric social treatment could be read as an attempt to challenge the status of medical auxiliaries and to substantiate claims to professional expertise and status.73 for psychiatrists , andrew scull has argued , the development and use of physical treatments was linked as much to questions of professional claims to expert knowledge , as to scientific advances . physical treatments , in short , embodied expert psychiatric knowledge and helped to sustain the status of the profession.74 shulamit ramon argued that psws were comfortable with their status as medical auxiliaries , followed the conceptual framework adopted by psychiatrists , adopted a pessimistic approach to working with adults , and embraced physical interventions with enthusiasm , but an examination of the apsw archives demonstrates that this was simply not the case.75 while some of the first psws to qualify suggested that the psw could assist psychiatrists , the apsw very rapidly sought to define the psw s distinctive sphere of expertise . in 1951 , for example , the apsw successfully resisted suggestions that psws should be registered as medical auxiliaries , claiming that such a designation would be inappropriate , as psws were social workers with roots in the social sciences.76 five years later , the apsw attacked the british medical association for evidence it had given to the working party on social workers . when the doctors emphasise so much a real sense of vocation and so little the acquisition of skills , they are perpetuating in the social field a state of affairs which would not be accepted in their own profession.77 within psychiatric social work , child guidance had long been seen as the most prestigious field of work because the psw could claim to be undertaking psychotherapeutic treatment with the mother of the child , often portrayed as a patient in her own right . it was in the unpromising environment of local authority mental health departments and psychiatric hospital work that psws moved beyond an attempt to adjust individuals to society to identify how social factors constrained recovery . in these fields , psws found themselves working with clients perceived to be bjpsw articles provide an insight into how psws working in this field sought to challenge these distinctions between care , cure and treatment . given the relatively small number of psws practising , the low circulation of the bjpsw , and the difficulty of ascertaining how representative individual contributors were of practice throughout the profession , these initiatives probably had little impact in practice . recalling his work with william mayer - gross in the late 1940s and early 1950s , psw cyril greenland described the scepticism he felt when mayer - gross expressed his belief that a cure for schizophrenia was imminent.78 the tantalising promise that the new physical therapies could cure mental illness proved a hollow illusion : although advocates of physical treatments , such as william sargant , emphasised their curative powers , many patients were , in practice , described as improved or relieved after physical treatments . psychiatrists made confident assertions regarding the somatic aetiology of mental illnesses despite having failed to identify an underlying biological cause : they devised physical treatments in a crude , empirical fashion and applied treatments in an equally haphazard fashion . indeed , many psychiatrists rationalised risky procedures of often limited or doubtful therapeutic value when treating chronic patients.79 the reports of psws working on the frontline of physical therapies in psychiatric hospitals illustrate that physical therapies had failed to remove the problems posed by enduring mental illness . the psychiatric social treatment approach advocated by margaret ferard vacillated uneasily between despondency and optimism . we might censure the pessimistic and , at times , disciplinary tone adopted in the articles written by psws working with people within hospitals or those discharged to the community , and suggest that psws perpetuated the image of the chronic , damaged mental patient and maintained traditional hierarchies of power . alternatively , we could interpret psychiatric social treatment as an inventive attempt to alleviate the difficulties experienced by people with enduring mental illness , whose symptoms had proved intractable in the face of physical therapies and whose needs were frequently neglected or marginalised by a paradigm of psychiatric practice keen to emphasis the curability of mental illness . the research undertaken for this paper was funded by the arts and humanities research council . i am grateful to david george , hilary marland , neil pemberton , mathew thomson , jonathan toms and mick worboys for their suggestions on earlier versions of this article , and would like to thank the anonymous referees for their helpful comments . participants at a social work history network meeting in 2008 also provided valuable feedback on an earlier version of this article .

seeking to align psychiatric practice with general medicine following the inauguration of the national health service , psychiatric hospitals in post - war britain deployed new treatments designed to induce somatic change , such as ect , leucotomy and sedatives . advocates of these treatments , often grouped together under the term physical therapies , expressed relief that the social problems encountered by patients could now be interpreted as symptomatic of underlying biological malfunction rather than as a cause of disorder that required treatment . drawing on the british journal of psychiatric social work , this article analyses the critique articulated by psychiatric social workers based within hospitals who sought to facilitate the social reintegration of patients following treatment . it explores the development of psychiatric social treatment , an approach devised by psychiatric social workers to meet the needs of people with enduring mental health problems in hospital and community settings that sought to alleviate distress and improve social functioning by changing an individual s social environment and interpersonal relationships . physical and social models of psychiatric treatment , this article argues , contested not only the aetiology of mental illness but also the nature of care , treatment and cure .

Introduction The Development of Psychiatric Social Work in Britain A Social Perspective on Physical Treatments Transforming the Benevolent Dustbin: Psychiatric Social Treatment and Community Care The Objectives of Physical and Social Treatments: Antithetical or Complementary? Conclusion: Treatment, Care, Recovery and Cure Acknowledgements

historian edward shorter viewed the introduction of the tranquiliser largactil in 1953 as a pivotal moment in psychiatric practice , initiating a revolution in psychiatry that transformed psychiatry from a branch of social work to a field that called for the most precise knowledge of pharmacology.1 in his 1967 autobiography , psychiatrist william sargant expressed similar sentiments , recalling how the introduction of largactil helped realise his dream of transforming psychiatric practice into a branch of general medicine . administer enough largactil to keep even the acutest schizophrenic tranquilised while electric shock treatment and other methods speeded their recovery.2 like shorter , sargant reinforced his point that psychiatry had progressed by drawing a comparison with earlier social work approaches to mental illness , inferring that psychiatric social work was obsolete . nowadays we may only need to prescribe four or five electric shock treatments , or a new course of some antidepressant drug.3 largactil belonged to a group of psychiatric therapies grouped together under the label of physical treatments . these treatment methods , which included insulin treatment , convulsion , malarial therapy , prefrontal leucotomy , sedatives and stimulants , were designed to alleviate psychological symptoms by inducing physiological change or by altering the structure of the brain.4 if we turn to accounts produced by practitioners of the relatively new profession of psychiatric social work disparaged in sargant s autobiography , we gain a different perspective on the impact of physical treatments on patients . based within a psychiatric hospital , one such psychiatric social worker ( hereafter psw ) madelene crump , found her work expanding when the new sedatives and tranquillisers of the 1950s , such as largactil , produced an increased number of cases deemed by psychiatrists to have recovered sufficiently to be discharged . commencing with an account of the development of psychiatric social work , the article will contend that psws , by virtue of their training and function , were uniquely placed to trace the repercussions of physical therapies through their work with discharged patients in the community . it explores how psws shared their concerns with colleagues via the british journal of psychiatric social work ( hereafter bjpsw ) , which was established in 1947 by the association of psychiatric social workers ( hereafter apsw ) publications sub - committee with the intention of providing a vehicle for the exchange of ideas regarding the methods of psychiatric social work.6 the bjpsw offered psws a space in which to forge a critical analysis of physical treatment methods . the article will then explore how some psws strove to assert their professional status and transcend their auxiliary function by arguing that the care and support they provided to patients and their families could fulfil needs unmet by physical treatments , drew upon a distinctive type of expertise , and could most accurately be conceptualised as psychiatric social treatment. this detailed analysis of specific cases prefaces a consideration of how the objectives of social and the article concludes by stressing the fluidity of the concepts of care , treatment , recovery and cure , tracing how the terms were mobilised in support of professional ideologies and aspirations . the roots of psychiatric social work can be traced back to the work of earlier charitable organisations that worked with those who experienced mental distress and their families within community settings.7 the growing acceptance of psychological explanations for people s behaviour and capabilities and the emergence of the mental hygiene movement also proved influential.8 social worker mary jarrett first developed professional training for social workers in psychiatric fields in 1914 at the boston psychopathic hospital , and psws became part of the team in the newly established child guidance clinics that aimed to prevent juvenile delinquency.9 these american developments dovetailed with the growing interest expressed in the problem child in britain.10 when the commonwealth fund agreed to finance the establishment of child guidance clinics in britain , it stressed the need to train social workers in a university setting . those who chose to do so worked either in child guidance clinics , where they undertook casework with mothers , or within psychiatric hospitals , where they compiled a social history of cases admitted and helped patients readjust following discharge from hospital.12 a report undertaken in 1951 found that only eight of the 331 psws working in britain were employed within the mental health departments of local authorities , where they provided support to people living in the community who experienced mental health problems , while 239 were employed in psychiatric social work in mental hospitals , general hospitals or child guidance clinics.13 eight years later , the younghusband report suggested that 325 psws would provide adequate coverage to local authority health services but noted that this field only employed 26 full - time psws.14 it was only by 1969 that the balance was redressed , with 257 psws engaged in community care , 264 in mental hospitals , and 259 working in child guidance clinics.15 with a membership dispersed geographically and occupationally , the apsw endeavoured to facilitate discussion amongst practitioners . from 1950 , the apsw distributed copies of the bjpsw to all subscribing members , facilitating the aspiration expressed by editor margaret ashdown that meetings of the association would discuss the content of the journal , stimulating the interest and initiative of the members.16 ashdown also felt the bjpsw might help publicise the achievements of the apsw to related professions , serving as a whispering gallery , by means of which our voices , which some of us feel to be so feeble , can be made to carry to our professional neighbours , without fear or strain.17 given the circulation of the initial edition of the bjpsw , these hopes appear misplaced : the initial print run of 1,000 copies in 1947 and 1948 had to be reduced to 600 copies by 1949 when it became clear that the apsw had over 200 copies left of each of the previous journals.18 membership of the apsw stood at only 398 by the end of 1948 . proponents of physical therapies , such as william sargant , claimed that mental illnesses had a somatic aetiology and wanted to bridge the gap between psychiatry and general medicine . in an introduction to physical methods of treatment , co - authored with fellow psychiatrist eliot slater nearly a decade before the introduction of largactil , sargant claimed that new treatment methods had transformed mental hospitals beyond recognition within a decade.19 physical treatments , asserted sargant and slater , produce their beneficial effects with greater speed and greater certainty than the older and more well - established psychotherapeutic methods.20 psychiatrists could now interpret the emotional distress and social problems experienced by patients as a product of underlying biological malfunction rather than a cause of their disorder . correspondingly , therapeutic practices designed to unpick the social roots of mental illness by delving into a patient s history or to alleviate the social consequences of mental distress could at best play a supporting role . although the board of control continued to regulate the mental health services until the passing of the 1959 mental health act , the nominal incorporation of psychiatric hospitals within the new national health service in 1948 presented an opportunity for psychiatrists to realign their professional activities with general medicine.21 this was also an era of rising patient numbers . overcrowding in mental hospitals was estimated nationally at fourteen per cent in 1950 ; at st andrew s hospital in norfolk , overcrowding had reached twenty - five and fifty per cent on men s and women s wards respectively between 1951 and 1957.22 as the resident population in mental hospitals peaked at 151,400 in 1954,23 many psychiatrists may well have been inclined to agree with sargant and slater regarding the incapacity of highly individual and time - consuming methods to deal with a large - scale problem , viewing physical treatments as more efficacious in terms of speed , convenience and certainty.24 other major psychiatric textbooks , such as clinical psychiatry , co - authored by william - mayer - gross , eliot slater and martin roth in 1954 , also emphasised the organic aetiology of mental illness and the value of physical therapies , largely dismissing the influence of social and psychological factors in mental distress.25 psw cyril greenland , who worked for mayer - gross between 1948 and 1955 , recalled his superior s conviction that since the causes of metal illness would sooner or later be revealed by the biological sciences , sociology and social work had a very limited role to play in psychiatry.26 positioned at the boundary between the hospital and the community , psws were less sanguine in their assessments of the efficacy of physical treatments . crump believed that despite mrs p s longing to have her children back she was never likely to be stable enough to establish and maintain a normal home for them.27 in her analysis of how different psychiatric professions understood mental illness , shulamit ramon used crump s article to evidence her assertion that amongst psws there was no serious debate on the psychiatric means of intervention , which she attributed to the unquestioning acceptance of psychiatric authority by the psw.28 crump , ramon claimed , expressed her enthusiasm at the impact of the new drugs. by examining the impact on relationships between family members , psws such as lane and crump were able to question , not just the treatment method , but also the criteria by which psychiatrists measured recovery , suggesting that physical treatments could have a detrimental impact on the lives of patients and their families . psws working in mental hospitals attempted to alleviate the social problems arising in the lives of patients who had undergone physical treatments . nevertheless , he believed that there was often much that could be done to help such people , but socially rather than medically.37 power , and other psws working in this field , developed a model of psychiatric social treatment that aimed to build on what was healthy within their clients by adjusting their social surroundings and interpersonal relations . they described how such an approach enabled them to enhance the lives of people with enduring mental health problems , many of whom had been discarded by psychiatrists as beyond the help of medical treatment , and to challenge the efficacy of a medical approach that did not consider people s social needs and their lives within a community . describing her work with clients diagnosed with paranoid schizophrenia in the community , margaret ferard , who was employed by a psychiatric hospital , coined the phrase psychiatric social treatment , to distinguish her work from psychiatric treatment carried out primarily from a medical standpoint.38 she argued that if a psw was in possession of a professional skill that she consciously employs with a therapeutic aim , it must logically follow that she is in fact carrying out treatment. in contrast to the rationale underpinning physical treatments which aimed to cure incipient cases , ferard defined the objective of such treatment as less ambitious , frankly palliative , designed to help the patient to fit into the community as well as possible in spite of his symptoms.39 ferard focused on assessing and optimising her clients degree of mental health , instead of concentrating on their illness so that she might enable her clients to adjust to society . to prevent the family s anxieties from damaging the social adjustment of her cases , ferard found herself acting as a safety valve for both client and family.40 by focusing on capabilities rather than symptoms , ferard was able to assist her clients to gain employment , and consequently , more independence.41 psw eugene heimler developed ferard s emphasis on maximising the healthy aspects of her cases and restoring their social functioning further . witnessing at first hand how a sense of futility and purposeless had destroyed people s mental health while a prisoner at auschwitz , buchenwald and troglitz , heimler wondered if mental distress could be alleviated if people were given a sense of purpose.42 he sought to adjust the environment to suit individuals , arguing that even people with apparently crippling delusions could lead normal lives if given conditions that suited them.43 heimler drew on psychoanalytic theory to argue that the present could be utilised as a therapeutic tool to induce people to adopt a new pattern of functioning which would assist them to feel differently about the past , explaining satisfaction can alleviate past frustrations.44 heimler extended his analysis to a study of the relationship between an individual s satisfaction levels and their ability to function socially . low satisfaction level scores indicated an individual s inability to function adequately in a social setting.46 by embracing the ability of the present to change the way a person may feel about negative events in the past , heimler adopted a more therapeutically optimistic attitude than ferard s palliative approach , attempting to sever the link between mental illness and the ability of an individual to function adequately in their community . thus , the psw studied the individual patient in the context of their family and social environment and advocated social psychiatric treatment . in some respects , psychiatrists and psychoanalysts seeking to rehabilitate and re - socialise soldiers suffering from neurosis at mill hill and northfield military hospitals experimented with group treatment methods , utilising interpersonal relations and the social environment of the hospital as a therapeutic tool . after the cessation of the second world war , some hospitals adopted this therapeutic approach , leading to the development of the concept of the therapeutic community.48 in practice , social and physical approaches to treatment frequently co - existed within hospitals , and the desirable clinical outcomes of physical and social treatments were often similar . in this sense , their work reflected a broader willingness to utilise the human sciences to understand , manage and enhance the behaviour of individuals and society.50 the american pioneer of psychiatric social work , mary jarrett , believed that psws were aptly trained to help maladjusted individuals adapt to the workplace . many such practitioners , he argued are totally misled by bogus ideas getting their information from social knowledge which is knowledge about societies and groups , not about individuals.57 attempting to adjust the individual suffering from mental illness so that he or she conformed to their social environment was a long - standing objective in mental healthcare and transcended the perceived boundaries of physical and social treatment approaches.58 as pressman argued , many psychiatrists who advocated physical treatments also sought to re - socialise patients they viewed as maladjusted or maladapted , measuring success in terms of a patient s conformity or adjustment to hospital and ward routines.59 the therapeutic approaches may have been at opposite poles , but the objective was virtually identical . the social approaches advocated by psws were thus not unique within the field of psychiatric practice but were out of step with the dominant therapeutic trends of the 1940s and 1950 : the effort to assimilate mental illness with physical illness and to transform asylums into hospitals through the use of physical treatments . in 1960 , the same year that the younghusband report was published , the apsw described psychiatric social work in a career pamphlet as a branch of social casework which is concerned with helping disturbed people and society adapt themselves to one another.60 as social therapist , the psw s role was now to mediate between the interests of the private individual and the wider public . it can be said that adjustment is necessary because reality has to be accepted , but the social workers would like to do a little adjustment of reality and society for a change.62 while william sargant complacently relegated psychiatric social work to the dustbin of history in his 1967 memoir , changes in mental healthcare policy and broader cultural developments had led to a reassessment of the place of social therapies in psychiatric practice . physical treatments , which aimed to facilitate the management of patients in overcrowded hospitals by inducing calmer behaviour , were of limited use for practitioners seeking to deinstitutionalise long - stay patients . social psychiatrists , such as john wing , argued that such patients could only be socially reintegrated through a programme of rehabilitation which focused on employment , family and social functioning.63 the medical psychiatric approach also came under attack from the anti - psychiatry movement , informed by broader social and cultural trends that favoured a social approach to mental disorder.64 joshua bierer , who founded the marlborough day hospital , argued that many mental disorders involved the breakdown of an individual s socialisation skills within the family , the workplace or general interpersonal relationships.65 he believed that mental hospitals contributed towards the de - socialisation of those who experienced mental distress and criticised the increasing specialisation within medicine and psychiatry : we know more and more about less and less ! drawing on interviews with health service users , peter barham and robert hayward demonstrated how the shortfall of specialised personnel and services affected the lives of people who experienced severe mental illness in the 1980s and 1990s.67 vaughan , for example , had lost possession of his flat during the four months he had been in hospital , but his doctor appeared unable or unwilling to recognise how vaughan s medical and social problems interacted . henry described how he believed that his diagnosis of schizophrenia would always make me a second - class citizen , despite the fact that his symptoms were largely under control.69 henry s account , barham and hayward asserted , illustrates not the natural consequences of mental illness , but the social consequences of becoming mentally ill the social pressures and constraints that have turned him into a person devoid of purpose and worth.70 addressing a meeting of the apsw in 1959 , child psychiatrist tom ratcliffe admonished his listeners for transcending their auxiliary role to provide interpretative analytical casework.71 there was a danger , he claimed , that the therapeutic approaches adopted by psws could become governed more by the training level and dare we say the professional ambitions of the therapist or caseworker , than by the level of therapy which is most appropriate to the client s needs and capacity.72 psws , ratcliffe implied , were medical auxiliaries responsible for providing care , not therapists who provided treatment . assertions that psws were practising psychiatric social treatment could be read as an attempt to challenge the status of medical auxiliaries and to substantiate claims to professional expertise and status.73 for psychiatrists , andrew scull has argued , the development and use of physical treatments was linked as much to questions of professional claims to expert knowledge , as to scientific advances . physical treatments , in short , embodied expert psychiatric knowledge and helped to sustain the status of the profession.74 shulamit ramon argued that psws were comfortable with their status as medical auxiliaries , followed the conceptual framework adopted by psychiatrists , adopted a pessimistic approach to working with adults , and embraced physical interventions with enthusiasm , but an examination of the apsw archives demonstrates that this was simply not the case.75 while some of the first psws to qualify suggested that the psw could assist psychiatrists , the apsw very rapidly sought to define the psw s distinctive sphere of expertise . in 1951 , for example , the apsw successfully resisted suggestions that psws should be registered as medical auxiliaries , claiming that such a designation would be inappropriate , as psws were social workers with roots in the social sciences.76 five years later , the apsw attacked the british medical association for evidence it had given to the working party on social workers . when the doctors emphasise so much a real sense of vocation and so little the acquisition of skills , they are perpetuating in the social field a state of affairs which would not be accepted in their own profession.77 within psychiatric social work , child guidance had long been seen as the most prestigious field of work because the psw could claim to be undertaking psychotherapeutic treatment with the mother of the child , often portrayed as a patient in her own right . recalling his work with william mayer - gross in the late 1940s and early 1950s , psw cyril greenland described the scepticism he felt when mayer - gross expressed his belief that a cure for schizophrenia was imminent.78 the tantalising promise that the new physical therapies could cure mental illness proved a hollow illusion : although advocates of physical treatments , such as william sargant , emphasised their curative powers , many patients were , in practice , described as improved or relieved after physical treatments . psychiatrists made confident assertions regarding the somatic aetiology of mental illnesses despite having failed to identify an underlying biological cause : they devised physical treatments in a crude , empirical fashion and applied treatments in an equally haphazard fashion . indeed , many psychiatrists rationalised risky procedures of often limited or doubtful therapeutic value when treating chronic patients.79 the reports of psws working on the frontline of physical therapies in psychiatric hospitals illustrate that physical therapies had failed to remove the problems posed by enduring mental illness . alternatively , we could interpret psychiatric social treatment as an inventive attempt to alleviate the difficulties experienced by people with enduring mental illness , whose symptoms had proved intractable in the face of physical therapies and whose needs were frequently neglected or marginalised by a paradigm of psychiatric practice keen to emphasis the curability of mental illness .

attention - deficit and hyperactivity - disorder ( adhd ) affects 58% of the worldwide childhood population , it has high comorbidity and it shares symptoms with other behavioral and emotional disorders ( boyle et al . , 2011 ; larson et al . , 2011 ) . adhd prevalence rates vary significantly between and within countries , and depend on the ascertainment method and criteria utilized . of particular concern is the marked variability in diagnostic rates of adhd in developed countries ( getahun et al . , 2013 ) . this may reflect increased awareness of teachers and parents to symptoms of adhd in communities with a focus on education and adequate health care . however , even within communities there is marked variability in rates of adhd due to the subjective nature of primarily descriptive ( symptom - based ) diagnostic procedures ( asherson et al . , 2012 ; polanczyk et al . , 2014 ; see also recent reports by the us center for disease control and prevention ) . such diagnostic heterogeneity and its poor reliability have hampered efforts to determine the pathophysiology of adhd ( morgan et al . , 2013 ) . attempts have been made to find neuroimaging - based biomarkers of adhd using structural magnetic resonance imaging ( johnston et al . 2013 ) , resting - state functional mri ( hoekzema et al . , 2014 ; tomasi and volkow , 2014 ) , fmri data acquired during a single cognitive task ( hale et al . , 2015 ; hart et al . , 2014a ; hart et al . , 2014b ) , or combinations of some of these techniques ( anderson et al . , 2014 ; these studies provide mixed insights regarding the possible underlying neurocognitive deficit of adhd , and they report moderate classification accuracies ( rarely exceeding 80% ) , insufficient for clinical diagnosis . adhd is characterized by behavioral symptoms and multiple cognitive deficits associated with context dependent abnormal patterns of neural activity distributed across multiple brain regions . consequently , adhd is unlikely to be characterized by localized structural brain abnormalities robust enough to be detected using available structural imaging techniques . it is also unlikely that significant functional brain abnormalities characterizing adhd would be evident regardless of the mental state of the test subject , during the scan ( castellanos and proal , 2012 ; hammer et al . , 2015 ) . key characteristics of adhd include poor working memory , greater reliance on external feedback , and abnormal reward processing . these are associated with altered patterns of activity in distinct brain networks : ( i ) attention and working memory , comprising the dorsolateral prefrontal cortex , parietal cortex and temporal cortices ( burgess et al . , 2010 ; ehlis et al . , 2008 ; vance et al . , 2007 ) ; ( ii ) executive functions and cognitive control ( including feedback processing and response selection ) , comprising the dorsal , medial and ventral frontal cortices ( booth et al . , 2005 ; clark et al . , 2007 ; sonuga - barke and fairchild , 2012 ) ; and ( iii ) reward - processing , comprising the orbitofrontal cortex , anterior cingulate cortex and basal ganglia ( del campo et al . , 2013 ; plichta et al . notably , there are substantial individual differences in reactions to reward and feedback manipulation in adhd ( demurie et al . , 2011 ; , 2015 ; hammer et al . , 2015 ; plichta and scheres , 2014 ; van der schaaf et al . , accordingly , we hypothesized that using fmri data from an ensemble of visuospatial working memory ( vswm ) tasks that differ in the motivational context ( determined by the availability of reward and feedback ) would increase the odds that abnormal patterns of brain activity would be reliably detected in a larger proportion of adhd cases . we expected this to enable more accurate detection of adhd cases than the accuracies obtained by using fmri data acquired from any single vswm task , or the accuracy based on the participant 's respective behavioral performance . given the central role of vswm in adhd , we compared fmri data from four distinct vswm tasks in boys with adhd and typically developed ( td ) boys . tasks differed in the availability of trial - by - trial feedback ( feedback versus no - feedback ) and the participant 's expectation for significant monetary reward ( large versus small ) . all tasks required tracking the spatial location of letters while ignoring the letters ' identity , and executing timely responses . the manipulation of feedback and reward provided different motivational contexts , each requiring somewhat distinct executive skills found to be impaired in children with adhd . we used a multimodal analysis based on relatively few brain regions of interest , discovered using an independent univariate analysis ( hammer et al . , 2015 ; morris et al . , 2012 ; mulligan et al . , 2011 ; thoma and henson , 2011 ) . using a sparse principal component analysis , we further reduced the number of variables that were provided as input to the classifier . this substantially limited the odds of discovering an overfitted adhd classification model , and simplified the interpretation of the discovered model . we used a logistic regression ( lr ) classifier , which directly models the class conditional probabilities for each case ( i.e. , calculating the predicted probability that a given child has adhd ) by attempting to find a model allowing a decisive classification of as many cases as possible . this attribute is important in clinical settings where we aim to find a model that allows classification with confidence ( nouretdinov et al . , 2011 ) . twenty boys with a diagnosis of adhd combined - type ( mean age in years = 10.42 sd = 0.80 ) and 20 typically developed ( td ) boys ( 10.96 0.91 ) participated in the experiment . participants gave their informed consent ( and parental consent ) in accordance with the policies of the institutional review board ( irb ) at northwestern university . at the time of the fmri scanning session , adhd youth were weaned off stimulant medication for at least 24 h ( 12 participants used prescribed stimulants on a regular basis during the time they participated in this study ) . adhd diagnoses were based on exceeding the clinical cut offs on the adhd rating scale ( dupaul et al . , 1998 ) and the semi - structured diagnostic interview , the kiddie schedule for affective disorders and schizophrenia for school aged children : present and lifetime ( k - sads - pl ) version ( kaufman et al . , 1997 ) . mean total adhd score was higher in the adhd group than in the td group ( supplemental table 1 ) . participants were excluded if they had been diagnosed with a neurological disorder or were treated medically for a comorbid psychiatric disorder . all participants were right - handed native english speakers with normal or corrected to normal vision . mean full - scale iq scores were within normal range in both groups , however the average iq in the adhd group was lower than the average iq in the td group ( supplemental table 1 ) , which is not uncommon in adhd studies ( e.g. , hart et al . , 2014a ) . each task was 48 trials long . before and after each 2-back task the participant performed a fixation task where he was asked to press a key whenever the fixation - cross changed its color ( which happened in 4/12 of the trials ) . e - prime 2.0 ( psychology software tools , inc . ) was used for stimuli presentation and for recording participants ' responses ( for more details about the experimental design , see hammer et al . , 2015 ) . the two independent factors in the study were reward size ( large - reward versus small - reward ) and presence of trial - by - trial feedback ( no - feedback versus feedback ) . in an earlier practice session , and at the beginning of the scanning session , each participant was instructed that the reward for each correct decision in the large - reward task was 10 times larger than in the small - reward task . in the trial - by - trial feedback task , each key - press was followed by either a green square ( indicating a correct decision ) or a red square ( indicating an incorrect decision ) presented in the center of the screen . in the no - feedback task , the participant was informed about his overall performance only after concluding the task ( fig . 1 ) . imaging data were acquired on a 3.0 tesla siemens tim trio scanner using a 12-channel head coil . a susceptibility weighted single - shot epi ( echo planar imaging ) method with bold ( blood oxygenation level - dependent ) was used for functional image acquisition with the following scan parameters : tr = 2000 ms , te = 20 ms , flip angle = 80 , matrix size = 128 120 , field of view = 220 206.3 mm , slice thickness = 3 mm ( 0.48 mm gap ) , and number of slices = 32 ( an effective functional voxel size of 2 2 4 mm ) . a total of 145 images ( trs ) a high resolution , t1 weighted 3d image was also acquired with the following parameters : tr = 2300 ms , te = 3.36 ms , flip angle = 9 matrix size = 256 256 , field of view = 256 mm , slice thickness = 1 mm , and number of slices = 160 . the acquisition of the anatomical scan took approximately 9 min . prior to the scanning session children this enabled confirming that the participant is capable of keeping his head still for the duration of the scanning session . to minimize head movements in the scanner , gaps between the participant 's head and the head - coil data analysis was performed using mathworks matlab , spm8 ( statistical parametric mapping , wellcome trust centre for neuroimaging , london , uk ) , and ibm spss . preprocessing involved : ( i ) slice timing ; ( ii ) realignment of all functional images to the 24th image . ( iii ) co - registration of the functional and anatomical images ; ( iv ) normalization of the t1 image to the mni305 template image , which is most commonly used also for analyzing fmri data of pediatric populations ( e.g. , burgund et al . ( v ) 4 4 8 mm full width half maximum ( fwhm ) gaussian kernel smoothing . ( vi ) we confirmed that movement was kept below 4 mm ( in any of the x , y , or z dimensions ) within a scan using the artrepair software . images ( up to 9 per scan ) were realigned in artrepair , using interpolated values from the two adjacent non - outlier images . for subsequent general linear model ( glm ) analyses , the excluded noisy images were deweighted . as reported in supplemental table 2 , the two groups did not differ in patterns of head movements , and the replacement of outlier images primarily enabled reducing the signal to noise ratio in the fmri data of both groups . in order to further reduce within scan variability in neural activity , only trials in which the participant responded correctly ( hit and correct rejection ) were modeled , with onset time - locked to the beginning of each trial ( calhoun et al . ( vii ) a high pass filter with a cut - off of 256 s was applied . structural and functional brain images were inspected and found not to have significant image artifacts . we confirmed that for each participant maximal head displacement ( the distance between the two most distant fmri images within a scan ) in all translational axes was no larger than the size of a voxel ( i.e. , 4 mm ) . there were no significant between - groups differences in any head translation or rotation axes , all p > 0.25 ( supplemental table 2 ) . we also confirmed that it is unlikely that head movements underlie the adhd classification results we got based on the brain activity data ( supplemental fig . 1 ) . feature detection was based on a univariate glm analysis , intended to identify functional brain regions of interest ( frois ) that showed significant activation or deactivation in all four vswm tasks , contrasted with all the fixation tasks , using the brain activity data of the adhd boys and td boys combined ( total of 40 participants ; see chu et al . , 2012 ; friston , 2012 for discussions regarding optimal sample size ) . we found eight frois showing significant activation ( vswm > fixation ) and eight frois showing significant deactivation ( fixation > vswm ) . we used a voxel threshold of p < 0.01 ( family wise error [ fwe ] corrected ) , and voxel cluster threshold of p < 0.01 ( minimum cluster size of 50 voxels ; fwe ) , where each cluster had a single significant peak at p < 0.05 ( fwe ) . an anatomical gray - matter mask ( using the talairach daemon brain atlas gray - matter mask , with dilate = 3 ) , and a sphere mask with a radius of 15 mm from each froi peak voxel , constrained the froi volume . these 16 rois likely reflect the vswm network in a broad childhood population , in varying contexts ( fig . for each participant we calculated the difference in mean beta values between each of the four vswm 2-back tasks and the mean of all fixation tasks in each of the 16 frois ( 2-back fixation ; fixation2-back ) . overall , given four vswm tasks and 16 frois , the initial number of features , characterizing each participant , was 64 ( see supplemental fig . 2 for activation profiles ) . given the relatively large initial number of features , and the high correlations between some of the features ( see supplemental fig . 3 for the correlation matrix ) , we reduced the dimensionality of the data by using a sparse principal component analysis ( spca ) . this resulted with a relatively small number of orthogonal principal components ( pcs ) , which together explained most of the variability in the data , recalculated based only on the few features with the highest loadings ( berthet and rigollet , 2013 ; ritchie et al . this procedure has three major advantages : ( i ) spca enabled reducing the number of variables fed to the classifier to a number substantially smaller than the number of participants ; ( ii ) recalculating the pcs based only on features with the highest loadings enabled excluding lower weight features that were likely to add mostly noise ; and ( iii ) having each pc being affected by relatively few features , where each feature affects at most one pc , enabled better determining the underlying neurocognitive mechanisms represented by each pc . we used sparse loading selection based on thresholding of the rotated loadings ( loading threshold > 0.6 ; absolute weights > 0.1 ) . loading rotation was based on the varimax rotation method ( lu and zhang , 2012 ; ma , 2013 ; qi and luo , 2015 ; sjstrand et al . , 2006 ) . the use of this threshold resulted with 39 features ( out of 64 ) affecting the first 10 pcs ( pcs with eigenvalues > 2 ) , where each feature affected a single pc ( fig . the first 10 pcs explained approximately 70% of the variability evident in the original 64 features . each of the remaining pcs explained less than 3% of the variability in the data . interestingly , we found each one of the first 10 pcs to reflect brain activity from several rois from the same vswm task , but not brain activity in a specific roi in several tasks . the exception was pc-2 , which was based on the left and right middle frontal gyri in the two vswm tasks without feedback . this supports our hypothesis that functional brain - imaging data from several distinct tasks is likely to add information useful for classification . for the learning of the adhd classification model the lr directly models the class conditional probabilities for each case , attempting to decisively classify as many cases as possible . a standard , 2013 ; ponce - alvarez et al . , 2012 ) was used for finding which of the 10 pcs significantly contributed to classification accuracy . in each cross validation iteration the lr classifier was trained based on the fmri data of 39 participants , and then the goodness of fit of the learned model was evaluated based on the correlation between the clinician diagnosis of the left - out participant ( adhd or td ) and the lr classifier predicted adhd probability for this participant . the predictive power from all iterations was averaged to determine which of the 10 pcs are with statistically significant predictive power , and for estimating the predictive power of the final model . the reporting of a classification model based only on significant pcs has two advantages : ( i ) such a model is less likely to be overfitted or biased due to being based on too many predictors ( pcs ) , and thus it provides a more conservative estimate of the classification accuracies that can be achieved with the data at hand and ( ii ) it further reduces the number of brain regions by which the two groups of interest ( i.e. , adhd vs. tds ) differ , enabling a more parsimonious characterization of the differences in neurocognitive mechanisms between the two groups ( see supplemental fig . 5 for an illustration of the data processing pipeline ) . running the lr with the leave - one - out cross validation showed a statistically significant contribution for adhd classification accuracies for only four pcs ( pc-2 , p < 0.02 ; pc-3 , p < 0.04 ; pc-4 , p < 0.04 ; pc-8 , p < 0.02 ) . excluding each one of these four pcs from the classification model substantially impaired the classification accuracies , whereas adding any of the other six pcs did not increase the classification accuracies . the classification accuracy of the model based on the four statistically significant pcs was 92.5% , with 95% sensitivity and 90% specificity . importantly , most of the classification decisions had high predicted probability values ( pp > 0.67 ) assigned to most adhd boys ( 75% ) , and low values ( pp < 0.33 ) assigned to most td boys ( 80% ; fig . 3 ) , indicating a model with an excellent fit ( omnibus test for model fit , (4 ) = 28.52 , p < 0.0001 ) . this accuracy level is not statistically different from perfect accuracy reflecting the clinician 's diagnosis ( 100% ) , p = 0.12 ( one - tailed fisher exact test ; testing the hypothesis a permutation test shows that the classification accuracies of the above - described adhd classification model are unlikely to be discovered by chance . in each permutation , labels of half the cases from each group were switched with the opposite group . the mean accuracy of 15 distinct permutations ( 62.0% ) was significantly lower than that of the adhd model ( 92.5% ) , p = 0.000 ( exact test ) ; t(14 ) = 14.97 , p < 0.0001 ( one - tailed , one - sample t - test ) . even the highest observed accuracy of a permuted - labels model was close to being significantly lower ( 15% ) than that of the adhd model , p = 0.06 ( one - tailed fisher exact test ; testing the hypothesis adhd model is better than the best permuted model ) . the mean correct decisive classification accuracies ( with pp < 0.33 or pp > 0.67 ) in the 15 permutations were 24.5% , versus 77.5% of the adhd model , p = 0.000 ( exact test ) , t(14 ) = 13.59 , p < 0.0001 ( one - tailed , one - sample t - test ; see also supplemental fig . 4 ) . notably , the four significant pcs encompassed in the adhd model are based on fmri data from all four vswm tasks : pc-2 is based on the two tasks without feedback ; pc-3 is based on the small - reward with feedback task ; pc-4 is based on the large - reward with feedback task ; and pc-8 is based on the small - reward with feedback task ( fig . 2 ) . in the following analysis , we further investigated the utility of using fmri data from multiple vswm tasks as compared with using fmri data from a single task . here we used the threshold of eigenvalue larger than one ( keeping pcs that together explained more than 70% of the variance in the data ) . adhd classification accuracies based on the fmri data from each single task ( fig . 4 ; table 1 ) were significantly lower than the classification accuracy of the model based on all four tasks ( fig . 3 ) . the exception was the classification accuracy based on the fmri data from the small - reward with feedback task , which was 80% . however , here the correct decisive classification accuracy ( with pp < 0.33 or pp > 0.67 ) based on the fmri data from the small - reward with feedback vswm task was 60% , which is significantly lower ( 17.5% ) than the four - task classification accuracy , p < 0.05 ( one - tailed fisher exact test ) . feeding the lr classifier with the behavioral data from all four tasks resulted in an overall accuracy level of 75% ( 70% sensitivity ; 80% specificity ; omnibus test for model fit , (4 ) = 12.37 , p < 0.01 ) . this overall accuracy level is significantly lower ( 17.5% ) than the accuracy based on the fmri data from the four tasks , p < 0.04 ( one - tailed fisher exact test ; testing the hypothesis fmri model is better than the behavioral model ) . moreover , here only 57.5% of the classification decisions were both accurate and decisive , with high predicted probability values of being a boy with adhd ( pp > 0.67 ) assigned to only 50% of the adhd boys , and low values ( pp < 0.33 ) assigned to only 65% of the td boys ( fig . this accuracy level is significantly lower ( 20% ) than the decisive classification accuracy based on the four significant pcs ( based on four tasks ) , p < 0.04 ( one - tailed fisher exact test ) . performance levels in the four tasks were highly correlated , where a boy that exhibited low performance in one task likely exhibited low performance in the other tasks . moreover , we found high correlations between the behavioral performances in the four vswm tasks and pc-2 ( fig . that is , the brain activity represented by pc-2 ( right and left mfg , tasks without feedback ) explains much of the observed variability in the behavioral data . on the other hand , the behavioral performances had low correlations with pc-3 , pc-4 and pc-8 , hence the additional diagnostic information provided by these pcs to the classification model . feeding the lr classifier with both the behavioral and fmri data ( 10 pcs ) resulted in exactly the same model as when feeding it with only the fmri data . we show that using a logistic regression ( lr ) classifier , which received as input brain imaging data that was acquired while children perform four distinct vswm tasks , enabled an adhd classification accuracy of 92.5% ( fig . 3 ) . classification accuracies that were based on the fmri data from all four tasks were significantly higher than those obtained using the fmri data from any single task . we found that the brain regions in which boys with adhd exhibited altered pattern of brain activity differed from one task to the other . the corresponding behavioral data from the four vswm tasks enabled an classification accuracy level of 75% ( fig . 5 ) , which is significantly lower than those obtained using the fmri data from the four tasks . participants who were misclassified based on the data from one vswm task were not necessarily misclassified when using the data from other vswm tasks ( fig . when the lr classifier was fed with the pcs calculated based on the four vswm tasks together , we observed a substantial improvement in the classification accuracy ( fig . this is consistent with the underlying theorem of ensemble - based classification ( rokach , 2010 ; klppel et al . , 2012 ) . as expected based on this theorem , classifying participants by using several measurements for each participant ( e.g. , fmri data from few distinct vswm tasks ) , where each measurement enables a better than chance classification accuracy , and where there is a substantial degree of independency between the measurements ( e.g. , distinct cognitive tasks ) , enabled higher classification accuracies compared to accuracies achieved by using the data from each single measurement . our findings are with important theoretical contribution , providing additional support to the idea that the manifestation of neurocognitive abnormalities in adhd is context dependent ( dovis et al . specifically , in the absence of trial - by - trial feedback , altered activity patterns characterizing adhd were most evident in the bilateral middle frontal gyri ( mfg ) , specifically the right - mfg ( see pc-2 weight in fig . 3 ; see feature weights in fig . the mfg is believed to play an executive role in the visuospatial working memory network , and a primary role in volitional ( top - down ) allocation of attention ( burgess et al . we found that in vswm tasks without feedback , boys with adhd exhibited lower levels of activity in the mfg as compared with td boys . this may indicate poor vswm and poor capacity in allocating attention to target stimuli in children with adhd , manifested when feedback is absent ( see cortese et al . the fmri data from the small - reward with feedback vswm task also had a considerable contribution to adhd classification ( pc-8 and pc-3 ; fig . 3 ; fig . here , boys with adhd exhibited altered brain activity in a network ( pc-8 ) that primarily included the bilateral orbitofrontal cortex ( bi - ofc ) and the left fusiform gyrus ( left - ffg ) . the ofc has been reported to play two primary roles that have potential relevance to the current task : together with the inferior frontal gyrus , the anterior insula , the superior temporal cortex and the temporoparietal junction , the ofc is part of the ventral attention network , acting as a bottom - up saliency detection system determining subjective and context - dependent susceptibility to unexpected salient stimuli ( corbetta et al . , 2008 ; the ofc was also found to be involved in the processing of reward - related information ( schoenbaum and roesch , 2005 ; pauli et al . , 2012 ) . it is suggested that the ofc is involved in monitoring which recent actions were rewarded , and predicting which future actions are most likely to be rewarded ( kahnt et al . , 2010 ) . the left - ffg was found to be involved in letter identification and reading ( mccandliss et al . , 2003 ; dehaene and cohen , 2011 ; mcnorgan et al . , 2013 here we found greater deactivation in these two brain regions in td boys , as compared to boys with adhd , primarily in the small - reward with feedback vswm task . this may indicate that boys with adhd fail in suppressing task irrelevant information ( letter identity in a task that requires monitoring the spatial location of letters , and visual feedback indicating insignificant reward ) . the features with significant loadings on pc-3 ( small - reward with feedback ) were the two frois in the right medial / superior prefrontal gyrus ( right - mefg- and right - mefg+ , fig . 2b ) , the left - mfg , the right superior temporal gyrus ( right - stg ) , the right anterior insula ( right - antins ) , and the right supramarginal gyrus . these brain regions are involved in feedback processing ( ferdinand and opitz , 2014 ) , bottom - up attention and in sensory integration ( prado et al . the loadings on pc-3 ( and pc-8 ) , indicate that in the small - reward with feedback condition children with adhd are likely to exhibit altered suppression of irrelevant visual features ( ffg ) , altered visuospatial processing ( precuneus ) , altered sensory integration ( stg and supramarginal ) , altered bottom - up attention control ( ofc ) , altered feedback processing and top - down attention control ( mefg and mfg ) , and altered synchronization between bottom - up and top - down attention control ( antins ) . the fmri data from the large - reward with feedback vswm task had the smallest contribution to adhd classification ( 57.5% accuracy , with only a trend toward a significant model fit ; fig . altered activity in adhd in this vswm task was evident in pc-4 ( fig . 3 ) , which included the right inferior parietal lobe ( right - ipl ) , the right - antins , the right - mefg ( the activated right - mefg froi ; see fig . 2 ) and the right precuneus . these frois partially overlap ( right - antins and right - mefg ) with those included in pc-3 ( small - reward with feedback ) . this implies that the right - antins and right - mefg are associated with altered feedback processing in adhd ( regardless of reward expectation ) . this is consistent with earlier findings showing that the right - antins and the right - mefg ( together with the anterior cingulate ) mediate between the central executive network and brain regions involved in risk / gain prediction , where response selection is required ( menon and udin , 2010 ; preuschoff et al . , 2008 ; early studies showed that children with adhd exhibit poor cognitive control associated with lower levels of neural activity in the anterior insula , as compared with tds ( cubillo et al . we show that using fmri data from four distinct vswm tasks enabled substantially better classification accuracies than the use of fmri data from a single vswm task . nevertheless , the current investigation was limited to the manipulation of feedback and reward in vswm tasks , where many characteristics of the four tasks were identical . it is possible that an even better adhd classification can be achieved by using other , perhaps more demanding cognitive tasks that require other cognitive skills impaired in adhd ( e.g. , tasks with specifically greater response inhibition demands ; booth et al . , 2005 ; rubia et al . , 2005 moreover , the duration of each vswm task we used here was 48 trials long ( ~100 s ) . it is possible that these tasks can be shortened ( e.g. , reduced to 32 , or even fewer trials ) without compromising classification accuracies . as part of the development of a practical diagnosis tool , future investigations should aim to identify an optimized ensemble of cognitive tasks that would yield the highest classification accuracies in the shortest scanning time possible . future investigation may also involve looking for an ensemble of tasks that enable differentiation between children with adhd from other clinical populations , with shared symptoms . the use of multi - task - based fmri data may also enable earlier diagnosis of adhd , prior to the onset of clear behavioral symptoms , which in turn may enable earlier intervention . in order to be of practical clinical use , task - based fmri diagnosis requires the scanned participants to accurately perform a cognitive task while keeping still throughout a relatively long scan . thus , a multi - task - based fmri diagnosis session may not be practical for very young children or individuals with severe cognitive deficits . however , mild adhd and moderate adhd are much more common and represents more of a diagnostic dilemma for clinicians . in contrast , severe or very early onset adhd would likely have a more distinct etiology . future developments should ideally involve an integrative use of multi - task - based fmri , resting - state fmri and structural imaging . this would likely enable an effective diagnosis of most clinical cases , and the detection of the onset of some clinical condition in early childhood . ideally , future imaging - based diagnostic tools may enable finer differentiation of adhd subgroups , assisting clinicians in customizing intervention . it is likely that similarly high ( or even higher ) classification accuracies can be achieved by using other machine - learning based classification methods . future studies should specifically explore machine - learning methods based on whole brain data , from multiple tasks , which automatically detect brain regions in which the targeted clinical population exhibits abnormal patterns of brain activity ( e.g. , ryali et al . we show that fmri data acquired while participants perform a few distinct cognitive tasks enables substantial improvement in the detection of adhd cases , as compared with the use of fmri data from a single task ( or corresponding behavioral data ) . showing that fmri data enables better classification accuracies than the corresponding behavioral data suggests that even adhd cases that exhibited normal - like vswm performance were likely to be characterized by substantially altered pattern of brain activation . this provides a proof - of - concept that scanning subjects while they perform an ensemble of distinct cognitive tasks is likely to payoff , enabling greater accuracies and higher confidence diagnosis of clinical cases . we suggest that this approach can be used for diagnosing other clinical populations , and possibly also for dissociating between distinct clinical populations who share behavioral symptoms . this would require using cognitive tasks that target the neurocognitive deficits characterizing the clinical condition of interest , or a battery of tasks that may enable dissociating between distinct neurocognitive abnormalities .

finding neurobiological markers for neurodevelopmental disorders , such as attention deficit and hyperactivity disorder ( adhd ) , is a major objective of clinicians and neuroscientists . we examined if functional magnetic resonance imaging ( fmri ) data from a few distinct visuospatial working memory ( vswm ) tasks enables accurately detecting cases with adhd . we tested 20 boys with adhd combined type and 20 typically developed ( td ) boys in four vswm tasks that differed in feedback availability ( feedback , no - feedback ) and reward size ( large , small ) . we used a multimodal analysis based on brain activity in 16 regions of interest , significantly activated or deactivated in the four vswm tasks ( based on the entire participants ' sample ) . dimensionality of the data was reduced into 10 principal components that were used as the input variables to a logistic regression classifier . fmri data from the four vswm tasks enabled a classification accuracy of 92.5% , with high predicted adhd probability values for most clinical cases , and low predicted adhd probabilities for most tds . this accuracy level was higher than those achieved by using the fmri data of any single task , or the respective behavioral data . this indicates that task - based fmri data acquired while participants perform a few distinct vswm tasks enables improved detection of clinical cases .

Introduction Materials and methods Results Discussion

attention - deficit and hyperactivity - disorder ( adhd ) affects 58% of the worldwide childhood population , it has high comorbidity and it shares symptoms with other behavioral and emotional disorders ( boyle et al . attempts have been made to find neuroimaging - based biomarkers of adhd using structural magnetic resonance imaging ( johnston et al . , 2014 ; tomasi and volkow , 2014 ) , fmri data acquired during a single cognitive task ( hale et al . , 2014b ) , or combinations of some of these techniques ( anderson et al . , 2014 ; these studies provide mixed insights regarding the possible underlying neurocognitive deficit of adhd , and they report moderate classification accuracies ( rarely exceeding 80% ) , insufficient for clinical diagnosis . key characteristics of adhd include poor working memory , greater reliance on external feedback , and abnormal reward processing . these are associated with altered patterns of activity in distinct brain networks : ( i ) attention and working memory , comprising the dorsolateral prefrontal cortex , parietal cortex and temporal cortices ( burgess et al . , accordingly , we hypothesized that using fmri data from an ensemble of visuospatial working memory ( vswm ) tasks that differ in the motivational context ( determined by the availability of reward and feedback ) would increase the odds that abnormal patterns of brain activity would be reliably detected in a larger proportion of adhd cases . we expected this to enable more accurate detection of adhd cases than the accuracies obtained by using fmri data acquired from any single vswm task , or the accuracy based on the participant 's respective behavioral performance . given the central role of vswm in adhd , we compared fmri data from four distinct vswm tasks in boys with adhd and typically developed ( td ) boys . tasks differed in the availability of trial - by - trial feedback ( feedback versus no - feedback ) and the participant 's expectation for significant monetary reward ( large versus small ) . we used a multimodal analysis based on relatively few brain regions of interest , discovered using an independent univariate analysis ( hammer et al . this substantially limited the odds of discovering an overfitted adhd classification model , and simplified the interpretation of the discovered model . we used a logistic regression ( lr ) classifier , which directly models the class conditional probabilities for each case ( i.e. twenty boys with a diagnosis of adhd combined - type ( mean age in years = 10.42 sd = 0.80 ) and 20 typically developed ( td ) boys ( 10.96 0.91 ) participated in the experiment . mean total adhd score was higher in the adhd group than in the td group ( supplemental table 1 ) . the two independent factors in the study were reward size ( large - reward versus small - reward ) and presence of trial - by - trial feedback ( no - feedback versus feedback ) . in an earlier practice session , and at the beginning of the scanning session , each participant was instructed that the reward for each correct decision in the large - reward task was 10 times larger than in the small - reward task . in the trial - by - trial feedback task , each key - press was followed by either a green square ( indicating a correct decision ) or a red square ( indicating an incorrect decision ) presented in the center of the screen . in the no - feedback task , the participant was informed about his overall performance only after concluding the task ( fig . a susceptibility weighted single - shot epi ( echo planar imaging ) method with bold ( blood oxygenation level - dependent ) was used for functional image acquisition with the following scan parameters : tr = 2000 ms , te = 20 ms , flip angle = 80 , matrix size = 128 120 , field of view = 220 206.3 mm , slice thickness = 3 mm ( 0.48 mm gap ) , and number of slices = 32 ( an effective functional voxel size of 2 2 4 mm ) . to minimize head movements in the scanner , gaps between the participant 's head and the head - coil data analysis was performed using mathworks matlab , spm8 ( statistical parametric mapping , wellcome trust centre for neuroimaging , london , uk ) , and ibm spss . ( iii ) co - registration of the functional and anatomical images ; ( iv ) normalization of the t1 image to the mni305 template image , which is most commonly used also for analyzing fmri data of pediatric populations ( e.g. ( vi ) we confirmed that movement was kept below 4 mm ( in any of the x , y , or z dimensions ) within a scan using the artrepair software . as reported in supplemental table 2 , the two groups did not differ in patterns of head movements , and the replacement of outlier images primarily enabled reducing the signal to noise ratio in the fmri data of both groups . we also confirmed that it is unlikely that head movements underlie the adhd classification results we got based on the brain activity data ( supplemental fig . feature detection was based on a univariate glm analysis , intended to identify functional brain regions of interest ( frois ) that showed significant activation or deactivation in all four vswm tasks , contrasted with all the fixation tasks , using the brain activity data of the adhd boys and td boys combined ( total of 40 participants ; see chu et al . we found eight frois showing significant activation ( vswm > fixation ) and eight frois showing significant deactivation ( fixation > vswm ) . we used a voxel threshold of p < 0.01 ( family wise error [ fwe ] corrected ) , and voxel cluster threshold of p < 0.01 ( minimum cluster size of 50 voxels ; fwe ) , where each cluster had a single significant peak at p < 0.05 ( fwe ) . an anatomical gray - matter mask ( using the talairach daemon brain atlas gray - matter mask , with dilate = 3 ) , and a sphere mask with a radius of 15 mm from each froi peak voxel , constrained the froi volume . for each participant we calculated the difference in mean beta values between each of the four vswm 2-back tasks and the mean of all fixation tasks in each of the 16 frois ( 2-back fixation ; fixation2-back ) . given the relatively large initial number of features , and the high correlations between some of the features ( see supplemental fig . 3 for the correlation matrix ) , we reduced the dimensionality of the data by using a sparse principal component analysis ( spca ) . this resulted with a relatively small number of orthogonal principal components ( pcs ) , which together explained most of the variability in the data , recalculated based only on the few features with the highest loadings ( berthet and rigollet , 2013 ; ritchie et al . we used sparse loading selection based on thresholding of the rotated loadings ( loading threshold > 0.6 ; absolute weights > 0.1 ) . loading rotation was based on the varimax rotation method ( lu and zhang , 2012 ; ma , 2013 ; qi and luo , 2015 ; sjstrand et al . the first 10 pcs explained approximately 70% of the variability evident in the original 64 features . each of the remaining pcs explained less than 3% of the variability in the data . interestingly , we found each one of the first 10 pcs to reflect brain activity from several rois from the same vswm task , but not brain activity in a specific roi in several tasks . the exception was pc-2 , which was based on the left and right middle frontal gyri in the two vswm tasks without feedback . in each cross validation iteration the lr classifier was trained based on the fmri data of 39 participants , and then the goodness of fit of the learned model was evaluated based on the correlation between the clinician diagnosis of the left - out participant ( adhd or td ) and the lr classifier predicted adhd probability for this participant . the reporting of a classification model based only on significant pcs has two advantages : ( i ) such a model is less likely to be overfitted or biased due to being based on too many predictors ( pcs ) , and thus it provides a more conservative estimate of the classification accuracies that can be achieved with the data at hand and ( ii ) it further reduces the number of brain regions by which the two groups of interest ( i.e. the classification accuracy of the model based on the four statistically significant pcs was 92.5% , with 95% sensitivity and 90% specificity . importantly , most of the classification decisions had high predicted probability values ( pp > 0.67 ) assigned to most adhd boys ( 75% ) , and low values ( pp < 0.33 ) assigned to most td boys ( 80% ; fig . this accuracy level is not statistically different from perfect accuracy reflecting the clinician 's diagnosis ( 100% ) , p = 0.12 ( one - tailed fisher exact test ; testing the hypothesis a permutation test shows that the classification accuracies of the above - described adhd classification model are unlikely to be discovered by chance . the mean accuracy of 15 distinct permutations ( 62.0% ) was significantly lower than that of the adhd model ( 92.5% ) , p = 0.000 ( exact test ) ; t(14 ) = 14.97 , p < 0.0001 ( one - tailed , one - sample t - test ) . the mean correct decisive classification accuracies ( with pp < 0.33 or pp > 0.67 ) in the 15 permutations were 24.5% , versus 77.5% of the adhd model , p = 0.000 ( exact test ) , t(14 ) = 13.59 , p < 0.0001 ( one - tailed , one - sample t - test ; see also supplemental fig . notably , the four significant pcs encompassed in the adhd model are based on fmri data from all four vswm tasks : pc-2 is based on the two tasks without feedback ; pc-3 is based on the small - reward with feedback task ; pc-4 is based on the large - reward with feedback task ; and pc-8 is based on the small - reward with feedback task ( fig . in the following analysis , we further investigated the utility of using fmri data from multiple vswm tasks as compared with using fmri data from a single task . here we used the threshold of eigenvalue larger than one ( keeping pcs that together explained more than 70% of the variance in the data ) . adhd classification accuracies based on the fmri data from each single task ( fig . 4 ; table 1 ) were significantly lower than the classification accuracy of the model based on all four tasks ( fig . the exception was the classification accuracy based on the fmri data from the small - reward with feedback task , which was 80% . however , here the correct decisive classification accuracy ( with pp < 0.33 or pp > 0.67 ) based on the fmri data from the small - reward with feedback vswm task was 60% , which is significantly lower ( 17.5% ) than the four - task classification accuracy , p < 0.05 ( one - tailed fisher exact test ) . feeding the lr classifier with the behavioral data from all four tasks resulted in an overall accuracy level of 75% ( 70% sensitivity ; 80% specificity ; omnibus test for model fit , (4 ) = 12.37 , p < 0.01 ) . this overall accuracy level is significantly lower ( 17.5% ) than the accuracy based on the fmri data from the four tasks , p < 0.04 ( one - tailed fisher exact test ; testing the hypothesis fmri model is better than the behavioral model ) . moreover , here only 57.5% of the classification decisions were both accurate and decisive , with high predicted probability values of being a boy with adhd ( pp > 0.67 ) assigned to only 50% of the adhd boys , and low values ( pp < 0.33 ) assigned to only 65% of the td boys ( fig . this accuracy level is significantly lower ( 20% ) than the decisive classification accuracy based on the four significant pcs ( based on four tasks ) , p < 0.04 ( one - tailed fisher exact test ) . moreover , we found high correlations between the behavioral performances in the four vswm tasks and pc-2 ( fig . that is , the brain activity represented by pc-2 ( right and left mfg , tasks without feedback ) explains much of the observed variability in the behavioral data . feeding the lr classifier with both the behavioral and fmri data ( 10 pcs ) resulted in exactly the same model as when feeding it with only the fmri data . we show that using a logistic regression ( lr ) classifier , which received as input brain imaging data that was acquired while children perform four distinct vswm tasks , enabled an adhd classification accuracy of 92.5% ( fig . classification accuracies that were based on the fmri data from all four tasks were significantly higher than those obtained using the fmri data from any single task . we found that the brain regions in which boys with adhd exhibited altered pattern of brain activity differed from one task to the other . the corresponding behavioral data from the four vswm tasks enabled an classification accuracy level of 75% ( fig . 5 ) , which is significantly lower than those obtained using the fmri data from the four tasks . participants who were misclassified based on the data from one vswm task were not necessarily misclassified when using the data from other vswm tasks ( fig . when the lr classifier was fed with the pcs calculated based on the four vswm tasks together , we observed a substantial improvement in the classification accuracy ( fig . , fmri data from few distinct vswm tasks ) , where each measurement enables a better than chance classification accuracy , and where there is a substantial degree of independency between the measurements ( e.g. , distinct cognitive tasks ) , enabled higher classification accuracies compared to accuracies achieved by using the data from each single measurement . specifically , in the absence of trial - by - trial feedback , altered activity patterns characterizing adhd were most evident in the bilateral middle frontal gyri ( mfg ) , specifically the right - mfg ( see pc-2 weight in fig . the mfg is believed to play an executive role in the visuospatial working memory network , and a primary role in volitional ( top - down ) allocation of attention ( burgess et al . we found that in vswm tasks without feedback , boys with adhd exhibited lower levels of activity in the mfg as compared with td boys . the fmri data from the small - reward with feedback vswm task also had a considerable contribution to adhd classification ( pc-8 and pc-3 ; fig . here , boys with adhd exhibited altered brain activity in a network ( pc-8 ) that primarily included the bilateral orbitofrontal cortex ( bi - ofc ) and the left fusiform gyrus ( left - ffg ) . , 2013 here we found greater deactivation in these two brain regions in td boys , as compared to boys with adhd , primarily in the small - reward with feedback vswm task . this may indicate that boys with adhd fail in suppressing task irrelevant information ( letter identity in a task that requires monitoring the spatial location of letters , and visual feedback indicating insignificant reward ) . 2b ) , the left - mfg , the right superior temporal gyrus ( right - stg ) , the right anterior insula ( right - antins ) , and the right supramarginal gyrus . these brain regions are involved in feedback processing ( ferdinand and opitz , 2014 ) , bottom - up attention and in sensory integration ( prado et al . the loadings on pc-3 ( and pc-8 ) , indicate that in the small - reward with feedback condition children with adhd are likely to exhibit altered suppression of irrelevant visual features ( ffg ) , altered visuospatial processing ( precuneus ) , altered sensory integration ( stg and supramarginal ) , altered bottom - up attention control ( ofc ) , altered feedback processing and top - down attention control ( mefg and mfg ) , and altered synchronization between bottom - up and top - down attention control ( antins ) . the fmri data from the large - reward with feedback vswm task had the smallest contribution to adhd classification ( 57.5% accuracy , with only a trend toward a significant model fit ; fig . , 2008 ; early studies showed that children with adhd exhibit poor cognitive control associated with lower levels of neural activity in the anterior insula , as compared with tds ( cubillo et al . we show that using fmri data from four distinct vswm tasks enabled substantially better classification accuracies than the use of fmri data from a single vswm task . nevertheless , the current investigation was limited to the manipulation of feedback and reward in vswm tasks , where many characteristics of the four tasks were identical . it is possible that an even better adhd classification can be achieved by using other , perhaps more demanding cognitive tasks that require other cognitive skills impaired in adhd ( e.g. as part of the development of a practical diagnosis tool , future investigations should aim to identify an optimized ensemble of cognitive tasks that would yield the highest classification accuracies in the shortest scanning time possible . future investigation may also involve looking for an ensemble of tasks that enable differentiation between children with adhd from other clinical populations , with shared symptoms . the use of multi - task - based fmri data may also enable earlier diagnosis of adhd , prior to the onset of clear behavioral symptoms , which in turn may enable earlier intervention . in order to be of practical clinical use , task - based fmri diagnosis requires the scanned participants to accurately perform a cognitive task while keeping still throughout a relatively long scan . thus , a multi - task - based fmri diagnosis session may not be practical for very young children or individuals with severe cognitive deficits . future developments should ideally involve an integrative use of multi - task - based fmri , resting - state fmri and structural imaging . this would likely enable an effective diagnosis of most clinical cases , and the detection of the onset of some clinical condition in early childhood . it is likely that similarly high ( or even higher ) classification accuracies can be achieved by using other machine - learning based classification methods . future studies should specifically explore machine - learning methods based on whole brain data , from multiple tasks , which automatically detect brain regions in which the targeted clinical population exhibits abnormal patterns of brain activity ( e.g. we show that fmri data acquired while participants perform a few distinct cognitive tasks enables substantial improvement in the detection of adhd cases , as compared with the use of fmri data from a single task ( or corresponding behavioral data ) . this would require using cognitive tasks that target the neurocognitive deficits characterizing the clinical condition of interest , or a battery of tasks that may enable dissociating between distinct neurocognitive abnormalities .

the number of the patients with heart failure is increasing in both developed and developing countries , with approximately 1%2% of adults in developed countries experiencing heart failure ( 2 ) . in iran , the number of these patients is estimated to be about 3,500 out of 100,000 ( 3 ) . meanwhile , about 50%55% of the patients with heart failure are women ( 4 ) , although most studies consider women less often ( 5 ) . women s experience of heart failure is different than that of men in some aspects . framingham s heart study showed that women report a higher effect of heart failure on their daily lives , and they also have a lower quality of life compared to men ( 6 ) . in fact , heart failure , with its potentially disabling nature , can affect daily life activities , and these patients lose their independency in their daily life activities and must rely on others for their self - care ( 7 ) . in this way , individuals ability to be involved in health - promoting behaviors as appropriate activities to improve health status and prevent the severe functional defect of the disease is influenced ( 6 ) . health - promoting behaviors are activities to maintain or increase the level of health and self - actualization and happiness among individuals ( 7 ) . these behaviors emphasize a positive pattern of life , which leads to an increase in health level and quality of life ( 8) . pender believes that healthy life behaviors include spiritual growth ; personal responsibility for health , sports , and nutrition ; interpersonal communications ; and tension management ( 9 ) . the effectiveness of health - promoting behaviors on the promotion of individuals health has been confirmed in various studies ( 10 ) . in fact , these behaviors should be considered the main strategies for maintaining and improving health ( 11 ) . enjezab et al.s study of health - promoting behaviors of middle - aged women in yazd showed a low level of these behaviors in components of physical activity and personal responsibility for health ( 12 ) . thanavaro , thanavaro , and delicath , in their study of women with chest pain ( 13 ) , and kheirjoo et al . , in their study of women with rheumatoid arthritis , also reported that health - promoting behaviors were not appropriate among these women ( 14 ) . on the other hand , physical and mental health inner strength is a factor for mental health and emotional well - being ( 15 ) and , consequently , a dynamic component of holistic healing ( 16 ) . people have the resources to survive , grow , and mature just as they have the abilities to resist adversities , endure suffering , and experience a good life despite harsh conditions . the ways people succeed in facing difficulties , such as prolonged illness , functional decline , grief , and loss , vary according to life circumstances and personal capacities and other resources . inner strength has been defined as an inner source of promoting well - being in human beings ( 17 ) . inner strength contains four concepts : knowing or anguish and searching , connectedness , engagement , and movement ( 18 ) . knowing and searching refers to passing over fear , vulnerability , and weakness to move from a feeling of fear and shock toward acceptance . connectedness is described as the development of supporting self - communication as well as communication with family and friends , and it acts as a spiritual power . finally , movement describes the dimension of movement , rest , activity , an honest self - evaluation , and physical and mental balance ( 19 ) . all humans have the potential and capacity to construct and empower themselves through inner strength ( 16 ) . inner strength exists in humans prior to the occurrence of challenging life events , such as chronic diseases , and a life - changing event or challenge can trigger its manifestation ( 18 ) . putnam studied the association between inner strength and health - promoting behaviors and its effects on quality of life among middle - aged women , reporting a positive association between these two factors . inner strength and health - promoting behaviors were also determined as predictive elements for quality of life ( 20 ) . in another study by dingley on inner strength in women with cancer , a strong association between inner strength and self - management nursing , as a profession , is committed to promoting health among society s members . the holistic approach in nursing is a reason for considering a human as a unique existence ; as such , one s physical , psychological , and mental health are not separable as they are combined in an integrated entity . well - being and various aspects of human health have a multi - dimensional and interactive association with each other ( 21 ) . the effective role of inner strength and health - promoting behaviors on the improvement of quality of life and obtaining of positive health results and self - management in chronic diseases has been demonstrated ( 20 ) . heart failure is one of the most fatal diseases in iranian women ( 22 ) , who play important roles by fulfilling their daily activities ; indeed , female seniors are more active than their male counterparts ( 23 ) . for these reasons , in the last two decades , increasing efforts have been made to evaluate and promote the well - being of iranian women with heart failure ; however , little is known in this regard ( 22 ) . thus , it seemed necessary to conduct a study related to subjects such as inner strength and health - promoting behaviors in women with heart failure in iranian society . this study aimed to determine the correlation between inner strength and health - promoting behaviors in women with heart failure referred to the hospitals affiliated with shahid beheshti university of medical sciences ( sbmu ) in tehran , the capital of iran . because these hospitals are affiliated with one of the most important and largest universities for education , treatment , and research in medical sciences , patients throughout the country in this cross - sectional study , five hospitals affiliated with sbmu with active cardiac clinics were selected through purposive sampling . one hundred forty - five qualified women with heart failure were included in the study through convenient sampling . inclusion criteria were age 40 years , a diagnosed recorded heart failure in the patient s file and medical records , class two or three heart failure , at least six weeks since disease diagnosis , ability to read and write in persian , and no history of mental diseases or the use of psychotropic medications . patients who changed their minds about completing the questionnaires while completing them were excluded from the study . the first was a personal characteristics questionnaire , a researcher - made 12-item questionnaire in which 11 items asked about age , marital status , level of education , spouse s level of education , number of children , occupation status , family monthly income , medical insurance coverage status , length of heart failure involvement , number of hospitalizations in the past year , and involvement in other physical diseases . participants completed these 11 items ; the remaining item , on the classification of the disease , was completed by the researcher . the second tool was a 27-item tool measuring inner strength in women with chronic diseases , designed by roux et al . in 2003 this tool investigates the dimensions of inner strength in four dimensions of knowing and searching with seven items , connectedness with seven items , engagement with six items , and movement with seven items ( 16 ) based on a five - point scale ( i.e. , absolutely agree , agree , somewhat agree , disagree , and absolutely disagree ) . possible points ranged from 27 to 135 , with higher points showing higher inner strength ( 19 ) . the health - promoting life profile ii ( hplp ii ) , the third tool , is a 52-item questionnaire designed by walker et al . in 1987 this tool measures the application of health - promoting behaviors in six subscales : nutrition ( nine items ) , physical activity ( eight items ) , personal accountability in healthcare ( nine items ) , tension management ( eight items ) , interpersonal communications ( nine items ) , and spiritual growth ( nine items ) . the points of health - promoting behavior range from 52 to 208 and can be separately calculated for each dimension ( 24 ) . the content validity index and content validity ratio and face validity were used to measure the tools validity in such way that the three - section questionnaires were distributed among three psychiatric nurses , three public health nurses , and four phds of nursing , all of whom were academic members of nursing schools of tehran universities of medical sciences ( tums ) and sbmu . content validity indexes for the inner strength questionnaire concerning association ( 0.97 ) , simplicity ( 0.96 ) , and clarity ( 0.97 ) were obtained . the content validity index for the whole hplp ii concerning association , simplicity , and clarity the content validity ratio for inner strength and hplp ii questionnaires were calculated as 0.93 and 0.95 , respectively . experts viewpoints of the content validity calculation were used to check the face validity of the questionnaire . in addition , viewpoints of five qualified patients concerning the understandability and appearance of appropriateness of the items were also considered . two methods of internal consistency , cronbach s alpha and test re - test , were used to calculate reliability . cronbach s alphas of inner strength questionnaire and hplp ii were =0.89 and =0.83 , respectively . to conduct test re - test , 15 qualified patients were selected and three questionnaires were given to them with a two - week interval and after completion . the pearson correlation coefficient for the inner strength questionnaire and hplp ii were calculated as r=0.87 and r=0.89 , respectively . to collect data , after getting written permission from the post - graduate department of sbmu , one of the researchers referred to the selected hospitals and obtained written permission from the hospital authorities . she then referred to the hospital clinics and , after introducing herself and explaining the research goals to the head of the clinic as well as coordinating with her , referred to female patients medical files in the cardiology clinic , identifying those patients with a recorded heart failure diagnosis in their files . finally , the qualified subjects were selected through convenient sampling . due to a lack of precise statistics for female patients with heart failure and given that there was no possibility of other sampling methods , convenient sampling was used . written consent was obtained from the subjects to participate in the study . for subjects convenience and to achieve more precise information from them , a quiet place in the clinic was considered for the completion of the questionnaires . for those subjects with lower educational level , data were analyzed using descriptive statistical tests and the pearson correlation coefficient to determine the association between data via ibm spss statistics version 20 ( ibm corp . , armonk , ny , usa ) . in this cross - sectional study , five hospitals affiliated with sbmu with active cardiac clinics were selected through purposive sampling . one hundred forty - five qualified women with heart failure were included in the study through convenient sampling . inclusion criteria were age 40 years , a diagnosed recorded heart failure in the patient s file and medical records , class two or three heart failure , at least six weeks since disease diagnosis , ability to read and write in persian , and no history of mental diseases or the use of psychotropic medications . patients who changed their minds about completing the questionnaires while completing them were excluded from the study . the first was a personal characteristics questionnaire , a researcher - made 12-item questionnaire in which 11 items asked about age , marital status , level of education , spouse s level of education , number of children , occupation status , family monthly income , medical insurance coverage status , length of heart failure involvement , number of hospitalizations in the past year , and involvement in other physical diseases . participants completed these 11 items ; the remaining item , on the classification of the disease , was completed by the researcher . the second tool was a 27-item tool measuring inner strength in women with chronic diseases , designed by roux et al . in 2003 ( 20 ) . this tool investigates the dimensions of inner strength in four dimensions of knowing and searching with seven items , connectedness with seven items , engagement with six items , and movement with seven items ( 16 ) based on a five - point scale ( i.e. , absolutely agree , agree , somewhat agree , disagree , and absolutely disagree ) . possible points ranged from 27 to 135 , with higher points showing higher inner strength ( 19 ) . the health - promoting life profile ii ( hplp ii ) , the third tool , is a 52-item questionnaire designed by walker et al . in 1987 this tool measures the application of health - promoting behaviors in six subscales : nutrition ( nine items ) , physical activity ( eight items ) , personal accountability in healthcare ( nine items ) , tension management ( eight items ) , interpersonal communications ( nine items ) , and spiritual growth ( nine items ) . the points of health - promoting behavior range from 52 to 208 and can be separately calculated for each dimension ( 24 ) . the content validity index and content validity ratio and face validity were used to measure the tools validity in such way that the three - section questionnaires were distributed among three psychiatric nurses , three public health nurses , and four phds of nursing , all of whom were academic members of nursing schools of tehran universities of medical sciences ( tums ) and sbmu . content validity indexes for the inner strength questionnaire concerning association ( 0.97 ) , simplicity ( 0.96 ) , and clarity ( 0.97 ) were obtained . the content validity index for the whole hplp ii concerning association , simplicity , and clarity was calculated as 0.98 . the content validity ratio for inner strength and hplp ii questionnaires were calculated as 0.93 and 0.95 , respectively . experts viewpoints of the content validity calculation were used to check the face validity of the questionnaire . in addition , viewpoints of five qualified patients concerning the understandability and appearance of appropriateness of the items were also considered . two methods of internal consistency , cronbach s alpha and test re - test , were used to calculate reliability . cronbach s alphas of inner strength questionnaire and hplp ii were =0.89 and =0.83 , respectively . to conduct test re - test , 15 qualified patients were selected and three questionnaires were given to them with a two - week interval and after completion . the pearson correlation coefficient for the inner strength questionnaire and hplp ii were calculated as r=0.87 and r=0.89 , respectively . to collect data , after getting written permission from the post - graduate department of sbmu , one of the researchers referred to the selected hospitals and obtained written permission from the hospital authorities . she then referred to the hospital clinics and , after introducing herself and explaining the research goals to the head of the clinic as well as coordinating with her , referred to female patients medical files in the cardiology clinic , identifying those patients with a recorded heart failure diagnosis in their files . finally , the qualified subjects were selected through convenient sampling . due to a lack of precise statistics for female patients with heart failure and given that there was no possibility of other sampling methods , convenient sampling was used . written consent was obtained from the subjects to participate in the study . for subjects convenience and to achieve more precise information from them , a quiet place in the clinic was considered for the completion of the questionnaires . for those subjects with lower educational level , data were analyzed using descriptive statistical tests and the pearson correlation coefficient to determine the association between data via ibm spss statistics version 20 ( ibm corp . , armonk , ny , usa ) . the 0.01 significance level was considered to obtain more accurate estimates . approximately 89.6% of the subjects were married , and 54.5% and 35.9% of subjects and their spouses , respectively , had primary school education . subjects mean number of children was 3.47 ( 1.61 ) , 78.6% of the subjects were homemakers , and 45.5% earned a family monthly income between 33 and 165 u.s . approximately 93.8% had medical insurance , and 51.7% and 48.3% were in the second and third classes of the disease , respectively . subjects mean length of heart failure was 15.29 ( 10.72 ) months , 51.7% had a history of hospitalization in the past year , and 48.3% had other physical diseases in addition to heart failure . the data also showed that 65.5% of the subjects had appropriate inner strength , 32.4% had moderate , and 2.1% had poor inner strength . means ( sd ) of obtained points in inner strength and its subscales are presented in table 1 . in addition , the results showed that 54.5% of the subjects had a moderate level of health - promoting behaviors and 4.1% had a poor level of health - promoting behaviors . means ( sd ) of calculated points in health - promoting behaviors and its subscales are presented in table 2 . the pearson correlation coefficient showed a direct and significant correlation between the variable of inner strength and general promotion behaviors and all dimensions of health - promoting behaviors ( table 3 ) . the correlation of inner strength with health - promoting behaviors was r=0.77 , revealing a direct correlation between two variables in such way that an increase or decrease in the inner strength variable would change health - promoting behaviors in the same way ( p=0.000 ) . the data also showed that all dimensions of inner strength ( except for knowing and searching with physical activity , the dimension of connectedness with personal accountability in healthcare , and the dimension of connectedness with physical activity ) had a direct significant association with health - promoting behaviors ( p=0.000 to p= 0.008 ) ( table 4 ) . this study examined the correlation between inner strength and health - promoting behaviors in women with heart failure referred to hospitals affiliated with sbmu . the results showed that the mean score of inner strength in women with heart failure was 92.6 ( 18.25 ) , indicating an appropriate level . dingley ( 19 ) reported a mean score of inner strength as 108.6 in women with breast cancer , which shows an excellent level . putnam reported a mean score of inner strength as 56.86 in middle - aged women , indicating a moderate level ( 20 ) . roux , dingley , and bush concluded that an important event in life can lead to the manifestation of inner strength ( 21 ) . inner strength exists prior to the occurrence of a challenge in life , and experiencing challenging events triggers the potential for and ability to access inner strength . in the present study and in dingley s ( 19 ) study , subjects experienced a specific challenge ( onset of the disease ) that acted as an effective factor in the development of inner strength and caused their inner strength to reach an appropriate level . meanwhile , in putnam s study , about three - fourths of participants were completely healthy and , consequently , could facilitate inner strength development ( 20 ) . among inner strength dimensions , subjects obtained higher points on the dimension of connectedness , which is consistent with dingley s results ( 19 ) . obtaining higher points on the dimension of connectedness among subjects can be due to the fact that , in cases of a disease , missing someone , sorrow , or a great change in life , individuals seek help from spiritual resources to adapt or fulfill their needs ( 25 ) . in putnam s study , participants obtained the lowest points on the dimension of connectedness ( 20 ) . this difference in results can be due to the cultural and religious differences in various societies . in iranian society , religion , spirituality , and one s spiritual relationship with god and the family as well as familial communications play a major role in individuals lives . iranians always - and especially in cases of problems - get support from their religious beliefs as an important source . on the other hand , family members try to make closer relationships with the member in trouble in order to solve the problem . the lowest obtained points on dimensions of inner strength were for movement , which might be due to women s limited knowledge of heart failure and the advantages of appropriate physical activity and movement . subjects high fear of worsening their condition through physical activity as well as their limited familiarity with relaxation activities , resulting in a physical and mental balance in the body , , 65.5% of subjects indicated an appropriate level of inner strength while other subjects had moderate and poor levels . in fact , not all of the individuals were necessarily empowered by inner strength after being exposed to life - challenging events like a chronic disease . this inner source of human strength is influenced by personal , external , and environmental factors ( 16 ) . empowering patients inner strength is achieved by helping them maintain a relationship with their family and friends and preserve a spiritual relationship as well as inquiring about the disease and having access to resources to answer the questions . encouraging patients to express their fears and detect their inner power , collaborating in decision making concerning treatment programs , accessing supportive resources , and focusing on achieving health instead of on the disease are other factors that help empower inner strength . in the present study , 34.5% of the subjects had a moderate or poor level of inner strength , revealing the need for nurses , authorities , and healthcare providers attention to help women with heart failure detect , maintain , and empower their inner strength . meanwhile , in enjezab et al.s study , middle - aged women in yazd obtained higher points in health - promoting behaviors ( 12 ) . although the inclusion criteria were a lack of any mental or speech problems , the participants health status was not determined whereas subjects in the present study had heart failure . as individuals health status affects the frequency of their participation in health - promoting behaviors , the difference between enjezab et al.s study and the present study seem reasonable ( 12 ) . pierce s study on women with heart failure ( 6 ) and beal et al.s study on women with fibromyalgia ( 26 ) revealed an appropriate level of health - promoting behaviors in these women . the difference in the level of health - promoting behaviors in various societies can result from personal , economic , social , and environmental factors , which affect individuals health status . social norms , mass media , national health policies , commercial activities , and environmental conditions affect individuals health - promoting behaviors ( 11 ) . unfortunately , in iran , health - promoting programs have not found their appropriate place in health and treatment services , yet most of the budget in this field is spent on treatment and medication ( 27 ) . based on the results of the present study , subjects obtained the highest point in interpersonal communications . in most studies conducted on lifestyles , high points have been reported for interpersonal support in both healthy individuals and those with chronic diseases ( 14 ) . based on their obtained points , the subjects were also at an appropriate level for the dimensions of spiritual growth and nutrition and at a moderate level for the dimensions of personal accountability in healthcare and tension management , which is in line with pierce s study on women with heart failure ( 6 ) . ( 14 ) examined on women with rheumatoid arthritis ( 14 ) and bea et al . examined women with fibromyalgia ( 26) in both studies , dimensions of spiritual growth , nutrition , and personal accountability in healthcare were at an appropriate level while the dimension of tension management was at a moderate level . with regard to the moderate level of personal accountability in healthcare among subjects in the present study , personal accountability in healthcare refers to the acceptance of the responsibility for one s health and administration of self - care . some patients with heart failure actually do not believe in the positive effect of their self - care behaviors in relieving their disease symptoms , which impairs their self - care behaviors ; these patients have no motivation for such behaviors ( 28 ) . in the present study , inner strength had a direct significant correlation with general health - promoting behaviors as well as all its dimensions , which is in line with putnam s ( 20 ) finding . inner strength is a factor for mental health and psychological well - being and a component of spirituality and is also considered as a dynamic element in holistic healing . the ability to face challenges and facilitate healing , make an appropriate change in life , feel self - control over one s condition , and have the possibility to maintain a balance between one s inner world and one s life experiences and the environment are the outcomes of inner strength ( 16 ) . stronger inner strength is associated with higher mental and spiritual health . given these findings , women with heart failure who have stronger inner strength , have a greater potential to adapt to a chronic disease condition and try to reach the highest level of health . health - promoting behaviors are people s behaviors or activities resulting from their desire to amend their health status ( 29 ) . the possibility of engaging in such behaviors in women with heart failure who have stronger inner strength is high , and the obtained positive significant association between inner strength and health - promoting behaviors in the subjects is reasonable . the results also showed that all dimensions of inner strength ( except for knowing and searching with physical activity and the dimensions of connectedness with personal accountability in healthcare and connectedness with physical activity ) have a direct significant correlation on dimensions of health - promoting behaviors . putnam also found a direct significant correlation between all dimensions of inner strength ( expect for knowing and searching with dimensions of personal accountability in healthcare , the dimension of connectedness with the dimension of physical activity , and the dimension of connectedness with the dimension of nutrition ) with dimensions of the health - promoting behaviors variable ( 20 ) , which is in line with the findings of the present study . another limitation was the small number of studies on inner strength and health - promoting behaviors involving patients with heart failure in iran and the world ; the authors tried to minimize this limitation by using related studies . this study aimed to determine the correlation between inner strength and health - promoting behaviors in women with heart failure . therefore , to enhance the level of health and well - being and lower the costs of care in women with heart failure , it seems essential for nurses to develop and empower patients inner strength . due to the limited number of studies on health promotion for patients with chronic disease , especially female patients in iran ,

introductioninner strength is a factor for mental health and well - being and , consequently , a dynamic component of holistic healing . health - promoting behaviors are appropriate activities to improve health status and prevent the progression of the functional defect resulting from heart failure . the present study aimed to determine the correlation between inner strength and health - promoting behaviors in women with heart failure referred to hospitals affiliated with shahid beheshti university of medical sciences ( sbmu ) in 2013.methodsin this cross - sectional study , 145 women with hearth failure were selected through convenient sampling from the clients referred to hospitals affiliated with sbmu . the data collection tool included a three - section questionnaire of personal characteristics , inner strength , and health - promoting life profile ii ( hplp ii ) . the data analysis used descriptive statistical tests and pearson correlation coefficient through spss version 20.resultsa direct significant correlation was found between inner strength and all dimensions of health - promoting behaviors and overall health - promoting behaviors ( p=0.000 ) as well as between all dimensions of inner strength ( except for the dimension of knowing and searching with physical activity and the dimension of connectedness with personal accountability in healthcare as well as connectedness with physical activity ) with health - promoting behaviors ( p=0.000 to p=0008).conclusionto improve the level of health and well - being and reduce the costs of care services in women with health failure , close attention should be paid to developing and empowering their inner strength .

1. Introduction 2. Material and Methods 2.1. Research design and setting 2.2. Inclusion and exclusion criteria 2.3. Instrument 2.4. Validity and reliability 2.5. Data collection 3. Results 4. Discussion 5. Study limitations 6. Conclusions

the number of the patients with heart failure is increasing in both developed and developing countries , with approximately 1%2% of adults in developed countries experiencing heart failure ( 2 ) . meanwhile , about 50%55% of the patients with heart failure are women ( 4 ) , although most studies consider women less often ( 5 ) . in fact , heart failure , with its potentially disabling nature , can affect daily life activities , and these patients lose their independency in their daily life activities and must rely on others for their self - care ( 7 ) . in this way , individuals ability to be involved in health - promoting behaviors as appropriate activities to improve health status and prevent the severe functional defect of the disease is influenced ( 6 ) . health - promoting behaviors are activities to maintain or increase the level of health and self - actualization and happiness among individuals ( 7 ) . the effectiveness of health - promoting behaviors on the promotion of individuals health has been confirmed in various studies ( 10 ) . enjezab et al.s study of health - promoting behaviors of middle - aged women in yazd showed a low level of these behaviors in components of physical activity and personal responsibility for health ( 12 ) . , in their study of women with rheumatoid arthritis , also reported that health - promoting behaviors were not appropriate among these women ( 14 ) . on the other hand , physical and mental health inner strength is a factor for mental health and emotional well - being ( 15 ) and , consequently , a dynamic component of holistic healing ( 16 ) . inner strength has been defined as an inner source of promoting well - being in human beings ( 17 ) . inner strength contains four concepts : knowing or anguish and searching , connectedness , engagement , and movement ( 18 ) . knowing and searching refers to passing over fear , vulnerability , and weakness to move from a feeling of fear and shock toward acceptance . connectedness is described as the development of supporting self - communication as well as communication with family and friends , and it acts as a spiritual power . finally , movement describes the dimension of movement , rest , activity , an honest self - evaluation , and physical and mental balance ( 19 ) . putnam studied the association between inner strength and health - promoting behaviors and its effects on quality of life among middle - aged women , reporting a positive association between these two factors . inner strength and health - promoting behaviors were also determined as predictive elements for quality of life ( 20 ) . in another study by dingley on inner strength in women with cancer , a strong association between inner strength and self - management nursing , as a profession , is committed to promoting health among society s members . the holistic approach in nursing is a reason for considering a human as a unique existence ; as such , one s physical , psychological , and mental health are not separable as they are combined in an integrated entity . well - being and various aspects of human health have a multi - dimensional and interactive association with each other ( 21 ) . the effective role of inner strength and health - promoting behaviors on the improvement of quality of life and obtaining of positive health results and self - management in chronic diseases has been demonstrated ( 20 ) . for these reasons , in the last two decades , increasing efforts have been made to evaluate and promote the well - being of iranian women with heart failure ; however , little is known in this regard ( 22 ) . thus , it seemed necessary to conduct a study related to subjects such as inner strength and health - promoting behaviors in women with heart failure in iranian society . this study aimed to determine the correlation between inner strength and health - promoting behaviors in women with heart failure referred to the hospitals affiliated with shahid beheshti university of medical sciences ( sbmu ) in tehran , the capital of iran . because these hospitals are affiliated with one of the most important and largest universities for education , treatment , and research in medical sciences , patients throughout the country in this cross - sectional study , five hospitals affiliated with sbmu with active cardiac clinics were selected through purposive sampling . one hundred forty - five qualified women with heart failure were included in the study through convenient sampling . inclusion criteria were age 40 years , a diagnosed recorded heart failure in the patient s file and medical records , class two or three heart failure , at least six weeks since disease diagnosis , ability to read and write in persian , and no history of mental diseases or the use of psychotropic medications . the first was a personal characteristics questionnaire , a researcher - made 12-item questionnaire in which 11 items asked about age , marital status , level of education , spouse s level of education , number of children , occupation status , family monthly income , medical insurance coverage status , length of heart failure involvement , number of hospitalizations in the past year , and involvement in other physical diseases . the second tool was a 27-item tool measuring inner strength in women with chronic diseases , designed by roux et al . in 2003 this tool investigates the dimensions of inner strength in four dimensions of knowing and searching with seven items , connectedness with seven items , engagement with six items , and movement with seven items ( 16 ) based on a five - point scale ( i.e. the health - promoting life profile ii ( hplp ii ) , the third tool , is a 52-item questionnaire designed by walker et al . in 1987 this tool measures the application of health - promoting behaviors in six subscales : nutrition ( nine items ) , physical activity ( eight items ) , personal accountability in healthcare ( nine items ) , tension management ( eight items ) , interpersonal communications ( nine items ) , and spiritual growth ( nine items ) . the points of health - promoting behavior range from 52 to 208 and can be separately calculated for each dimension ( 24 ) . the content validity index and content validity ratio and face validity were used to measure the tools validity in such way that the three - section questionnaires were distributed among three psychiatric nurses , three public health nurses , and four phds of nursing , all of whom were academic members of nursing schools of tehran universities of medical sciences ( tums ) and sbmu . content validity indexes for the inner strength questionnaire concerning association ( 0.97 ) , simplicity ( 0.96 ) , and clarity ( 0.97 ) were obtained . the content validity index for the whole hplp ii concerning association , simplicity , and clarity the content validity ratio for inner strength and hplp ii questionnaires were calculated as 0.93 and 0.95 , respectively . cronbach s alphas of inner strength questionnaire and hplp ii were =0.89 and =0.83 , respectively . the pearson correlation coefficient for the inner strength questionnaire and hplp ii were calculated as r=0.87 and r=0.89 , respectively . to collect data , after getting written permission from the post - graduate department of sbmu , one of the researchers referred to the selected hospitals and obtained written permission from the hospital authorities . she then referred to the hospital clinics and , after introducing herself and explaining the research goals to the head of the clinic as well as coordinating with her , referred to female patients medical files in the cardiology clinic , identifying those patients with a recorded heart failure diagnosis in their files . finally , the qualified subjects were selected through convenient sampling . due to a lack of precise statistics for female patients with heart failure and given that there was no possibility of other sampling methods , convenient sampling was used . for subjects convenience and to achieve more precise information from them , a quiet place in the clinic was considered for the completion of the questionnaires . for those subjects with lower educational level , data were analyzed using descriptive statistical tests and the pearson correlation coefficient to determine the association between data via ibm spss statistics version 20 ( ibm corp . in this cross - sectional study , five hospitals affiliated with sbmu with active cardiac clinics were selected through purposive sampling . one hundred forty - five qualified women with heart failure were included in the study through convenient sampling . inclusion criteria were age 40 years , a diagnosed recorded heart failure in the patient s file and medical records , class two or three heart failure , at least six weeks since disease diagnosis , ability to read and write in persian , and no history of mental diseases or the use of psychotropic medications . the first was a personal characteristics questionnaire , a researcher - made 12-item questionnaire in which 11 items asked about age , marital status , level of education , spouse s level of education , number of children , occupation status , family monthly income , medical insurance coverage status , length of heart failure involvement , number of hospitalizations in the past year , and involvement in other physical diseases . the second tool was a 27-item tool measuring inner strength in women with chronic diseases , designed by roux et al . this tool investigates the dimensions of inner strength in four dimensions of knowing and searching with seven items , connectedness with seven items , engagement with six items , and movement with seven items ( 16 ) based on a five - point scale ( i.e. the health - promoting life profile ii ( hplp ii ) , the third tool , is a 52-item questionnaire designed by walker et al . in 1987 this tool measures the application of health - promoting behaviors in six subscales : nutrition ( nine items ) , physical activity ( eight items ) , personal accountability in healthcare ( nine items ) , tension management ( eight items ) , interpersonal communications ( nine items ) , and spiritual growth ( nine items ) . the points of health - promoting behavior range from 52 to 208 and can be separately calculated for each dimension ( 24 ) . the content validity index and content validity ratio and face validity were used to measure the tools validity in such way that the three - section questionnaires were distributed among three psychiatric nurses , three public health nurses , and four phds of nursing , all of whom were academic members of nursing schools of tehran universities of medical sciences ( tums ) and sbmu . content validity indexes for the inner strength questionnaire concerning association ( 0.97 ) , simplicity ( 0.96 ) , and clarity ( 0.97 ) were obtained . the content validity index for the whole hplp ii concerning association , simplicity , and clarity was calculated as 0.98 . the content validity ratio for inner strength and hplp ii questionnaires were calculated as 0.93 and 0.95 , respectively . cronbach s alphas of inner strength questionnaire and hplp ii were =0.89 and =0.83 , respectively . the pearson correlation coefficient for the inner strength questionnaire and hplp ii were calculated as r=0.87 and r=0.89 , respectively . to collect data , after getting written permission from the post - graduate department of sbmu , one of the researchers referred to the selected hospitals and obtained written permission from the hospital authorities . she then referred to the hospital clinics and , after introducing herself and explaining the research goals to the head of the clinic as well as coordinating with her , referred to female patients medical files in the cardiology clinic , identifying those patients with a recorded heart failure diagnosis in their files . finally , the qualified subjects were selected through convenient sampling . due to a lack of precise statistics for female patients with heart failure and given that there was no possibility of other sampling methods , convenient sampling was used . for subjects convenience and to achieve more precise information from them , a quiet place in the clinic was considered for the completion of the questionnaires . for those subjects with lower educational level , data were analyzed using descriptive statistical tests and the pearson correlation coefficient to determine the association between data via ibm spss statistics version 20 ( ibm corp . the data also showed that 65.5% of the subjects had appropriate inner strength , 32.4% had moderate , and 2.1% had poor inner strength . in addition , the results showed that 54.5% of the subjects had a moderate level of health - promoting behaviors and 4.1% had a poor level of health - promoting behaviors . means ( sd ) of calculated points in health - promoting behaviors and its subscales are presented in table 2 . the pearson correlation coefficient showed a direct and significant correlation between the variable of inner strength and general promotion behaviors and all dimensions of health - promoting behaviors ( table 3 ) . the correlation of inner strength with health - promoting behaviors was r=0.77 , revealing a direct correlation between two variables in such way that an increase or decrease in the inner strength variable would change health - promoting behaviors in the same way ( p=0.000 ) . the data also showed that all dimensions of inner strength ( except for knowing and searching with physical activity , the dimension of connectedness with personal accountability in healthcare , and the dimension of connectedness with physical activity ) had a direct significant association with health - promoting behaviors ( p=0.000 to p= 0.008 ) ( table 4 ) . this study examined the correlation between inner strength and health - promoting behaviors in women with heart failure referred to hospitals affiliated with sbmu . the results showed that the mean score of inner strength in women with heart failure was 92.6 ( 18.25 ) , indicating an appropriate level . dingley ( 19 ) reported a mean score of inner strength as 108.6 in women with breast cancer , which shows an excellent level . roux , dingley , and bush concluded that an important event in life can lead to the manifestation of inner strength ( 21 ) . in the present study and in dingley s ( 19 ) study , subjects experienced a specific challenge ( onset of the disease ) that acted as an effective factor in the development of inner strength and caused their inner strength to reach an appropriate level . meanwhile , in putnam s study , about three - fourths of participants were completely healthy and , consequently , could facilitate inner strength development ( 20 ) . among inner strength dimensions , subjects obtained higher points on the dimension of connectedness , which is consistent with dingley s results ( 19 ) . obtaining higher points on the dimension of connectedness among subjects can be due to the fact that , in cases of a disease , missing someone , sorrow , or a great change in life , individuals seek help from spiritual resources to adapt or fulfill their needs ( 25 ) . in putnam s study , participants obtained the lowest points on the dimension of connectedness ( 20 ) . in iranian society , religion , spirituality , and one s spiritual relationship with god and the family as well as familial communications play a major role in individuals lives . the lowest obtained points on dimensions of inner strength were for movement , which might be due to women s limited knowledge of heart failure and the advantages of appropriate physical activity and movement . subjects high fear of worsening their condition through physical activity as well as their limited familiarity with relaxation activities , resulting in a physical and mental balance in the body , , 65.5% of subjects indicated an appropriate level of inner strength while other subjects had moderate and poor levels . empowering patients inner strength is achieved by helping them maintain a relationship with their family and friends and preserve a spiritual relationship as well as inquiring about the disease and having access to resources to answer the questions . encouraging patients to express their fears and detect their inner power , collaborating in decision making concerning treatment programs , accessing supportive resources , and focusing on achieving health instead of on the disease are other factors that help empower inner strength . in the present study , 34.5% of the subjects had a moderate or poor level of inner strength , revealing the need for nurses , authorities , and healthcare providers attention to help women with heart failure detect , maintain , and empower their inner strength . meanwhile , in enjezab et al.s study , middle - aged women in yazd obtained higher points in health - promoting behaviors ( 12 ) . although the inclusion criteria were a lack of any mental or speech problems , the participants health status was not determined whereas subjects in the present study had heart failure . as individuals health status affects the frequency of their participation in health - promoting behaviors , the difference between enjezab et al.s study and the present study seem reasonable ( 12 ) . pierce s study on women with heart failure ( 6 ) and beal et al.s study on women with fibromyalgia ( 26 ) revealed an appropriate level of health - promoting behaviors in these women . the difference in the level of health - promoting behaviors in various societies can result from personal , economic , social , and environmental factors , which affect individuals health status . social norms , mass media , national health policies , commercial activities , and environmental conditions affect individuals health - promoting behaviors ( 11 ) . unfortunately , in iran , health - promoting programs have not found their appropriate place in health and treatment services , yet most of the budget in this field is spent on treatment and medication ( 27 ) . based on the results of the present study , subjects obtained the highest point in interpersonal communications . based on their obtained points , the subjects were also at an appropriate level for the dimensions of spiritual growth and nutrition and at a moderate level for the dimensions of personal accountability in healthcare and tension management , which is in line with pierce s study on women with heart failure ( 6 ) . examined women with fibromyalgia ( 26) in both studies , dimensions of spiritual growth , nutrition , and personal accountability in healthcare were at an appropriate level while the dimension of tension management was at a moderate level . with regard to the moderate level of personal accountability in healthcare among subjects in the present study , personal accountability in healthcare refers to the acceptance of the responsibility for one s health and administration of self - care . some patients with heart failure actually do not believe in the positive effect of their self - care behaviors in relieving their disease symptoms , which impairs their self - care behaviors ; these patients have no motivation for such behaviors ( 28 ) . in the present study , inner strength had a direct significant correlation with general health - promoting behaviors as well as all its dimensions , which is in line with putnam s ( 20 ) finding . inner strength is a factor for mental health and psychological well - being and a component of spirituality and is also considered as a dynamic element in holistic healing . the ability to face challenges and facilitate healing , make an appropriate change in life , feel self - control over one s condition , and have the possibility to maintain a balance between one s inner world and one s life experiences and the environment are the outcomes of inner strength ( 16 ) . given these findings , women with heart failure who have stronger inner strength , have a greater potential to adapt to a chronic disease condition and try to reach the highest level of health . health - promoting behaviors are people s behaviors or activities resulting from their desire to amend their health status ( 29 ) . the possibility of engaging in such behaviors in women with heart failure who have stronger inner strength is high , and the obtained positive significant association between inner strength and health - promoting behaviors in the subjects is reasonable . the results also showed that all dimensions of inner strength ( except for knowing and searching with physical activity and the dimensions of connectedness with personal accountability in healthcare and connectedness with physical activity ) have a direct significant correlation on dimensions of health - promoting behaviors . putnam also found a direct significant correlation between all dimensions of inner strength ( expect for knowing and searching with dimensions of personal accountability in healthcare , the dimension of connectedness with the dimension of physical activity , and the dimension of connectedness with the dimension of nutrition ) with dimensions of the health - promoting behaviors variable ( 20 ) , which is in line with the findings of the present study . another limitation was the small number of studies on inner strength and health - promoting behaviors involving patients with heart failure in iran and the world ; the authors tried to minimize this limitation by using related studies . this study aimed to determine the correlation between inner strength and health - promoting behaviors in women with heart failure . therefore , to enhance the level of health and well - being and lower the costs of care in women with heart failure , it seems essential for nurses to develop and empower patients inner strength .

transitional care refers to actions to ensure the coordination and continuity of care for patients as they transfer between locations of care . care transitions are particularly important for the 145 million americans who live with chronic illness and receive care from multiple providers in various settings , often with noncommunicating medical record systems . poor transitions can fragment care , resulting in conflicting recommendations , increased medical error and duplication , and inadequate information to both patients and caregivers . an effective measure of care transition quality the most widely used measure of care transition quality is the care transitions measure ( ctm15 ) . the 4 ctm domains , derived from patient focus groups , are ( 1 ) information transfer , ( 2 ) patient and caregiver preparation , ( 3 ) support for selfmanagement , and ( 4 ) empowerment to assert preferences . the labels for the retained domains differ a little from these : ( 1 ) critical understanding , ( 2 ) preferences important , ( 3 ) management preparation , and ( 4 ) care plan . all 15 items in the ctm15 use a 4point scale with responses ranging from strongly disagree to strongly agree . the items are scored by summing the responses ( between 1 and 4 ) followed by linear transformation to a 0to100 range . the ctm15 has been shown to discriminate between patients who did and did not have a subsequent emergency department visit or rehospitalization for their index condition and between health care facilities with different levels of system integration . a 3item version of the ctm was found to explain 88% of the variance in the full measure ; the ctm3 has been demonstrated to have the same ability to detect group differences as the longer version in earlier studies . the short form includes items from 2 of the 4 originally identified domains ( critical understanding and preferences are important ) . the developers argue that the information lost by using the ctm3 measure is small compared with the reduction in response burden . the ctm3 measures the extent to which the hospital staff accomplished essential care processes in preparing the patient for discharge and participating in posthospital selfcare activities . therefore , it is advised that survey data collection be administered between 48 hours and 6 weeks postdischarge . the ctm3 was endorsed by the national quality forum and included in the consumer assessment of healthcare providers and systems ( cahps ) hospital survey in 2010 . as with most selfreport questionnaires currently used in health research , the ctm is scored using a classic sum score transformed on a 0to100 scale . while this approach is widely used , moresophisticated approaches to scale scoring based on item response theory ( irt ) are gaining popularity , since the irt method uses more of the available information in each item , thus , theoretically , irt can produce a more discriminating score based on the same underlying data than a sum score . in previous studies , comparisons of sum versus irtbased scoring of patientreported measures have produced mixed results ; that is , the benefits of irt scoring differ and are really important for some scales but not others . to the best of our knowledge the development and initial validation of the ctm15 and ctm3 are well documented ; however , these studies were conducted on relatively small samples of less than 250 people . given that the ctm3 is now included among the core questions designed to provide a standardized survey instrument and data collection methodology for measuring patients ' perspectives on hospital care as part of a national quality of care initiative , examining ctm measure performance in an independent largescale study is warranted . the aim of this report is to conduct a psychometric analysis of ctm items and evaluate the dimensionality , internal consistency , and construct validity of ctm15 and ctm3 . we also explore the effect of irtbased scale scoring on measurement ability and compare the performance of the simple summation and irtbased scoring approaches head to head . transitions , risks , and actions in coronary events center for outcomes research and education ( tracecore ) is a large , multiracial cohort of adult patients hospitalized with acute coronary syndromes ( acs ) from 6 hospitals in massachusetts and georgia . a total of 2300 patients were enrolled in the study and were approached for computerassisted telephone followup interviews at 1 , 3 , 6 , and 12 months post acs hospitalization . interviews focus on individual patient quality of life , rehospitalization , behavioral , and psychosocial characteristics . this study relies on data from the 1545 patients who completed both baseline and 1month followup interviews , including the ctm15 . the study has been approved by the university of massachusetts medical school institutional review board and all participants gave informed consent for participation . we used ctm15 data from the 1month followup interview and demographic characteristics and a selfreported health status measure collected at the baseline interview . other data collected at 1 month include selfreported questions on rehospitalization , emergency department visits and symptom development postdischarge along with 3 independent questions evaluating transition quality ( access to medical records , prescheduled followup appointments , and knowing who to contact if symptoms worsen ) . classic test theory psychometric analyses were performed to provide an overall understanding of item and scale quality . we computed itemlevel statistics , including mean scores , proportions of response categories , and itemtotal correlations . items with a negative or low ( < 0.2 ) item total correlation have undesirable discriminating power and should be flagged for further study . we also examined scalelevel score distribution , ceiling and floor effects , and internal consistency ( cronbach 's ) . dimensionality analyses were conducted to examine the factor structure of the ctm items in the tracecore cohort . the evidence of factor structure how many latent factors are needed in explaining the variance of item responses can be used to both justify the use of a single scale score as currently reported in the ctm and lay the groundwork for satisfying the unidimensionality assumption for further advanced psychometric modeling ( eg , irt ) . confirmatory factor analyses were conducted in the framework of structural equation modeling using the software mplus . the polychoric correlation matrix and weighted least squares with adjustments for mean and variance estimation ordered categorical data were used . in addition to a model replicating the ctm 's reported 4factor structure , we tested a unidimensional model and a bifactor model ( items loading on 1 general factor and 4 secondary factors ) . the model data fit was evaluated by examining the magnitude and patterns of item factor loadings , as well as the commonly used fit statistics compared with established thresholds ( eg , the comparative fit index ( cfi ) 0.95 , root mean square error of approximation ( rmsea ) 0.08 ) . the residual matrix and modification indices were used to detect any local item dependency , which can potentially arise when a subset of items have the same phrasing or format and thus are correlated with each other after the primary factor is controlled for . it was hypothesized that the ctm items present essential unidimensionality with a dominant factor , namely , that the general factor has uniformly high loadings on all items and explains the majority of total item covariance . modern measurement theory assumes that each response option has a specific relationship with the underlying construct , so a unique response can be selected for a different interval of the scale . if this assumption is violated , individual response options do not provide unique information and therefore can be collapsed before fitting an irt model . we inspected item characteristic curves for outoforder or nondiscriminating response options by using the testgraf program . good items should have response choice categories with unequivocal and unique relationships to the latent trait appearing in rank order . visual inspection of these item characteristic curve graphs provides insight into whether an item 's response choice categories overlap and require rescaling ( or collapsing across response choice categories ) to achieve distinct categories . to conduct the irt analyses with the ctm the model fit for all items was evaluated using irtfit macro for sas ( sas institute ) . validity analyses were conducted to evaluate the ability of ctm scores ( ctm15 , ctm3 , ctmirt ) to discriminate patients of different transitional care quality and health status using previously reported variables . specifically , the knowngroups method of construct validation was used to compare groups using 1way anova and t tests . groups compared were formed on the basis of demographic characteristics and patients ' responses to nonctm questions assessing care transition quality processes ( eg , access to medical records at discharge , scheduled followup visits with a health care provider ) , health care use at 1 month postdischarge ( emergency department visits , rehospitalization ) , health status scores , and symptom trajectories postdischarge . transitions , risks , and actions in coronary events center for outcomes research and education ( tracecore ) is a large , multiracial cohort of adult patients hospitalized with acute coronary syndromes ( acs ) from 6 hospitals in massachusetts and georgia . a total of 2300 patients were enrolled in the study and were approached for computerassisted telephone followup interviews at 1 , 3 , 6 , and 12 months post acs hospitalization . interviews focus on individual patient quality of life , rehospitalization , behavioral , and psychosocial characteristics . this study relies on data from the 1545 patients who completed both baseline and 1month followup interviews , including the ctm15 . the study has been approved by the university of massachusetts medical school institutional review board and all participants gave informed consent for participation . we used ctm15 data from the 1month followup interview and demographic characteristics and a selfreported health status measure collected at the baseline interview . other data collected at 1 month include selfreported questions on rehospitalization , emergency department visits and symptom development postdischarge along with 3 independent questions evaluating transition quality ( access to medical records , prescheduled followup appointments , and knowing who to contact if symptoms worsen ) . classic test theory psychometric analyses were performed to provide an overall understanding of item and scale quality . we computed itemlevel statistics , including mean scores , proportions of response categories , and itemtotal correlations . items with a negative or low ( < 0.2 ) item total correlation have undesirable discriminating power and should be flagged for further study . we also examined scalelevel score distribution , ceiling and floor effects , and internal consistency ( cronbach 's ) . dimensionality analyses were conducted to examine the factor structure of the ctm items in the tracecore cohort . the evidence of factor structure how many latent factors are needed in explaining the variance of item responses can be used to both justify the use of a single scale score as currently reported in the ctm and lay the groundwork for satisfying the unidimensionality assumption for further advanced psychometric modeling ( eg , irt ) . confirmatory factor analyses were conducted in the framework of structural equation modeling using the software mplus . the polychoric correlation matrix and weighted least squares with adjustments for mean and variance estimation ordered categorical data were used . in addition to a model replicating the ctm 's reported 4factor structure , we tested a unidimensional model and a bifactor model ( items loading on 1 general factor and 4 secondary factors ) . the model data fit was evaluated by examining the magnitude and patterns of item factor loadings , as well as the commonly used fit statistics compared with established thresholds ( eg , the comparative fit index ( cfi ) 0.95 , root mean square error of approximation ( rmsea ) 0.08 ) . the residual matrix and modification indices were used to detect any local item dependency , which can potentially arise when a subset of items have the same phrasing or format and thus are correlated with each other after the primary factor is controlled for . it was hypothesized that the ctm items present essential unidimensionality with a dominant factor , namely , that the general factor has uniformly high loadings on all items and explains the majority of total item covariance . modern measurement theory assumes that each response option has a specific relationship with the underlying construct , so a unique response can be selected for a different interval of the scale . if this assumption is violated , individual response options do not provide unique information and therefore can be collapsed before fitting an irt model . we inspected item characteristic curves for outoforder or nondiscriminating response options by using the testgraf program . good items should have response choice categories with unequivocal and unique relationships to the latent trait appearing in rank order . visual inspection of these item characteristic curve graphs provides insight into whether an item 's response choice categories overlap and require rescaling ( or collapsing across response choice categories ) to achieve distinct categories . to conduct the irt analyses with the ctm the model fit for all items was evaluated using irtfit macro for sas ( sas institute ) . validity analyses were conducted to evaluate the ability of ctm scores ( ctm15 , ctm3 , ctmirt ) to discriminate patients of different transitional care quality and health status using previously reported variables . specifically , the knowngroups method of construct validation was used to compare groups using 1way anova and t tests . groups compared were formed on the basis of demographic characteristics and patients ' responses to nonctm questions assessing care transition quality processes ( eg , access to medical records at discharge , scheduled followup visits with a health care provider ) , health care use at 1 month postdischarge ( emergency department visits , rehospitalization ) , health status scores , and symptom trajectories postdischarge . we compared the 3 scores by using relative validity coefficients . classic test theory psychometric analyses were performed to provide an overall understanding of item and scale quality . we computed itemlevel statistics , including mean scores , proportions of response categories , and itemtotal correlations . items with a negative or low ( < 0.2 ) item total correlation have undesirable discriminating power and should be flagged for further study . we also examined scalelevel score distribution , ceiling and floor effects , and internal consistency ( cronbach 's ) . dimensionality analyses were conducted to examine the factor structure of the ctm items in the tracecore cohort . the evidence of factor structure how many latent factors are needed in explaining the variance of item responses can be used to both justify the use of a single scale score as currently reported in the ctm and lay the groundwork for satisfying the unidimensionality assumption for further advanced psychometric modeling ( eg , irt ) . confirmatory factor analyses were conducted in the framework of structural equation modeling using the software mplus . the polychoric correlation matrix and weighted least squares with adjustments for mean and variance estimation ordered categorical data were used . in addition to a model replicating the ctm 's reported 4factor structure , we tested a unidimensional model and a bifactor model ( items loading on 1 general factor and 4 secondary factors ) . the model data fit was evaluated by examining the magnitude and patterns of item factor loadings , as well as the commonly used fit statistics compared with established thresholds ( eg , the comparative fit index ( cfi ) 0.95 , root mean square error of approximation ( rmsea ) 0.08 ) . the residual matrix and modification indices were used to detect any local item dependency , which can potentially arise when a subset of items have the same phrasing or format and thus are correlated with each other after the primary factor is controlled for . it was hypothesized that the ctm items present essential unidimensionality with a dominant factor , namely , that the general factor has uniformly high loadings on all items and explains the majority of total item covariance . modern measurement theory assumes that each response option has a specific relationship with the underlying construct , so a unique response can be selected for a different interval of the scale . if this assumption is violated , individual response options do not provide unique information and therefore can be collapsed before fitting an irt model . we inspected item characteristic curves for outoforder or nondiscriminating response options by using the testgraf program . good items should have response choice categories with unequivocal and unique relationships to the latent trait appearing in rank order . visual inspection of these item characteristic curve graphs provides insight into whether an item 's response choice categories overlap and require rescaling ( or collapsing across response choice categories ) to achieve distinct categories . to conduct the irt analyses with the ctm the model fit for all items was evaluated using irtfit macro for sas ( sas institute ) . validity analyses were conducted to evaluate the ability of ctm scores ( ctm15 , ctm3 , ctmirt ) to discriminate patients of different transitional care quality and health status using previously reported variables . specifically , the knowngroups method of construct validation was used to compare groups using 1way anova and t tests . groups compared were formed on the basis of demographic characteristics and patients ' responses to nonctm questions assessing care transition quality processes ( eg , access to medical records at discharge , scheduled followup visits with a health care provider ) , health care use at 1 month postdischarge ( emergency department visits , rehospitalization ) , health status scores , and symptom trajectories postdischarge . study participants were predominantly white ( 79% ) and male ( 67% ) , reporting a mean age of 62 years . relatively few subjects reported having a college education or higher ( 27% ) or annual household income greater than $ 75 000 ( 29% ) . selfreported health status was normally distributed across the 5 response categories from poor to excellent ( table 1 ) . baseline characteristics of tracecore participants ctm indicates care transitions measure ; tracecore , transitions , risks , and actions in coronary events center for outcomes research and education . itemlevel review ( table 2 ) revealed sample means that were slightly higher ( average item mean 3.2 , range 2.9 to 3.3 ) than item means reported in earlier development and validation reports ( average item mean 3.1 , range 2.9 to 3.2 ) , yet , as reported in table 3 , these means were lower than those reported in recent publications ( eg , average item mean 3.7 , range 3.6 to 3.9 ) . in our study , the ctm15 scale demonstrated good internal consistency ( cronbach 's 0.95 ) that is comparable to previous studies ( cronbach 's 0.90 to 0.95 ) ; however , the ctm15 had a ceiling effect of 8.7% , substantially higher than the 1.1% reported in the original reports and similar to the 10% ceiling effect levels reported in some later studies . none of our participants scored at the floor ( at the lowest possible score ) ; indeed , very few ( 5.5% ) scored in the lower half of the scale 's theoretical range ( < 50 ) . ctm15 summary data : development and validation studies ctm indicates care transitions measure ; tracecore , transitions , risks , and actions in coronary events center for outcomes research and education . of the sample , of the sample , 40% had a value of 66.7 . among the 15 ctm item responses ( table 2 ) , nineteen percent ( n=288 ) of all participants selected agree as a response to all 15 questions , while strongly disagree , do n't know , and refused were least selected , across all items . dimensionality analyses of the ctm in our study population supports use of the single score assessment and provided evidence that the scale is sufficiently unidimensional for irt analysis . the bifactor model fit the data best ( cfi=0.981 , tuckerlevis index ( tli)=0.995 , rmsea=0.10 ) with the general factor having higher loadings on most of the items and explaining most of the total item covariance ( see table 4 ) . review of the residual matrix did not detect any items with local dependency ( had the lowest score ) , further supporting the essential unidimensionality of the data and the applicability of irt methods . confirmatory factor analyses model results cfi indicates comparative fit index ; rmsea , root mean square error of approximation , tli , tuckerlewis index . review of the item characteristic curves revealed that all 15 items had some nondiscriminating response options that should be collapsed . for 3 items , we collapsed the 4 response categories into 2 ( agree versus disagree ) , and for the rest of the items , the response options of disagree and strongly disagree were collapsed into 1 . the results of our irt parameter estimates and item fit are presented in table 5 . the fit index suggested that 5 items did not fit the model well , but the violations were minor . these item parameters were used to calculate an irtbased score for the 15 items of the ctm ( denoted ctmirt ) . a scatterplot of the 2 measures ( figure ) revealed that the relationship followed the expected sshaped curve for higher scores , but for scores below 40 , the scatter is rather wide , reflecting the scarcity of information available for estimating the irt parameters in this range . irt parameters and item fit for ctm items ctm indicates care transitions measure ; irt , item response theory . an anova ( table 6 ) was used to evaluate the ability of ctm to differentiate scores among different patient groups . as in previous studies , the ctm15 score was usually lower for patients who reported poor general health , problems with independent care transition indicators , and postdischarge rehospitalization and emergency department visits . in our study population , in contrast to the original reports , men and younger patients reported better ctm scores for most indicators , the differences in the group mean scores were small ( 2 to 3 points ; less than onefifth of an sd ) . known group validity results for the long ( ctm15 ) , short ( ctm3 ) , and irtbased ( ctm15 irt ) scores ctm indicates care transitions measure ; ed , emergency department ; irt , item response theory ; rv , relative validity . overall , the ctm3 was able to detect differences in selected groups , as well as the ctm15 , for many patient characteristics , selfreported health , and selfreport care transition indicator subgroups . however , the ctm3 slightly inflated patient care transition scores , compared with the ctm15 ; this difference was observed primarily for small subgroups . in this sample , the short measure failed to detect differences between patients with and without selfreported rehospitalization and emergency department visits . as expected , the sd of item responses was larger for the ctm3 than for the ctm15 . all relative validity coefficients for comparing the ctmirt with the traditional ctm15 were close to , and not significantly different from , 1.0 , suggesting that the irt scoring approach did not improve measurement precision . itemlevel review ( table 2 ) revealed sample means that were slightly higher ( average item mean 3.2 , range 2.9 to 3.3 ) than item means reported in earlier development and validation reports ( average item mean 3.1 , range 2.9 to 3.2 ) , yet , as reported in table 3 , these means were lower than those reported in recent publications ( eg , average item mean 3.7 , range 3.6 to 3.9 ) . in our study , the ctm15 scale demonstrated good internal consistency ( cronbach 's 0.95 ) that is comparable to previous studies ( cronbach 's 0.90 to 0.95 ) ; however , the ctm15 had a ceiling effect of 8.7% , substantially higher than the 1.1% reported in the original reports and similar to the 10% ceiling effect levels reported in some later studies . none of our participants scored at the floor ( at the lowest possible score ) ; indeed , very few ( 5.5% ) scored in the lower half of the scale 's theoretical range ( < 50 ) . ctm15 summary data : development and validation studies ctm indicates care transitions measure ; tracecore , transitions , risks , and actions in coronary events center for outcomes research and education . of the sample , of the sample , 40% had a value of 66.7 . among the 15 ctm item responses ( table 2 ) , the most frequently selected was nineteen percent ( n=288 ) of all participants selected agree as a response to all 15 questions , while strongly disagree , do n't know , and refused were least selected , across all items . dimensionality analyses of the ctm in our study population supports use of the single score assessment and provided evidence that the scale is sufficiently unidimensional for irt analysis . the bifactor model fit the data best ( cfi=0.981 , tuckerlevis index ( tli)=0.995 , rmsea=0.10 ) with the general factor having higher loadings on most of the items and explaining most of the total item covariance ( see table 4 ) . review of the residual matrix did not detect any items with local dependency ( had the lowest score ) , further supporting the essential unidimensionality of the data and the applicability of irt methods . confirmatory factor analyses model results cfi indicates comparative fit index ; rmsea , root mean square error of approximation , tli , tuckerlewis index . review of the item characteristic curves revealed that all 15 items had some nondiscriminating response options that should be collapsed . for 3 items , we collapsed the 4 response categories into 2 ( agree versus disagree ) , and for the rest of the items , the response options of disagree and strongly disagree were collapsed into 1 . the results of our irt parameter estimates and item fit are presented in table 5 . the fit index suggested that 5 items did not fit the model well , but the violations were minor . these item parameters were used to calculate an irtbased score for the 15 items of the ctm ( denoted ctmirt ) . a scatterplot of the 2 measures ( figure ) revealed that the relationship followed the expected sshaped curve for higher scores , but for scores below 40 , the scatter is rather wide , reflecting the scarcity of information available for estimating the irt parameters in this range . irt parameters and item fit for ctm items ctm indicates care transitions measure ; irt , item response theory . an anova ( table 6 ) was used to evaluate the ability of ctm to differentiate scores among different patient groups . as in previous studies , the ctm15 score was usually lower for patients who reported poor general health , problems with independent care transition indicators , and postdischarge rehospitalization and emergency department visits . in our study population , in contrast to the original reports , men and younger patients reported better ctm scores . for most indicators , the differences in the group mean scores were small ( 2 to 3 points ; less than onefifth of an sd ) . known group validity results for the long ( ctm15 ) , short ( ctm3 ) , and irtbased ( ctm15 irt ) scores ctm indicates care transitions measure ; ed , emergency department ; irt , item response theory ; rv , relative validity . overall , the ctm3 was able to detect differences in selected groups , as well as the ctm15 , for many patient characteristics , selfreported health , and selfreport care transition indicator subgroups . however , the ctm3 slightly inflated patient care transition scores , compared with the ctm15 ; this difference was observed primarily for small subgroups . in this sample , the short measure failed to detect differences between patients with and without selfreported rehospitalization and emergency department visits . relative validity coefficients suggest that this underperformance is not statistically significant . as expected , the sd of item responses was larger for the ctm3 than for the ctm15 . all relative validity coefficients for comparing the ctmirt with the traditional ctm15 were close to , and not significantly different from , 1.0 , suggesting that the irt scoring approach did not improve measurement precision . the major findings of this work of this study are related to the psychometric characteristics of the ctm and the application of irt methods for improvement of measurement precisions . the basic psychometric characteristics of the ctm15 in our data were similar to those previously reported ; itemlevel means were comparable to those previously reported and the measure had good internal consistency and reliability . however , we also identified some undesirable characteristics of the ctm15 scale score , which may influence the measurement of care transitions as a performance measure and future multivariable analyses . the distribution of ctm15 summary score was severely leftskewed , due to a substantial ceiling effect and clustering of high summary transition scores . moreover , the 4 response options do not provide unique information ; 3 ctm items may be better assessed using a binary response . these problems can be partially explained by strong acquiescence bias ( the tendency of respondents to agree with statements of opinion regardless of content ) , which is common with the agree / disagree format used . understanding the source does not fix the problem , however , since the highly skewed , clustered responses lead to scores with little variance , making it hard even with rescaling to discriminate levels of care transition quality . in our knowngroups validity analyses , the ctm15 found statistically significant hypothesized differences supporting findings from earlier reports ; however , the magnitude of differences ( 2 to 3 points on a 0to100 scale , with an sd of 16 ) observed for many of these tests , was small . in the absence of established guidelines for minimally important differences , we would typically only consider differences to be practically meaningful if they are 0.5 sd or greater . the problems discussed here with the ctm15 apply as well or more to its short form , the ctm3 . this is of particular concern given that the ctm3 is included in the cahps hospital survey , used to judge hospital quality . explorations of the validity of the ctm3 based on the knowngroups validity method had mixed results that raise questions about its ability to detect differences in care transition quality . our results complement those of a recent study of the ctm15 , which also identified some gaps in measurement performance for assessing the quality of care transitions in a complex population of older rehabilitation patients . while the ctm was found to be reliable , the authors noted that the construct validity and utility of the measure could be improved . qualitative data in their study revealed the arbitrary nature of the choice that some patients make when selecting a response category between agree and strongly agree . strongly agreei guess i just do n't want you to think i 'm not listening by choosing strongly agree all the time ! in addition , focusgroup respondents identified some aspects of transitional care that are not included in the ctm , such as , building a relationship , and effectively communicating with , one 's clinician , raising questions as to the ctm 's content validity . the authors also noted problems associated with the large proportion of participants responding in agreement , which aligns with reports from the ctm 's developers that most patients agreed with each ctm question ( range 69% to 94% ) . in this study , we also explored the possibility that the application of irt scoring may improve the measurement properties of the ctm15 . theoretically irt scoring can often improve measurement precision ; however , as indicated by nonsignificant relative validity coefficients , irt scoring did not improve the ctm15 . this is the first attempt to apply irt scoring to the ctm of which we are aware . several reports on the use of irt or rasch scoring approach to patient reported measures developed in the classical test theory framework have produced mixed results . when irt was used to score the short form36 physical function scale modest gains were observed , of these , the strongest gains were demonstrated in the most clinically dissimilar groups . in sensitivity studies of the same measure , irt scores were more sensitive for the general population across 7 countries , while results for the short form36 in patients with epilepsy were mixed depending on external criteria . considerable gains in precision were reported for the rasch scoring of the oxford hip score questionnaire . precision was also improved using irtbased scoring for the upper limb subscale of the motor assessment scale , particularly in the scale 's extreme ranges . an irtbased scoring approach improved the sensitivity of the visual function index , but produced no gains for crosssectional comparisons . no improvement in precision was observed for the health assessment questionnaire and the eortc qlqc30 scales . possible causes for this have been discussed previously and could be related to the misfit of some items to the irt model , the fact that the measure was developed within the classic test theory framework and not specifically for the irt , and the existence of several parallel items with highly similar content . unique to the ctm was acquiescence bias , in which up to 30% of respondents selected the same response option ( agree or strongly agree ) for all 15 items , leading to extreme clustering of the scale . for these respondents , irt scoring can not improve over a traditional sum scored approach . with the tight clustering of scale scores in the upper end of the scale , it is possible that irt scoring failed to capture any additional information for the respondents . these findings are also in line with previous reports that irt gains are potentially largest at the extremes of a construct 's range . finally , it is worth noting that while the ctm15 is a patientreported measure , it aims to evaluate the patient 's experience with a care process ; it does not relate to any of health , quality of life , or physical functioning . we used a large and diverse sample to conduct indepth psychometric evaluation using both classic and modern analytic methods . to the best of our knowledge , this is the most comprehensive psychometric evaluation of the ctm . our rich data set will also allow for future evaluation of relationship of the ctm with important clinical variables . while our sample is the largest one on which ctm15 psychometric analyses have been reported , it is still limited to a particular patient population and geographic regions . good psychometric practice would require validation in samples that are significantly different from the ones previously evaluated . our study participants who completed the measure reported on average higher level of education , better health , and higher income compared with all patients enrolled at baseline in the study . these differences may have biased the average ctm15 scores , leading to an underestimation of proportions of patients who report poor transitions and an overestimation of the ceiling effect in the measure . given the diverse sample on which the analyses were completed , however , the general conclusions of the psychometric evaluation are most likely robust . the major findings of this work of this study are related to the psychometric characteristics of the ctm and the application of irt methods for improvement of measurement precisions . the basic psychometric characteristics of the ctm15 in our data were similar to those previously reported ; itemlevel means were comparable to those previously reported and the measure had good internal consistency and reliability . however , we also identified some undesirable characteristics of the ctm15 scale score , which may influence the measurement of care transitions as a performance measure and future multivariable analyses . the distribution of ctm15 summary score was severely leftskewed , due to a substantial ceiling effect and clustering of high summary transition scores . moreover , the 4 response options do not provide unique information ; 3 ctm items may be better assessed using a binary response . these problems can be partially explained by strong acquiescence bias ( the tendency of respondents to agree with statements of opinion regardless of content ) , which is common with the agree / disagree format used . understanding the source does not fix the problem , however , since the highly skewed , clustered responses lead to scores with little variance , making it hard even with rescaling to discriminate levels of care transition quality . in our knowngroups validity analyses , the ctm15 found statistically significant hypothesized differences supporting findings from earlier reports ; however , the magnitude of differences ( 2 to 3 points on a 0to100 scale , with an sd of 16 ) observed for many of these tests , was small . in the absence of established guidelines for minimally important differences , we would typically only consider differences to be practically meaningful if they are 0.5 sd or greater . the problems discussed here with the ctm15 apply as well or more to its short form , the ctm3 . this is of particular concern given that the ctm3 is included in the cahps hospital survey , used to judge hospital quality . explorations of the validity of the ctm3 based on the knowngroups validity method had mixed results that raise questions about its ability to detect differences in care transition quality . our results complement those of a recent study of the ctm15 , which also identified some gaps in measurement performance for assessing the quality of care transitions in a complex population of older rehabilitation patients . while the ctm was found to be reliable , the authors noted that the construct validity and utility of the measure could be improved . qualitative data in their study revealed the arbitrary nature of the choice that some patients make when selecting a response category between agree and strongly agree . strongly agreei guess i just do n't want you to think i 'm not listening by choosing strongly agree all the time ! in addition , focusgroup respondents identified some aspects of transitional care that are not included in the ctm , such as , building a relationship , and effectively communicating with , one 's clinician , raising questions as to the ctm 's content validity . the authors also noted problems associated with the large proportion of participants responding in agreement , which aligns with reports from the ctm 's developers that most patients agreed with each ctm question ( range 69% to 94% ) . in this study , we also explored the possibility that the application of irt scoring may improve the measurement properties of the ctm15 . theoretically irt scoring can often improve measurement precision ; however , as indicated by nonsignificant relative validity coefficients , irt scoring did not improve the ctm15 . this is the first attempt to apply irt scoring to the ctm of which we are aware . several reports on the use of irt or rasch scoring approach to patient reported measures developed in the classical test theory framework have produced mixed results . when irt was used to score the short form36 physical function scale modest gains were observed , of these , the strongest gains were demonstrated in the most clinically dissimilar groups . in sensitivity studies of the same measure , irt scores were more sensitive for the general population across 7 countries , while results for the short form36 in patients with epilepsy were mixed depending on external criteria . considerable gains in precision were reported for the rasch scoring of the oxford hip score questionnaire . precision was also improved using irtbased scoring for the upper limb subscale of the motor assessment scale , particularly in the scale 's extreme ranges . an irtbased scoring approach improved the sensitivity of the visual function index , but produced no gains for crosssectional comparisons . no improvement in precision was observed for the health assessment questionnaire and the eortc qlqc30 scales . possible causes for this have been discussed previously and could be related to the misfit of some items to the irt model , the fact that the measure was developed within the classic test theory framework and not specifically for the irt , and the existence of several parallel items with highly similar content . unique to the ctm was acquiescence bias , in which up to 30% of respondents selected the same response option ( agree or strongly agree ) for all 15 items , leading to extreme clustering of the scale . for these respondents , irt scoring can not improve over a traditional sum scored approach . with the tight clustering of scale scores in the upper end of the scale , it is possible that irt scoring failed to capture any additional information for the respondents . these findings are also in line with previous reports that irt gains are potentially largest at the extremes of a construct 's range . finally , it is worth noting that while the ctm15 is a patientreported measure , it aims to evaluate the patient 's experience with a care process ; it does not relate to any of health , quality of life , or physical functioning . we used a large and diverse sample to conduct indepth psychometric evaluation using both classic and modern analytic methods . to the best of our knowledge , our rich data set will also allow for future evaluation of relationship of the ctm with important clinical variables . while our sample is the largest one on which ctm15 psychometric analyses have been reported , it is still limited to a particular patient population and geographic regions . good psychometric practice would require validation in samples that are significantly different from the ones previously evaluated . our study participants who completed the measure reported on average higher level of education , better health , and higher income compared with all patients enrolled at baseline in the study . these differences may have biased the average ctm15 scores , leading to an underestimation of proportions of patients who report poor transitions and an overestimation of the ceiling effect in the measure . given the diverse sample on which the analyses were completed , however , the general conclusions of the psychometric evaluation are most likely robust . our findings have important implications for analyzing and interpreting ctm scores . to the extent that ctm score distributions are both clustered around certain values and skewed , it is important that appropriate analytic techniques are used in the analyses of ctm scores . if the linear scoring of the ctm15 is not supported by available data , it would be more important to use analytic approaches for categorical data . results from the ctm short form may have even a higher ceiling effect and conclusions , thus it may not always correspond to results of the ctm15 as previously suggested . the strong acquiescence bias of the measure also suggests that ctm results may be representing an overly optimistic view of care transition quality . to increase the utility of the ctm , it will be important to determine whether observed magnitudes of difference ( 2 to 3 points on the ctm scale ) between groups are clinically important using recommended triangulation approaches . future work should also aim to improve care transition quality assessments by constructing a measure that fully reflects what patients care about in the area of care transition , and that is less prone to acquiescence bias . in summary , we identified some psychometric challenges in both the long and short forms of the ctm that could not be improved with irtbased scoring . combined with accumulating evidence of existing gaps in the ctm 's content validity , this study suggests that ctm scores should be interpreted with caution and may not be a sufficiently sensitive tool for detecting meaningful improvements in the quality of transitional care .

backgroundthe quality of transitional care is associated with important health outcomes such as rehospitalization and costs . the widely used care transitions measure ( ctm15 ) was developed with a classic test theory approach ; its short version ( ctm3 ) was included in the cahps hospital survey . we conducted a psychometric evaluation of both measures and explored whether item response theory ( irt ) could produce a more precise measure.methods and resultsas part of the transitions , risks , and actions in coronary events center for outcomes research and education , 1545 participants were interviewed during an acute coronary syndrome hospitalization , providing information on general health status ( short form36 ) , ctm15 , health utilization , and care process questions at 1 month postdischarge . we used classic and irt analyses and compared the measurement precision of ctm15 , ctm3 , and ctmirt based score using relative validity.participants were 79% nonhispanic white and 67% male , with an average age of 62 years . the ctm15 had good internal consistency ( cronbach 's =0.95 ) but demonstrated acquiescence bias ( 8.7% participants responded strongly agree and 19% responded agree to all items ) and limited score variability . these problems were more pronounced for the ctm3 . the ctm15 differentiated between patient groups defined by selfreported health status , health care utilization , and care transition process indicators . differences between groups were small ( 2 to 3 points ) . there was no gain in measurement precision from irt scoring . the ctm3 was not significantly lower for patients reporting rehospitalization or emergency department visits.conclusionwe identified psychometric challenges of the ctm , which may limit its value in research and practice . these results are in line with emerging evidence of gaps in the validity of the measure .

Introduction Methods Sample Measures Analytic Plan Classic Test Theory Psychometric Analyses Dimensionality Analyses IRT Modeling Validity Analyses Results Classic Test Theory Psychometric Analyses IRT Modeling Validity Analyses Discussion Major Findings Strengths and Limitations Conclusions and Implications

an effective measure of care transition quality the most widely used measure of care transition quality is the care transitions measure ( ctm15 ) . all 15 items in the ctm15 use a 4point scale with responses ranging from strongly disagree to strongly agree . a 3item version of the ctm was found to explain 88% of the variance in the full measure ; the ctm3 has been demonstrated to have the same ability to detect group differences as the longer version in earlier studies . the ctm3 was endorsed by the national quality forum and included in the consumer assessment of healthcare providers and systems ( cahps ) hospital survey in 2010 . while this approach is widely used , moresophisticated approaches to scale scoring based on item response theory ( irt ) are gaining popularity , since the irt method uses more of the available information in each item , thus , theoretically , irt can produce a more discriminating score based on the same underlying data than a sum score . the aim of this report is to conduct a psychometric analysis of ctm items and evaluate the dimensionality , internal consistency , and construct validity of ctm15 and ctm3 . transitions , risks , and actions in coronary events center for outcomes research and education ( tracecore ) is a large , multiracial cohort of adult patients hospitalized with acute coronary syndromes ( acs ) from 6 hospitals in massachusetts and georgia . we used ctm15 data from the 1month followup interview and demographic characteristics and a selfreported health status measure collected at the baseline interview . other data collected at 1 month include selfreported questions on rehospitalization , emergency department visits and symptom development postdischarge along with 3 independent questions evaluating transition quality ( access to medical records , prescheduled followup appointments , and knowing who to contact if symptoms worsen ) . we also examined scalelevel score distribution , ceiling and floor effects , and internal consistency ( cronbach 's ) . the evidence of factor structure how many latent factors are needed in explaining the variance of item responses can be used to both justify the use of a single scale score as currently reported in the ctm and lay the groundwork for satisfying the unidimensionality assumption for further advanced psychometric modeling ( eg , irt ) . it was hypothesized that the ctm items present essential unidimensionality with a dominant factor , namely , that the general factor has uniformly high loadings on all items and explains the majority of total item covariance . to conduct the irt analyses with the ctm the model fit for all items was evaluated using irtfit macro for sas ( sas institute ) . validity analyses were conducted to evaluate the ability of ctm scores ( ctm15 , ctm3 , ctmirt ) to discriminate patients of different transitional care quality and health status using previously reported variables . groups compared were formed on the basis of demographic characteristics and patients ' responses to nonctm questions assessing care transition quality processes ( eg , access to medical records at discharge , scheduled followup visits with a health care provider ) , health care use at 1 month postdischarge ( emergency department visits , rehospitalization ) , health status scores , and symptom trajectories postdischarge . transitions , risks , and actions in coronary events center for outcomes research and education ( tracecore ) is a large , multiracial cohort of adult patients hospitalized with acute coronary syndromes ( acs ) from 6 hospitals in massachusetts and georgia . other data collected at 1 month include selfreported questions on rehospitalization , emergency department visits and symptom development postdischarge along with 3 independent questions evaluating transition quality ( access to medical records , prescheduled followup appointments , and knowing who to contact if symptoms worsen ) . we also examined scalelevel score distribution , ceiling and floor effects , and internal consistency ( cronbach 's ) . the evidence of factor structure how many latent factors are needed in explaining the variance of item responses can be used to both justify the use of a single scale score as currently reported in the ctm and lay the groundwork for satisfying the unidimensionality assumption for further advanced psychometric modeling ( eg , irt ) . validity analyses were conducted to evaluate the ability of ctm scores ( ctm15 , ctm3 , ctmirt ) to discriminate patients of different transitional care quality and health status using previously reported variables . groups compared were formed on the basis of demographic characteristics and patients ' responses to nonctm questions assessing care transition quality processes ( eg , access to medical records at discharge , scheduled followup visits with a health care provider ) , health care use at 1 month postdischarge ( emergency department visits , rehospitalization ) , health status scores , and symptom trajectories postdischarge . we also examined scalelevel score distribution , ceiling and floor effects , and internal consistency ( cronbach 's ) . the evidence of factor structure how many latent factors are needed in explaining the variance of item responses can be used to both justify the use of a single scale score as currently reported in the ctm and lay the groundwork for satisfying the unidimensionality assumption for further advanced psychometric modeling ( eg , irt ) . to conduct the irt analyses with the ctm the model fit for all items was evaluated using irtfit macro for sas ( sas institute ) . validity analyses were conducted to evaluate the ability of ctm scores ( ctm15 , ctm3 , ctmirt ) to discriminate patients of different transitional care quality and health status using previously reported variables . groups compared were formed on the basis of demographic characteristics and patients ' responses to nonctm questions assessing care transition quality processes ( eg , access to medical records at discharge , scheduled followup visits with a health care provider ) , health care use at 1 month postdischarge ( emergency department visits , rehospitalization ) , health status scores , and symptom trajectories postdischarge . study participants were predominantly white ( 79% ) and male ( 67% ) , reporting a mean age of 62 years . baseline characteristics of tracecore participants ctm indicates care transitions measure ; tracecore , transitions , risks , and actions in coronary events center for outcomes research and education . in our study , the ctm15 scale demonstrated good internal consistency ( cronbach 's 0.95 ) that is comparable to previous studies ( cronbach 's 0.90 to 0.95 ) ; however , the ctm15 had a ceiling effect of 8.7% , substantially higher than the 1.1% reported in the original reports and similar to the 10% ceiling effect levels reported in some later studies . ctm15 summary data : development and validation studies ctm indicates care transitions measure ; tracecore , transitions , risks , and actions in coronary events center for outcomes research and education . among the 15 ctm item responses ( table 2 ) , nineteen percent ( n=288 ) of all participants selected agree as a response to all 15 questions , while strongly disagree , do n't know , and refused were least selected , across all items . for 3 items , we collapsed the 4 response categories into 2 ( agree versus disagree ) , and for the rest of the items , the response options of disagree and strongly disagree were collapsed into 1 . these item parameters were used to calculate an irtbased score for the 15 items of the ctm ( denoted ctmirt ) . irt parameters and item fit for ctm items ctm indicates care transitions measure ; irt , item response theory . as in previous studies , the ctm15 score was usually lower for patients who reported poor general health , problems with independent care transition indicators , and postdischarge rehospitalization and emergency department visits . in our study population , in contrast to the original reports , men and younger patients reported better ctm scores for most indicators , the differences in the group mean scores were small ( 2 to 3 points ; less than onefifth of an sd ) . known group validity results for the long ( ctm15 ) , short ( ctm3 ) , and irtbased ( ctm15 irt ) scores ctm indicates care transitions measure ; ed , emergency department ; irt , item response theory ; rv , relative validity . overall , the ctm3 was able to detect differences in selected groups , as well as the ctm15 , for many patient characteristics , selfreported health , and selfreport care transition indicator subgroups . however , the ctm3 slightly inflated patient care transition scores , compared with the ctm15 ; this difference was observed primarily for small subgroups . in this sample , the short measure failed to detect differences between patients with and without selfreported rehospitalization and emergency department visits . all relative validity coefficients for comparing the ctmirt with the traditional ctm15 were close to , and not significantly different from , 1.0 , suggesting that the irt scoring approach did not improve measurement precision . in our study , the ctm15 scale demonstrated good internal consistency ( cronbach 's 0.95 ) that is comparable to previous studies ( cronbach 's 0.90 to 0.95 ) ; however , the ctm15 had a ceiling effect of 8.7% , substantially higher than the 1.1% reported in the original reports and similar to the 10% ceiling effect levels reported in some later studies . ctm15 summary data : development and validation studies ctm indicates care transitions measure ; tracecore , transitions , risks , and actions in coronary events center for outcomes research and education . among the 15 ctm item responses ( table 2 ) , the most frequently selected was nineteen percent ( n=288 ) of all participants selected agree as a response to all 15 questions , while strongly disagree , do n't know , and refused were least selected , across all items . for 3 items , we collapsed the 4 response categories into 2 ( agree versus disagree ) , and for the rest of the items , the response options of disagree and strongly disagree were collapsed into 1 . irt parameters and item fit for ctm items ctm indicates care transitions measure ; irt , item response theory . as in previous studies , the ctm15 score was usually lower for patients who reported poor general health , problems with independent care transition indicators , and postdischarge rehospitalization and emergency department visits . for most indicators , the differences in the group mean scores were small ( 2 to 3 points ; less than onefifth of an sd ) . known group validity results for the long ( ctm15 ) , short ( ctm3 ) , and irtbased ( ctm15 irt ) scores ctm indicates care transitions measure ; ed , emergency department ; irt , item response theory ; rv , relative validity . overall , the ctm3 was able to detect differences in selected groups , as well as the ctm15 , for many patient characteristics , selfreported health , and selfreport care transition indicator subgroups . in this sample , the short measure failed to detect differences between patients with and without selfreported rehospitalization and emergency department visits . all relative validity coefficients for comparing the ctmirt with the traditional ctm15 were close to , and not significantly different from , 1.0 , suggesting that the irt scoring approach did not improve measurement precision . the basic psychometric characteristics of the ctm15 in our data were similar to those previously reported ; itemlevel means were comparable to those previously reported and the measure had good internal consistency and reliability . however , we also identified some undesirable characteristics of the ctm15 scale score , which may influence the measurement of care transitions as a performance measure and future multivariable analyses . these problems can be partially explained by strong acquiescence bias ( the tendency of respondents to agree with statements of opinion regardless of content ) , which is common with the agree / disagree format used . in our knowngroups validity analyses , the ctm15 found statistically significant hypothesized differences supporting findings from earlier reports ; however , the magnitude of differences ( 2 to 3 points on a 0to100 scale , with an sd of 16 ) observed for many of these tests , was small . the problems discussed here with the ctm15 apply as well or more to its short form , the ctm3 . this is of particular concern given that the ctm3 is included in the cahps hospital survey , used to judge hospital quality . explorations of the validity of the ctm3 based on the knowngroups validity method had mixed results that raise questions about its ability to detect differences in care transition quality . our results complement those of a recent study of the ctm15 , which also identified some gaps in measurement performance for assessing the quality of care transitions in a complex population of older rehabilitation patients . while the ctm was found to be reliable , the authors noted that the construct validity and utility of the measure could be improved . in addition , focusgroup respondents identified some aspects of transitional care that are not included in the ctm , such as , building a relationship , and effectively communicating with , one 's clinician , raising questions as to the ctm 's content validity . the authors also noted problems associated with the large proportion of participants responding in agreement , which aligns with reports from the ctm 's developers that most patients agreed with each ctm question ( range 69% to 94% ) . in this study , we also explored the possibility that the application of irt scoring may improve the measurement properties of the ctm15 . theoretically irt scoring can often improve measurement precision ; however , as indicated by nonsignificant relative validity coefficients , irt scoring did not improve the ctm15 . in sensitivity studies of the same measure , irt scores were more sensitive for the general population across 7 countries , while results for the short form36 in patients with epilepsy were mixed depending on external criteria . possible causes for this have been discussed previously and could be related to the misfit of some items to the irt model , the fact that the measure was developed within the classic test theory framework and not specifically for the irt , and the existence of several parallel items with highly similar content . unique to the ctm was acquiescence bias , in which up to 30% of respondents selected the same response option ( agree or strongly agree ) for all 15 items , leading to extreme clustering of the scale . with the tight clustering of scale scores in the upper end of the scale , it is possible that irt scoring failed to capture any additional information for the respondents . finally , it is worth noting that while the ctm15 is a patientreported measure , it aims to evaluate the patient 's experience with a care process ; it does not relate to any of health , quality of life , or physical functioning . to the best of our knowledge , this is the most comprehensive psychometric evaluation of the ctm . our rich data set will also allow for future evaluation of relationship of the ctm with important clinical variables . our study participants who completed the measure reported on average higher level of education , better health , and higher income compared with all patients enrolled at baseline in the study . these differences may have biased the average ctm15 scores , leading to an underestimation of proportions of patients who report poor transitions and an overestimation of the ceiling effect in the measure . the basic psychometric characteristics of the ctm15 in our data were similar to those previously reported ; itemlevel means were comparable to those previously reported and the measure had good internal consistency and reliability . however , we also identified some undesirable characteristics of the ctm15 scale score , which may influence the measurement of care transitions as a performance measure and future multivariable analyses . these problems can be partially explained by strong acquiescence bias ( the tendency of respondents to agree with statements of opinion regardless of content ) , which is common with the agree / disagree format used . in our knowngroups validity analyses , the ctm15 found statistically significant hypothesized differences supporting findings from earlier reports ; however , the magnitude of differences ( 2 to 3 points on a 0to100 scale , with an sd of 16 ) observed for many of these tests , was small . the problems discussed here with the ctm15 apply as well or more to its short form , the ctm3 . this is of particular concern given that the ctm3 is included in the cahps hospital survey , used to judge hospital quality . explorations of the validity of the ctm3 based on the knowngroups validity method had mixed results that raise questions about its ability to detect differences in care transition quality . our results complement those of a recent study of the ctm15 , which also identified some gaps in measurement performance for assessing the quality of care transitions in a complex population of older rehabilitation patients . in addition , focusgroup respondents identified some aspects of transitional care that are not included in the ctm , such as , building a relationship , and effectively communicating with , one 's clinician , raising questions as to the ctm 's content validity . the authors also noted problems associated with the large proportion of participants responding in agreement , which aligns with reports from the ctm 's developers that most patients agreed with each ctm question ( range 69% to 94% ) . in this study , we also explored the possibility that the application of irt scoring may improve the measurement properties of the ctm15 . theoretically irt scoring can often improve measurement precision ; however , as indicated by nonsignificant relative validity coefficients , irt scoring did not improve the ctm15 . in sensitivity studies of the same measure , irt scores were more sensitive for the general population across 7 countries , while results for the short form36 in patients with epilepsy were mixed depending on external criteria . precision was also improved using irtbased scoring for the upper limb subscale of the motor assessment scale , particularly in the scale 's extreme ranges . possible causes for this have been discussed previously and could be related to the misfit of some items to the irt model , the fact that the measure was developed within the classic test theory framework and not specifically for the irt , and the existence of several parallel items with highly similar content . unique to the ctm was acquiescence bias , in which up to 30% of respondents selected the same response option ( agree or strongly agree ) for all 15 items , leading to extreme clustering of the scale . with the tight clustering of scale scores in the upper end of the scale , it is possible that irt scoring failed to capture any additional information for the respondents . finally , it is worth noting that while the ctm15 is a patientreported measure , it aims to evaluate the patient 's experience with a care process ; it does not relate to any of health , quality of life , or physical functioning . to the best of our knowledge , our rich data set will also allow for future evaluation of relationship of the ctm with important clinical variables . our study participants who completed the measure reported on average higher level of education , better health , and higher income compared with all patients enrolled at baseline in the study . these differences may have biased the average ctm15 scores , leading to an underestimation of proportions of patients who report poor transitions and an overestimation of the ceiling effect in the measure . the strong acquiescence bias of the measure also suggests that ctm results may be representing an overly optimistic view of care transition quality . to increase the utility of the ctm , it will be important to determine whether observed magnitudes of difference ( 2 to 3 points on the ctm scale ) between groups are clinically important using recommended triangulation approaches . future work should also aim to improve care transition quality assessments by constructing a measure that fully reflects what patients care about in the area of care transition , and that is less prone to acquiescence bias . combined with accumulating evidence of existing gaps in the ctm 's content validity , this study suggests that ctm scores should be interpreted with caution and may not be a sufficiently sensitive tool for detecting meaningful improvements in the quality of transitional care .

optic neuritis ( on ) refers to inflammation of the optic nerve and is caused by inflammatory demyelination of the optic nerve , by infection , or by non - specific inflammation . the main clinical manifestations include pain during eye movement , sudden vision loss in one or both eyes , visual field defects , a relative afferent pupillary obstacle , and papilledema . clinically , on leads to lesions of the optic nerve axons and apoptosis of retinal ganglion cells . on can occur in patients with multiple sclerosis ( ms ) or neuromyelitis optica ( nmo ) . studies have estimated a prevalence of 5 cases per 100 000 individuals per year in central europe . although treatment of the inflammation results in good eyesight recovery in many patients with on , vision does not return to normal in others , and may be accompanied by abnormal color vision and visual field defects . on is commonly considered a retinal disease , but a previous study demonstrated that patients with on presented abnormalities in the visual cortex . currently , optical coherence tomography ( oct ) and visual evoked potential ( vep ) are the most important methods for diagnosis of on . oct is a noninvasive , high - resolution method that measures the thickness of the retinal nerve fiber layer . a previous study found that patients with ms had thinning of the retinal nerve fiber layer . vep is an important clinical test and has been used in studying patients with on and demyelinating diseases . the vep features of on are amplitude decrease and prolonged latency , which reflect nerve axon lesions and apoptosis of retinal ganglion cells . the significant correlation of multifocal vep ( mfvep ) latency suggests a role for demyelination in promoting axonal loss . a previous study showed that patients with on still had mfvep amplitude delays present in many locations , even if they recovered near - normal vision sensitivity . diffusion tensor imaging can accurately measure fractional anisotropy ( fa ) and mean diffusivity of the visual pathway . a previous study showed that axial diffusivity ( ad ) of affected nerves decreases during acute on and that this ad reduction correlates with the extent of axonal loss . another study reported that patients with idiopathic demyelinating on showed decreased mean fa in the affected nerves . in addition , a previous study using fmri found that patients with on had reduced functional connectivity within the visual system [ . although these findings showed neuronal morphological changes in the on , there was far less evidence for changes in the neuromechanism of brain in patients with on . vbm is a fully automated , whole - brain measurement technique that compares voxel - wise , between - group differences in local brain morphology . the vbm method has been successfully used to investigate mechanisms of diseases such as alzheimer s disease , obsessive compulsive disorder , and glaucoma . a previous study found that patients with neuromyelitis optica ( nmo ) showed white matter ( wm ) atrophy in areas such as the optic chiasm , pons , cerebellum , and frontal gyrus . another study reported that patients with nmo showed wm atrophy in the corpus callosum and optic radiations , and gray matter ( gm ) atrophy in the thalamus and prefrontal cortex . although these findings showed both wm and gm atrophy in patients with on , few studies have combined the vbm method with vep . to the best of our knowledge the current study is the first to explore the changes in wm and gm in on and their relationship with vep . twelve patients with on ( 4 males , 8 females ) were recruited from the ophthalmology department of the first affiliated hospital of nanchang university . inclusion criteria were : 1 ) acute vision loss with or without eye pain ; 2 ) visual field abnormalities associated with damage to nerve fibers ; 3 ) patients with relative pupillary conduction block or abnormal vep ; 4 ) no clinical or laboratory evidence of compression , ischemic , toxic , genetic , metabolic , or invasive optic neuropathy ; 5 ) no acute vision loss due to retinal disease , alternative eye disease , or disease of the nervous system ; 6 ) no treatment with any drugs before resting - state fmri scanning ; 7 ) no obvious abnormality in the brain parenchyma on head mri scans ( including cerebral infarction , cerebral hemorrhage , cerebral hemangioma , and cerebral tumors ) ; 8) no history of congenital or acquired diseases such as psychiatric disorder , hypertension , diabetes mellitus , or coronary artery disease , and no addictions such as heroin , smoking , or alcohol ; 9 ) no receipt of organ transplantation ; and 10 ) moderate body shape and weight . twelve healthy controls ( hcs ) ( 4 males , 8 females ) , who were age- , sex- , and education status - matched to the patients with on , were also recruited for this study . all hcs met the following criteria : 1 ) no abnormalities in visual pathways or brain parenchyma on head mri scans ; 2 ) no ocular disease , naked eye , the corrected visual acuity > 1.0 ; 3 ) normal nervous system and mental status , with no headaches ; and 4 ) no contraindications for mri . the study was authorized by the ethics committee of the first affiliated hospital of nanchang university . for each subject , the study protocol and procedure were fully explained and consent was obtained . all the methods of this research followed the declaration of helsinki and conformed to the principles of medical ethics . participants were scanned on a 3-tesla mr scanner ( trio , siemens , germany ) with a 12-channel head coil . high - resolution t1-weighted transversal images covering the whole brain were acquired with a magnetization - prepared rapid gradient echo ( mp - rage ) sequence : 176 slices with section thickness of 1.0 mm ; echo time=2.26 ms ; repetition time=1900 ms ; field of view=215230 mm . a neuroradiologist evaluated all scans for gross structural abnormalities . functional data were classified by use of mricro software ( www.mricro.com ) to eliminate incomplete data . structural images were processed with the voxel - based morphometry toolbox ( vbm8 ) ( http://dbm.neuro.uni-jena.de/vbm8/ ) implemented in statistical parametric mapping ( spm8 ) ( wellcome department of imaging neuroscience , london , uk ) running on matlab 7.9.0 ( r2009b ; the mathworks , inc , natick , ma , usa ) . vbm is a whole - brain , unbiased , semi - automated technique for characterizing regional cerebral differences in structural magnetic resonance images . segmentation of individual brains into gray matter , white matter , and cerebrospinal fluid was based on vbm8 using the default estimation options ( very light bias regularization , 60 mm cut - off for estimating the gaussian smoothness of bias in image intensity ; icbm [ international consortium for brain mapping ] european template for initial affine transformation ) . spatial normalization into the montreal neurological institute ( mni ) standard space was done by the high - dimensional dartel ( diffeomorphic anatomical registration through exponentiated lie algebra ) approach implemented in vbm8 . we used dartel to produce gray and white matter templates and used the generated template for all participants standardized gray matter and white matter . finally , the modulated volumes were smoothed with a 6-mm full - width - at - half - maximum ( fwhm ) gaussian kernel . normalized , modulated , smoothed images were submitted to group - level analyses . a general linear model ( glm ) analysis was performed with the spm8 ( http://www.fil.ion.ucl.ac.uk/spm ) toolkit to investigate the group differences in gm and wm between patients with on and healthy controls after controlling for the effects of age and sex . the significance level was set at p<0.05 , gaussian random field ( grf ) theory corrected , minimum z>2.3 . statistically significant voxels were superimposed on the standardization of 3dt1wi ( 3-dimensional magnetization prepared rapid acquisition gradient echo sequences ) to generate a color drawing . voxel threshold was selected for 20 neighboring voxels to analyze the area of gm atrophy in patients with on . for vep stimulation analysis all subjects underwent pattern - reversal vep stimulation ( retlport electrophysiological instrument , roland , brandenburg , germany ) in a dark and quiet room . three active skin electrodes were placed on the scalp along the midline ( over the inion ) and on lateral positions ( right and left ) . all patients underwent monocular recording with the untested eye covered . using stimulus mode with pattern - reversal vep stimulation , the parameters were set as : stimulus frequency=1.0 hz and 100 hz ; interphase=500 ms ; number of stimulations=100 ; average screen brightness=5 cd / m ; spatial frequency=50 ms / s ; and contrast ratio=90% . amplitude and latency vep values were studied at different angular dimensions of the stimulus ( 120 , 60 , and 15 degrees for stimuli with small , medium , and large spatial frequencies of stimulation , respectively ) . veps were characterized by a series of n75 , p100 , and n135 peaks , each characterized by a specific amplitude and latency . the vep decreased amplitude and prolonged latency , which reflect nerve axon lesions and apoptosis of retinal ganglion cells . for visual tests analysis first , the corneal curvature of all subjects was calculated , then we used the lens measurement instrument to detect the refractive diopter . second , subjects were kept standing 5 meters from vision chart and the gaze was kept parallel with the vision chart 1.0 . third , according to the diopter number , we chose the best lens to correct the vision . twelve patients with on ( 4 males , 8 females ) were recruited from the ophthalmology department of the first affiliated hospital of nanchang university . inclusion criteria were : 1 ) acute vision loss with or without eye pain ; 2 ) visual field abnormalities associated with damage to nerve fibers ; 3 ) patients with relative pupillary conduction block or abnormal vep ; 4 ) no clinical or laboratory evidence of compression , ischemic , toxic , genetic , metabolic , or invasive optic neuropathy ; 5 ) no acute vision loss due to retinal disease , alternative eye disease , or disease of the nervous system ; 6 ) no treatment with any drugs before resting - state fmri scanning ; 7 ) no obvious abnormality in the brain parenchyma on head mri scans ( including cerebral infarction , cerebral hemorrhage , cerebral hemangioma , and cerebral tumors ) ; 8) no history of congenital or acquired diseases such as psychiatric disorder , hypertension , diabetes mellitus , or coronary artery disease , and no addictions such as heroin , smoking , or alcohol ; 9 ) no receipt of organ transplantation ; and 10 ) moderate body shape and weight . twelve healthy controls ( hcs ) ( 4 males , 8 females ) , who were age- , sex- , and education status - matched to the patients with on , were also recruited for this study . all hcs met the following criteria : 1 ) no abnormalities in visual pathways or brain parenchyma on head mri scans ; 2 ) no ocular disease , naked eye , the corrected visual acuity > 1.0 ; 3 ) normal nervous system and mental status , with no headaches ; and 4 ) no contraindications for mri . the study was authorized by the ethics committee of the first affiliated hospital of nanchang university . for each subject , the study protocol and procedure were fully explained and consent was obtained . all the methods of this research followed the declaration of helsinki and conformed to the principles of medical ethics . participants were scanned on a 3-tesla mr scanner ( trio , siemens , germany ) with a 12-channel head coil . high - resolution t1-weighted transversal images covering the whole brain were acquired with a magnetization - prepared rapid gradient echo ( mp - rage ) sequence : 176 slices with section thickness of 1.0 mm ; echo time=2.26 ms ; repetition time=1900 ms ; field of view=215230 mm . a neuroradiologist evaluated all scans for gross structural abnormalities . functional data were classified by use of mricro software ( www.mricro.com ) to eliminate incomplete data . structural images were processed with the voxel - based morphometry toolbox ( vbm8 ) ( http://dbm.neuro.uni-jena.de/vbm8/ ) implemented in statistical parametric mapping ( spm8 ) ( wellcome department of imaging neuroscience , london , uk ) running on matlab 7.9.0 ( r2009b ; the mathworks , inc , natick , ma , usa ) . vbm is a whole - brain , unbiased , semi - automated technique for characterizing regional cerebral differences in structural magnetic resonance images . segmentation of individual brains into gray matter , white matter , and cerebrospinal fluid was based on vbm8 using the default estimation options ( very light bias regularization , 60 mm cut - off for estimating the gaussian smoothness of bias in image intensity ; icbm [ international consortium for brain mapping ] european template for initial affine transformation ) . spatial normalization into the montreal neurological institute ( mni ) standard space was done by the high - dimensional dartel ( diffeomorphic anatomical registration through exponentiated lie algebra ) approach implemented in vbm8 . we used dartel to produce gray and white matter templates and used the generated template for all participants standardized gray matter and white matter . finally , the modulated volumes were smoothed with a 6-mm full - width - at - half - maximum ( fwhm ) gaussian kernel . a general linear model ( glm ) analysis was performed with the spm8 ( http://www.fil.ion.ucl.ac.uk/spm ) toolkit to investigate the group differences in gm and wm between patients with on and healthy controls after controlling for the effects of age and sex . the significance level was set at p<0.05 , gaussian random field ( grf ) theory corrected , minimum z>2.3 . statistically significant voxels were superimposed on the standardization of 3dt1wi ( 3-dimensional magnetization prepared rapid acquisition gradient echo sequences ) to generate a color drawing . voxel threshold was selected for 20 neighboring voxels to analyze the area of gm atrophy in patients with on . for vep stimulation analysis all subjects underwent pattern - reversal vep stimulation ( retlport electrophysiological instrument , roland , brandenburg , germany ) in a dark and quiet room . three active skin electrodes were placed on the scalp along the midline ( over the inion ) and on lateral positions ( right and left ) . all patients underwent monocular recording with the untested eye covered . using stimulus mode with pattern - reversal vep stimulation , the parameters were set as : stimulus frequency=1.0 hz and 100 hz ; interphase=500 ms ; number of stimulations=100 ; average screen brightness=5 cd / m ; spatial frequency=50 ms / s ; and contrast ratio=90% . amplitude and latency vep values were studied at different angular dimensions of the stimulus ( 120 , 60 , and 15 degrees for stimuli with small , medium , and large spatial frequencies of stimulation , respectively ) . veps were characterized by a series of n75 , p100 , and n135 peaks , each characterized by a specific amplitude and latency . the vep decreased amplitude and prolonged latency , which reflect nerve axon lesions and apoptosis of retinal ganglion cells . for visual tests analysis we combined the snellen vision chart with artificial optometry . first , the corneal curvature of all subjects was calculated , then we used the lens measurement instrument to detect the refractive diopter . second , subjects were kept standing 5 meters from vision chart and the gaze was kept parallel with the vision chart 1.0 . third , according to the diopter number , we chose the best lens to correct the vision . if the subjects had no refractive errors , we recorded the unaided eye vision . there were no obvious differences in weight ( p=0.749 ) , age ( p=0.827 ) , or height ( p=0.719 ) between the patients with on and the hcs . there were significant differences between the patients with on and the hcs for best - corrected va - right ( p<0.001 ) and best - corrected va - left ( p=0.001 ) . there were marked differences between the ons and the hcs in latency of the vep in the right eye ( p=0.001 ) , latency of the vep in the left eye ( p<0.001 ) , amplitudes of the vep in the right eye ( p<0.001 ) , and amplitudes of the vep in the left eye ( p=0.008 ) . compared with the hcs , patients with on had significantly decreased gm volume ( gmv ) in the brain regions of the left postcentral gyrus , left inferior frontal gyrus , left anterior cingulate , right middle frontal gyrus , left middle frontal gyrus , and right inferior parietal lobule ( figure 1 [ blue ] , table 2 ) . patients with on also had decreased wm volume ( wmv ) in the brain regions of the left middle frontal gyrus , right superior frontal gyrus , left precentral gyrus and right inferior parietal lobule , and increased wmv in the left fusiform gyrus and left inferior parietal lobule ( figure 2 , table 3 ) . in addition , we showed the mean of altered gmv values and wmv values between the on group and hcs ( figure 3 ) . in the on group , the vep latency of the right eye showed a positive correlation with the wmv signal value of the left fusiform gyrus ( r=0.726 , p=0.008 ) and a negative correlation with the gmv signal value of the right inferior parietal lobule ( r=0.611 , p=0.035 ) . duration of on showed a negative correlation with the wmv signal value of the right superior frontal gyrus ( r=0.662 , p=0.019 ) . the best - corrected va of the right eye showed a negative correlation with the wmvs signal value of the left middle frontal gyrus ( r=0.704 , p=0.011 ) . there were no obvious differences in weight ( p=0.749 ) , age ( p=0.827 ) , or height ( p=0.719 ) between the patients with on and the hcs . there were significant differences between the patients with on and the hcs for best - corrected va - right ( p<0.001 ) and best - corrected va - left ( p=0.001 ) . there were marked differences between the ons and the hcs in latency of the vep in the right eye ( p=0.001 ) , latency of the vep in the left eye ( p<0.001 ) , amplitudes of the vep in the right eye ( p<0.001 ) , and amplitudes of the vep in the left eye ( p=0.008 ) . compared with the hcs , patients with on had significantly decreased gm volume ( gmv ) in the brain regions of the left postcentral gyrus , left inferior frontal gyrus , left anterior cingulate , right middle frontal gyrus , left middle frontal gyrus , and right inferior parietal lobule ( figure 1 [ blue ] , table 2 ) . patients with on also had decreased wm volume ( wmv ) in the brain regions of the left middle frontal gyrus , right superior frontal gyrus , left precentral gyrus and right inferior parietal lobule , and increased wmv in the left fusiform gyrus and left inferior parietal lobule ( figure 2 , table 3 ) . in addition , we showed the mean of altered gmv values and wmv values between the on group and hcs ( figure 3 ) . in the on group , the vep latency of the right eye showed a positive correlation with the wmv signal value of the left fusiform gyrus ( r=0.726 , p=0.008 ) and a negative correlation with the gmv signal value of the right inferior parietal lobule ( r=0.611 , p=0.035 ) . duration of on showed a negative correlation with the wmv signal value of the right superior frontal gyrus ( r=0.662 , p=0.019 ) . the best - corrected va of the right eye showed a negative correlation with the wmvs signal value of the left middle frontal gyrus ( r=0.704 , p=0.011 ) . to the best of our knowledge , our study is the first to investigate changes in wm and gm in patients with on using a vbm approach . we found markedly decreased gmv in the brain regions of the left postcentral gyrus , left inferior frontal gyrus , left anterior cingulate , right middle frontal gyrus , left middle frontal gyrus , and right inferior parietal lobule in patients with on . in addition , we observed that patients with on had significantly decreased wmv values in the brain regions of the left middle frontal gyrus , right superior frontal gyrus , left precentral gyrus , and right inferior parietal lobule , whereas they had significantly increased wmv values in the cluster of the left fusiform gyrus and left inferior parietal lobule . furthermore , we observed that the vep latency of the right eye in patients with on had a positive correlation with the wmv signal value of the left fusiform gyrus ( r=0.726 , p=0.008 ) and a negative correlation with the gmv signal value of the right inferior parietal lobule ( r=0.611 , p=0.035 ) . we also found that the duration of on had a negative correlation with the wmv signal value of the right superior frontal gyrus ( r=0.662 , p=0.019 ) . the best - corrected va of the right eye had a negative correlation with the wmv signal value of the left middle frontal gyrus ( r=0.704 , p=0.011 ) . compared with the hcs the postcentral gyrus is delimited by the central sulcus anteriorly and the postcentral sulcus posteriorly ; this area is the somatosensory cortex in the human brain . previous studies have identified many diseases that lead to postcentral gyrus dysfunction , such as parkinson disease , alzheimer disease , and multiple sclerosis . furthermore , duan et al . , using a voxel - based morphometry method , found that patients with nmo had decreased wmv in the right postcentral gyrus . in support of these findings , we also found that patients with on had significantly decreased gmv in the left postcentral gyrus . moreover , we found that patients with on had decreased gmv in the inferior frontal gyrus and bilateral middle frontal gyrus . a previous study demonstrated that the frontal eye fields are causally involved in the attentional top - down control of anticipatory alpha power in the contralateral visual system . in addition , the inferior frontal gyrus is related to emotional and cognitive empathy , while the middle frontal gyrus is associated with the processing of language . many diseases lead to middle frontal gyrus dysfunction , including schizophrenia and attention deficit hyperactivity disorder ( adhd ) . liu et al . found that patients with nmo had significantly increased amplitude of low - frequency fluctuations ( alff ) in the middle frontal gyrus . however , liang et al . showed that patients with nmo had decreased regional homogeneity ( reho ) in the left medial frontal gyrus . in support of these findings , we found significant regions of atrophy in the inferior frontal gyrus and bilateral middle frontal gyrus in patients with on . we therefore speculate that on may lead to dysfunction of the frontal gyrus , while deficit in the frontal gyrus may reflect the damage to eye motion and cognition in patients with on . we found that the vep latency of the right eye in patients with on correlated negatively with the gmv signal value of the right inferior parietal lobule ( r=0.611 , p=0.035 ) , which agrees with a previous study demonstrating that patients with on had prolonged latency . the prolonged vep latency in on reflects the severity of on ; therefore , the decreased gmv signal values of the right inferior parietal lobule may be related to the severity of on . compared with the hcs , patients with on had significantly decreased wmv values in the brain regions of the left middle frontal gyrus , right superior frontal gyrus , left precentral gyrus , and right inferior parietal lobule . many brain function areas are involved in the default mode network ( dmn ) , including the posterior and anterior cingulate cortices , inferior parietal cortex , medial temporal lobes , and medial frontal cortex . network , which shows higher activity at rest and tends to have a negative correlation with activity in task - positive networks . liang et al . found that patients with nmo had decreased reho in the left medial frontal gyrus . demonstrated that patients with nmo had decreased wmv in the left middle and medial frontal gyrus , the right superior frontal gyrus , and the bilateral inferior and superior parietal lobules . in support of these findings , we found that patients with on had decreased wmv values in the left middle frontal gyrus and right inferior parietal lobule , which may reflect dysfunction of the dmn in the patients with on . furthermore , the duration of on correlated negatively with the wmv signal value of the right superior frontal gyrus ( r=0.662 , p=0.019 ) , and we speculate that the longer the duration of on , the more serious the injury to the dmn . we also found that the best - corrected va of the right eye correlated negatively with wmv signal value of the left middle frontal gyrus ( r=0.704 , p=0.011 ) . we speculate that the decreased wmv signal value of the left middle frontal gyrus may reflect the damage to va in on . interestingly , we found significantly increased wmv values regions in the left fusiform gyrus and left inferior parietal lobule in patients with on . the fusiform gyrus is the fusiform face area , which participates in face processing and social cognition . a previous study showed that visual stimuli can cause increases in blood flow in the contralateral posterior fusiform gyrus . the increased wmv areas in the left fusiform gyrus may be related to compensation for the damaged visual function in patients with on . furthermore , we observed that the vep latency of the right eye in on correlated positively with the wmv signal value of the left fusiform gyrus ( r=0.726 , p=0.008 ) . the increased vep latency in on reflects the severity of on , and thus the increased wmv values of the fusiform gyrus may relate to the severity of on . , we found that patients with on had higher wmv values in the left inferior parietal lobule and reduced wmv values in the right inferior parietal lobule . thus , the higher wmv values in the left inferior parietal lobule may reflect functional reorganization to compensate for the damaged areas in patients with on . first , the sample size was too small , which may have affected the accuracy of the results . second , there were some differences in the duration of on , and we were not able to entirely separate sex differences and age differences . in future studies we found that patients with on had abnormal wm and gm involvement in regional brain changes , which correlated with the vep latency of the eye . . however , there are some limitations to our study , such as the relatively small sample size , the use of a single center , and the lack of comparison between patients before and after treatment . in future studies we will use other methods to evaluate the changes in brain function in patients with on .

backgroundthe aim of this study was to investigate potential morphological alterations of gray and white matter in patients with optic neuritis ( on ) and their relationship with behavioral performance , using voxel - based morphometry ( vbm).material / methodstwelve ( 4 males , 8 females ) patients with on and 12 ( 4 males , 8 females ) age- , sex- , and education - matched healthy controls ( hcs ) underwent magnetic resonance imaging ( mri ) . imaging data were analyzed using two - sample t tests to identify group differences in gray and white matter volume ( gmv , wmv ) . correlation analysis was used to explore relationships between observed gmv and wmv of different areas and visual evoked potential ( vep ) in on.resultscompared with hcs , on patients had : significantly decreased gmv in the left postcentral gyrus , left inferior frontal gyrus , left anterior cingulate , left and right middle frontal gyrus , and right inferior parietal lobule ; decreased wmv in the left middle frontal gyrus , right superior frontal gyrus , left precentral gyrus and right inferior parietal lobule ; and increased wmv in the left fusiform gyrus and left inferior parietal lobule . vep latency of the right eye in on correlated positively with wmv signal value of the left fusiform gyrus ( r=0.726 , p=0.008 ) , and negatively with gmv signal value of the right inferior parietal lobule ( r=0.611 , p=0.035 ) . duration of on correlated negatively with wmv signal value of the right superior frontal gyrus ( r=0.662 , p=0.019 ) , while best - corrected visual acuity ( va ) of the right eye correlated negatively with wmv signal value of the left middle frontal gyrus ( r=0.704 , p=0.011).conclusionsthese results suggest significant brain involvement in on , which may reflect the underlying pathologic mechanism . correlational results demonstrate that vep in on is closely associated with wmv and gmv atrophy in many brain regions .

Background Material and Methods Subjects Structural MRI parameters Image processing Statistical analysis Results Behavioral results Gray and white matter differences Correlation analysis Discussion Conclusions

optic neuritis ( on ) refers to inflammation of the optic nerve and is caused by inflammatory demyelination of the optic nerve , by infection , or by non - specific inflammation . clinically , on leads to lesions of the optic nerve axons and apoptosis of retinal ganglion cells . although treatment of the inflammation results in good eyesight recovery in many patients with on , vision does not return to normal in others , and may be accompanied by abnormal color vision and visual field defects . on is commonly considered a retinal disease , but a previous study demonstrated that patients with on presented abnormalities in the visual cortex . currently , optical coherence tomography ( oct ) and visual evoked potential ( vep ) are the most important methods for diagnosis of on . a previous study found that patients with ms had thinning of the retinal nerve fiber layer . vep is an important clinical test and has been used in studying patients with on and demyelinating diseases . a previous study showed that patients with on still had mfvep amplitude delays present in many locations , even if they recovered near - normal vision sensitivity . a previous study showed that axial diffusivity ( ad ) of affected nerves decreases during acute on and that this ad reduction correlates with the extent of axonal loss . although these findings showed neuronal morphological changes in the on , there was far less evidence for changes in the neuromechanism of brain in patients with on . vbm is a fully automated , whole - brain measurement technique that compares voxel - wise , between - group differences in local brain morphology . the vbm method has been successfully used to investigate mechanisms of diseases such as alzheimer s disease , obsessive compulsive disorder , and glaucoma . a previous study found that patients with neuromyelitis optica ( nmo ) showed white matter ( wm ) atrophy in areas such as the optic chiasm , pons , cerebellum , and frontal gyrus . another study reported that patients with nmo showed wm atrophy in the corpus callosum and optic radiations , and gray matter ( gm ) atrophy in the thalamus and prefrontal cortex . although these findings showed both wm and gm atrophy in patients with on , few studies have combined the vbm method with vep . to the best of our knowledge the current study is the first to explore the changes in wm and gm in on and their relationship with vep . twelve patients with on ( 4 males , 8 females ) were recruited from the ophthalmology department of the first affiliated hospital of nanchang university . inclusion criteria were : 1 ) acute vision loss with or without eye pain ; 2 ) visual field abnormalities associated with damage to nerve fibers ; 3 ) patients with relative pupillary conduction block or abnormal vep ; 4 ) no clinical or laboratory evidence of compression , ischemic , toxic , genetic , metabolic , or invasive optic neuropathy ; 5 ) no acute vision loss due to retinal disease , alternative eye disease , or disease of the nervous system ; 6 ) no treatment with any drugs before resting - state fmri scanning ; 7 ) no obvious abnormality in the brain parenchyma on head mri scans ( including cerebral infarction , cerebral hemorrhage , cerebral hemangioma , and cerebral tumors ) ; 8) no history of congenital or acquired diseases such as psychiatric disorder , hypertension , diabetes mellitus , or coronary artery disease , and no addictions such as heroin , smoking , or alcohol ; 9 ) no receipt of organ transplantation ; and 10 ) moderate body shape and weight . twelve healthy controls ( hcs ) ( 4 males , 8 females ) , who were age- , sex- , and education status - matched to the patients with on , were also recruited for this study . all hcs met the following criteria : 1 ) no abnormalities in visual pathways or brain parenchyma on head mri scans ; 2 ) no ocular disease , naked eye , the corrected visual acuity > 1.0 ; 3 ) normal nervous system and mental status , with no headaches ; and 4 ) no contraindications for mri . structural images were processed with the voxel - based morphometry toolbox ( vbm8 ) ( http://dbm.neuro.uni-jena.de/vbm8/ ) implemented in statistical parametric mapping ( spm8 ) ( wellcome department of imaging neuroscience , london , uk ) running on matlab 7.9.0 ( r2009b ; the mathworks , inc , natick , ma , usa ) . we used dartel to produce gray and white matter templates and used the generated template for all participants standardized gray matter and white matter . a general linear model ( glm ) analysis was performed with the spm8 ( http://www.fil.ion.ucl.ac.uk/spm ) toolkit to investigate the group differences in gm and wm between patients with on and healthy controls after controlling for the effects of age and sex . voxel threshold was selected for 20 neighboring voxels to analyze the area of gm atrophy in patients with on . twelve patients with on ( 4 males , 8 females ) were recruited from the ophthalmology department of the first affiliated hospital of nanchang university . inclusion criteria were : 1 ) acute vision loss with or without eye pain ; 2 ) visual field abnormalities associated with damage to nerve fibers ; 3 ) patients with relative pupillary conduction block or abnormal vep ; 4 ) no clinical or laboratory evidence of compression , ischemic , toxic , genetic , metabolic , or invasive optic neuropathy ; 5 ) no acute vision loss due to retinal disease , alternative eye disease , or disease of the nervous system ; 6 ) no treatment with any drugs before resting - state fmri scanning ; 7 ) no obvious abnormality in the brain parenchyma on head mri scans ( including cerebral infarction , cerebral hemorrhage , cerebral hemangioma , and cerebral tumors ) ; 8) no history of congenital or acquired diseases such as psychiatric disorder , hypertension , diabetes mellitus , or coronary artery disease , and no addictions such as heroin , smoking , or alcohol ; 9 ) no receipt of organ transplantation ; and 10 ) moderate body shape and weight . twelve healthy controls ( hcs ) ( 4 males , 8 females ) , who were age- , sex- , and education status - matched to the patients with on , were also recruited for this study . all hcs met the following criteria : 1 ) no abnormalities in visual pathways or brain parenchyma on head mri scans ; 2 ) no ocular disease , naked eye , the corrected visual acuity > 1.0 ; 3 ) normal nervous system and mental status , with no headaches ; and 4 ) no contraindications for mri . the study was authorized by the ethics committee of the first affiliated hospital of nanchang university . structural images were processed with the voxel - based morphometry toolbox ( vbm8 ) ( http://dbm.neuro.uni-jena.de/vbm8/ ) implemented in statistical parametric mapping ( spm8 ) ( wellcome department of imaging neuroscience , london , uk ) running on matlab 7.9.0 ( r2009b ; the mathworks , inc , natick , ma , usa ) . segmentation of individual brains into gray matter , white matter , and cerebrospinal fluid was based on vbm8 using the default estimation options ( very light bias regularization , 60 mm cut - off for estimating the gaussian smoothness of bias in image intensity ; icbm [ international consortium for brain mapping ] european template for initial affine transformation ) . we used dartel to produce gray and white matter templates and used the generated template for all participants standardized gray matter and white matter . a general linear model ( glm ) analysis was performed with the spm8 ( http://www.fil.ion.ucl.ac.uk/spm ) toolkit to investigate the group differences in gm and wm between patients with on and healthy controls after controlling for the effects of age and sex . voxel threshold was selected for 20 neighboring voxels to analyze the area of gm atrophy in patients with on . three active skin electrodes were placed on the scalp along the midline ( over the inion ) and on lateral positions ( right and left ) . there were no obvious differences in weight ( p=0.749 ) , age ( p=0.827 ) , or height ( p=0.719 ) between the patients with on and the hcs . there were significant differences between the patients with on and the hcs for best - corrected va - right ( p<0.001 ) and best - corrected va - left ( p=0.001 ) . there were marked differences between the ons and the hcs in latency of the vep in the right eye ( p=0.001 ) , latency of the vep in the left eye ( p<0.001 ) , amplitudes of the vep in the right eye ( p<0.001 ) , and amplitudes of the vep in the left eye ( p=0.008 ) . compared with the hcs , patients with on had significantly decreased gm volume ( gmv ) in the brain regions of the left postcentral gyrus , left inferior frontal gyrus , left anterior cingulate , right middle frontal gyrus , left middle frontal gyrus , and right inferior parietal lobule ( figure 1 [ blue ] , table 2 ) . patients with on also had decreased wm volume ( wmv ) in the brain regions of the left middle frontal gyrus , right superior frontal gyrus , left precentral gyrus and right inferior parietal lobule , and increased wmv in the left fusiform gyrus and left inferior parietal lobule ( figure 2 , table 3 ) . in the on group , the vep latency of the right eye showed a positive correlation with the wmv signal value of the left fusiform gyrus ( r=0.726 , p=0.008 ) and a negative correlation with the gmv signal value of the right inferior parietal lobule ( r=0.611 , p=0.035 ) . duration of on showed a negative correlation with the wmv signal value of the right superior frontal gyrus ( r=0.662 , p=0.019 ) . the best - corrected va of the right eye showed a negative correlation with the wmvs signal value of the left middle frontal gyrus ( r=0.704 , p=0.011 ) . there were no obvious differences in weight ( p=0.749 ) , age ( p=0.827 ) , or height ( p=0.719 ) between the patients with on and the hcs . there were significant differences between the patients with on and the hcs for best - corrected va - right ( p<0.001 ) and best - corrected va - left ( p=0.001 ) . there were marked differences between the ons and the hcs in latency of the vep in the right eye ( p=0.001 ) , latency of the vep in the left eye ( p<0.001 ) , amplitudes of the vep in the right eye ( p<0.001 ) , and amplitudes of the vep in the left eye ( p=0.008 ) . compared with the hcs , patients with on had significantly decreased gm volume ( gmv ) in the brain regions of the left postcentral gyrus , left inferior frontal gyrus , left anterior cingulate , right middle frontal gyrus , left middle frontal gyrus , and right inferior parietal lobule ( figure 1 [ blue ] , table 2 ) . patients with on also had decreased wm volume ( wmv ) in the brain regions of the left middle frontal gyrus , right superior frontal gyrus , left precentral gyrus and right inferior parietal lobule , and increased wmv in the left fusiform gyrus and left inferior parietal lobule ( figure 2 , table 3 ) . in the on group , the vep latency of the right eye showed a positive correlation with the wmv signal value of the left fusiform gyrus ( r=0.726 , p=0.008 ) and a negative correlation with the gmv signal value of the right inferior parietal lobule ( r=0.611 , p=0.035 ) . duration of on showed a negative correlation with the wmv signal value of the right superior frontal gyrus ( r=0.662 , p=0.019 ) . the best - corrected va of the right eye showed a negative correlation with the wmvs signal value of the left middle frontal gyrus ( r=0.704 , p=0.011 ) . to the best of our knowledge , our study is the first to investigate changes in wm and gm in patients with on using a vbm approach . we found markedly decreased gmv in the brain regions of the left postcentral gyrus , left inferior frontal gyrus , left anterior cingulate , right middle frontal gyrus , left middle frontal gyrus , and right inferior parietal lobule in patients with on . in addition , we observed that patients with on had significantly decreased wmv values in the brain regions of the left middle frontal gyrus , right superior frontal gyrus , left precentral gyrus , and right inferior parietal lobule , whereas they had significantly increased wmv values in the cluster of the left fusiform gyrus and left inferior parietal lobule . furthermore , we observed that the vep latency of the right eye in patients with on had a positive correlation with the wmv signal value of the left fusiform gyrus ( r=0.726 , p=0.008 ) and a negative correlation with the gmv signal value of the right inferior parietal lobule ( r=0.611 , p=0.035 ) . we also found that the duration of on had a negative correlation with the wmv signal value of the right superior frontal gyrus ( r=0.662 , p=0.019 ) . the best - corrected va of the right eye had a negative correlation with the wmv signal value of the left middle frontal gyrus ( r=0.704 , p=0.011 ) . compared with the hcs the postcentral gyrus is delimited by the central sulcus anteriorly and the postcentral sulcus posteriorly ; this area is the somatosensory cortex in the human brain . previous studies have identified many diseases that lead to postcentral gyrus dysfunction , such as parkinson disease , alzheimer disease , and multiple sclerosis . , using a voxel - based morphometry method , found that patients with nmo had decreased wmv in the right postcentral gyrus . in support of these findings , we also found that patients with on had significantly decreased gmv in the left postcentral gyrus . moreover , we found that patients with on had decreased gmv in the inferior frontal gyrus and bilateral middle frontal gyrus . in addition , the inferior frontal gyrus is related to emotional and cognitive empathy , while the middle frontal gyrus is associated with the processing of language . found that patients with nmo had significantly increased amplitude of low - frequency fluctuations ( alff ) in the middle frontal gyrus . showed that patients with nmo had decreased regional homogeneity ( reho ) in the left medial frontal gyrus . in support of these findings , we found significant regions of atrophy in the inferior frontal gyrus and bilateral middle frontal gyrus in patients with on . we therefore speculate that on may lead to dysfunction of the frontal gyrus , while deficit in the frontal gyrus may reflect the damage to eye motion and cognition in patients with on . we found that the vep latency of the right eye in patients with on correlated negatively with the gmv signal value of the right inferior parietal lobule ( r=0.611 , p=0.035 ) , which agrees with a previous study demonstrating that patients with on had prolonged latency . the prolonged vep latency in on reflects the severity of on ; therefore , the decreased gmv signal values of the right inferior parietal lobule may be related to the severity of on . compared with the hcs , patients with on had significantly decreased wmv values in the brain regions of the left middle frontal gyrus , right superior frontal gyrus , left precentral gyrus , and right inferior parietal lobule . many brain function areas are involved in the default mode network ( dmn ) , including the posterior and anterior cingulate cortices , inferior parietal cortex , medial temporal lobes , and medial frontal cortex . found that patients with nmo had decreased reho in the left medial frontal gyrus . demonstrated that patients with nmo had decreased wmv in the left middle and medial frontal gyrus , the right superior frontal gyrus , and the bilateral inferior and superior parietal lobules . in support of these findings , we found that patients with on had decreased wmv values in the left middle frontal gyrus and right inferior parietal lobule , which may reflect dysfunction of the dmn in the patients with on . furthermore , the duration of on correlated negatively with the wmv signal value of the right superior frontal gyrus ( r=0.662 , p=0.019 ) , and we speculate that the longer the duration of on , the more serious the injury to the dmn . we also found that the best - corrected va of the right eye correlated negatively with wmv signal value of the left middle frontal gyrus ( r=0.704 , p=0.011 ) . we speculate that the decreased wmv signal value of the left middle frontal gyrus may reflect the damage to va in on . interestingly , we found significantly increased wmv values regions in the left fusiform gyrus and left inferior parietal lobule in patients with on . the fusiform gyrus is the fusiform face area , which participates in face processing and social cognition . the increased wmv areas in the left fusiform gyrus may be related to compensation for the damaged visual function in patients with on . furthermore , we observed that the vep latency of the right eye in on correlated positively with the wmv signal value of the left fusiform gyrus ( r=0.726 , p=0.008 ) . the increased vep latency in on reflects the severity of on , and thus the increased wmv values of the fusiform gyrus may relate to the severity of on . , we found that patients with on had higher wmv values in the left inferior parietal lobule and reduced wmv values in the right inferior parietal lobule . thus , the higher wmv values in the left inferior parietal lobule may reflect functional reorganization to compensate for the damaged areas in patients with on . first , the sample size was too small , which may have affected the accuracy of the results . second , there were some differences in the duration of on , and we were not able to entirely separate sex differences and age differences . in future studies we found that patients with on had abnormal wm and gm involvement in regional brain changes , which correlated with the vep latency of the eye . in future studies we will use other methods to evaluate the changes in brain function in patients with on .

airway hyperresponsiveness ( ahr ) is defined as an excessive response to an aerosolized drug provocation that elicits little or no response in a normal person . this feature , at some point in time , appears to distinguish most patients with asthma , and underlies the rationale for bronchoprovocation testing [ 1 - 3 ] . bronchial challenge tests by inhalation of aerosolized methacholine ( mch ) are recommended by both the american thoracic society ( ats ) and the european respiratory society ( ers ) . differences in the reaction on provocative concentrations or provocative doses of methacholine causing a fall in fev1 by 20% and/or an increase in sraw by 100% derived by the dosimeter method or the tidal breathing method are still in intensive discussion [ 6 - 8 ] . the procedure was compared to the ats standard protocol of five different concentrations modified from the standard procedures of chai et al and ryan et al . detection of airway narrowing in anglo - american countries commonly relies on forced spirometry ( fev1 ) . a fall in fev1 20% following mch inhalation is widely used to determine the degree of ahr . fev1 , however , is largely depending on the subject 's cooperation , his compliance , and motivation . in addition inspiration to tlc and forced expiratory maneuvers widen the airway caliber and thus lower respiratory airflow flow resistance . slas et al showed that airways inflammation plays an essential role in responses to deep inhalations in healthy subjects and patients with bronchial asthma causing bronchodilatation . bronchidilatation by deep inhalation may however be of only minor importance in patients with chronic obstructive pulmonary disease ( copd ) and increased airway resistance . in contrast , for body plethysmographic measurements of specific airway resistance ( sraw ) , or specific conductance ( sgaw ) , which have lately been standardized and recommended by the ' deutsche atemwegsliga ' , only quiet breathing is required with a minimum of cooperation . the major part of airways resistance is localized in the first 8 to 12 bronchial generations and less in peripheral lungs . the peripheral lung , however , is incorporated in sraw or sgaw by their volume related component . an increase of sraw of 200% from baseline value and its absolute value larger than 2.0 kpa s as well as a fall in sgaw 40% from baseline with an absolute value below 0.5 1/(kpa s ) are recommended by both international societies , ats and ers , to determine ahr . ats and ers consider both spirometry ( fev1 ) and body plethysmography ( sreff and sgeff ) for bronchoprovocation testing ; comparative studies are , however , lacking . in the present study , we compared both methods in order to evaluate the concordance of forced spirometry and body plethysmography , concerning their clinical value during mch challenge testing in patients with confirmed bronchial asthma cumulative mch challenges test were performed in 155 patients ( 82 males , 73 females ) with a mean age of 50.4 15.9 years ( range 18 - 84 years ) , 170 10 cm height ( range 154 - 194 cm ) and a mean bmi of 28 6.2 kg / m(range 16.8 - 48.9 ) during a clinical assessment of patients with chronic cough or suspected mild asthma referred to a 205-bed - center for pulmonology and thoracic surgery prospectively . none of the patients had any cardiac or pulmonary disease other than suspected asthma and none were receiving medication for upper or lower airway disease , other than inhaled short acting bronchodilators . inclusion criteria : > 18 yr of age , non - smoker , no respiratory infection or exacerbation of asthma within the preceding 6 weeks ; fev1 70% pred and 1.5 l , no previous treatment with oral or inhaled steroids . subjects were interviewed by a pulmonologist , and asked to refrain from using caffeine - containing beverages , and gave informed written consent to the study . methacholine chloride was dissolved in isotonic saline to a final concentration of 16 mg / ml . this standard mch concentration was used at all provocation steps . the patients were advised to breathe tidal and slow without exceeding a maximal flow of 0.5 l / s . at inhalation after a time of 0.2 s , the nebulizer generated an aerosol bolus for defined periods of time ( 0.29 s , 0.59 s and 0.94 s ) . the mch challenge procedure had the following design : after measuring baseline values and an initial inhalation of aerosolized isotonic saline , maximal 5 provocation steps ( 0.025 , 0.050 , 0.100 , 0.201 , and 0.401 mg ) were performed . the aps provocation system ( cardinal health , hchberg , germany ) uses the effective and reliable nebulizer from medicaid with an aerodynamic mass median diameter of particles of mmad 3.2 m , which is optimal size for effective sedimentation of the aerosol in the lung . the cumulative inhaled doses after each step of inhalation ( 0.025 , 0.075 , 0.175 , 0.376 , and 0.777 mg mch ) were obtained by taking two breaths with mch boluses at step one and two , 4 breaths at step 3 , 8 breaths at step 4 , and 10 breaths at step 5 . classification of ahr was performed in four steps : severe ( mch dose < 0.05 mg ) , moderate ( 0.05 mg < mch dose < 0.20 mg ) mild ( 0.20 < mch dose < 0.65 mg ) , and normal responsive ( mch dose > 0.65 mg ) . specific airway resistance ( sraw ) , specific conductance ( sgaw ) , airway resistance ( raw ) , conductance ( gaw ) and intrathoraxic gas volume ( itgv ) were recorded by body plethysmography ( masterscreen body , cardinal health , hchberg , germany ) . in the combined measurements , the forced spirometric maneuver , the door of the plethysmograph was opened for this test . according to the recommendations of the manufactures classification of body plethysmography based on the current recommendations of criee at al . at rest , after saline inhalation and after each step of mch inhalation body plethysmography was measured 2 minutes after mch inhalation , with determination of reff , sreff , rtot , srtot , sgeff and sgtot , and itgv . an increase of sraw of 200% from 100% baseline value and its absolute value larger than 2.0 kpa s or a fall in sgaw 40% and its absolute value lower than 0.5 1/(kpa s ) are recommended as thresholds to determine airways hyperresponsiveness . at baseline and after crossing the pd threshold in body plethysmography ( pd+100 raw , sraw , ) forced spirometric maneuvers were performed and fev1 , fvc , and fev1 % fvc were determined and the pd-20 fev1 extrapolated . because the challenge needed to be stopped as soon as patients showed significant symptoms based on airway narrowing and assessed via pd+100 sraw , most spirometric datasets were left incomplete with some parameters not reaching the diagnostic threshold pd-20 fev1 . utilizing the regression analysis of the recorded airway resistance parameters taking the metacholine dose as the covariate , we could extrapolate to missing spirometric values and interpolate the estimated metacholine dose when crossing the threshold between two consecutive measurements . regression was done in a simple 3-way process of transforming data into linear space , doing linear regression and back transformation , where linear regression was performed by built - in statistical functions of an oracle10 g xe database . quality checking was done by calculating the mean goodness - of - fit for each parameter which yielded the following results : using regression analysis of the recorded airway parameters taking the mch dose as the covariate we could extrapolate to missing values and interpolate the estimated mch dose when crossing the threshold of -20% for fev1 , between two consecutive measurements . the regression analysis resulted in high values for the ' goodness of fit ' ( 0.83 > r < 0.95 ) . the required pd+100 mch doses for airway , specific airway resistance ( rtot , reff , srtot , and sreff ) , and specific conductance ( sgaw ) were compared to pd-20 of fev1 . cumulative mch challenges test were performed in 155 patients ( 82 males , 73 females ) with a mean age of 50.4 15.9 years ( range 18 - 84 years ) , 170 10 cm height ( range 154 - 194 cm ) and a mean bmi of 28 6.2 kg / m(range 16.8 - 48.9 ) during a clinical assessment of patients with chronic cough or suspected mild asthma referred to a 205-bed - center for pulmonology and thoracic surgery prospectively . none of the patients had any cardiac or pulmonary disease other than suspected asthma and none were receiving medication for upper or lower airway disease , other than inhaled short acting bronchodilators . inclusion criteria : > 18 yr of age , non - smoker , no respiratory infection or exacerbation of asthma within the preceding 6 weeks ; fev1 70% pred and 1.5 l , no previous treatment with oral or inhaled steroids . subjects were interviewed by a pulmonologist , and asked to refrain from using caffeine - containing beverages , and gave informed written consent to the study . methacholine chloride was dissolved in isotonic saline to a final concentration of 16 mg / ml . this standard mch concentration was used at all provocation steps . the patients were advised to breathe tidal and slow without exceeding a maximal flow of 0.5 l / s . at inhalation after a time of 0.2 s , the nebulizer generated an aerosol bolus for defined periods of time ( 0.29 s , 0.59 s and 0.94 s ) . the mch challenge procedure had the following design : after measuring baseline values and an initial inhalation of aerosolized isotonic saline , maximal 5 provocation steps ( 0.025 , 0.050 , 0.100 , 0.201 , and 0.401 mg ) were performed . the aps provocation system ( cardinal health , hchberg , germany ) uses the effective and reliable nebulizer from medicaid with an aerodynamic mass median diameter of particles of mmad 3.2 m , which is optimal size for effective sedimentation of the aerosol in the lung . the cumulative inhaled doses after each step of inhalation ( 0.025 , 0.075 , 0.175 , 0.376 , and 0.777 mg mch ) were obtained by taking two breaths with mch boluses at step one and two , 4 breaths at step 3 , 8 breaths at step 4 , and 10 breaths at step 5 . classification of ahr was performed in four steps : severe ( mch dose < 0.05 mg ) , moderate ( 0.05 mg < mch dose < 0.20 mg ) mild ( 0.20 < mch dose < 0.65 mg ) , and normal responsive ( mch dose > 0.65 mg ) . specific airway resistance ( sraw ) , specific conductance ( sgaw ) , airway resistance ( raw ) , conductance ( gaw ) and intrathoraxic gas volume ( itgv ) were recorded by body plethysmography ( masterscreen body , cardinal health , hchberg , germany ) . in the combined measurements , the forced spirometric maneuver , the door of the plethysmograph was opened for this test . according to the recommendations of the manufactures classification of body plethysmography based on the current recommendations of criee at al . at rest , after saline inhalation and after each step of mch inhalation body plethysmography was measured 2 minutes after mch inhalation , with determination of reff , sreff , rtot , srtot , sgeff and sgtot , and itgv . an increase of sraw of 200% from 100% baseline value and its absolute value larger than 2.0 kpa s or a fall in sgaw 40% and its absolute value lower than 0.5 1/(kpa s ) are recommended as thresholds to determine airways hyperresponsiveness . at baseline and after crossing the pd threshold in body plethysmography ( pd+100 raw , sraw , ) forced spirometric maneuvers were performed and fev1 , fvc , and fev1 % fvc were determined and the pd-20 fev1 extrapolated . because the challenge needed to be stopped as soon as patients showed significant symptoms based on airway narrowing and assessed via pd+100 sraw , most spirometric datasets were left incomplete with some parameters not reaching the diagnostic threshold pd-20 fev1 . utilizing the regression analysis of the recorded airway resistance parameters taking the metacholine dose as the covariate , we could extrapolate to missing spirometric values and interpolate the estimated metacholine dose when crossing the threshold between two consecutive measurements . regression was done in a simple 3-way process of transforming data into linear space , doing linear regression and back transformation , where linear regression was performed by built - in statistical functions of an oracle10 g xe database . quality checking was done by calculating the mean goodness - of - fit for each parameter which yielded the following results : using regression analysis of the recorded airway parameters taking the mch dose as the covariate we could extrapolate to missing values and interpolate the estimated mch dose when crossing the threshold of -20% for fev1 , between two consecutive measurements . the regression analysis resulted in high values for the ' goodness of fit ' ( 0.83 > r < 0.95 ) . the required pd+100 mch doses for airway , specific airway resistance ( rtot , reff , srtot , and sreff ) , and specific conductance ( sgaw ) were compared to pd-20 of fev1 . under baseline conditions , the mean values of respiratory parameters of males with mild asthma were in the normal range for sreff ( mean 0.77 0.036 kpa s , range 0.26 - 1.6 ) , fev1 ( 3.51 0.90 l , range 0.95 - 5.55 , or 95% according eccs reference values ) and sgeff ( mean 1.53 0.63 1/kpa s ) ( table 1 ) . the mean baseline values for females corresponded to eccs reference values for fev1 ( 2.45 0.60 l , range 1.23 - 4.16 l , or 95.5% predicted ) and to plethysmographically measured sreff ( 0.835 0.306 kpa s , range 0.12 - 1.65 ) and sgeff ( mean 1.37 0.63 1/kpa s ) . baseline respiratory parameters of patients with mild asthma included in the study . according to pd+100 sreff , 25 patients suffered from severe ahr , in 47 a moderate ahr was found , in 41 patients a mild airway ahr , and in another 41 patients normal responses with an increase in sreff < 2.0 kpa / l s or < 100% . so a total of 113 patients ( 75% ) showed positive mch challenges based on sreff ( figure 1 ) . number of patients with mch responses , classified as severe , moderate , mild , and normal , based on pd+100 for sreff , srtot , reff , rtot , pd-40 for sgeff and sgrtot and pd-20 for fev1 . regarding pd-40 sgeff with a decrease of 40% as the threshold value , 28 patients had severe , 58 patients moderate , and 45 mild ahr , whereas only 19 patients had normal responses , so 88% of the patients had positive test results by sgeff . taking a fall in fev1 > 20% of baseline value ( pd-20 fev1 ) as a criterion , only 6 patients had severe ahr , 23 had moderate responses , 21 had mild responses , and the majority of 104 patients had no response . so , 50 from the 154 patients ( 32% ) had a positive test result with fev1 ( figure 2 ) . accuracy of responses classified as severe , moderate , mild and normal , based on pd+100 for sreff , srtot , reff , rtot , pd-40 for sgeff and sgrtot , and pd-20 for fev1 . the mean mch - dose for diagnosis of severe ahr was 0.026 0.012 mg mch . moderate ahr was diagnosed after step 3 or 4 , at a mean cumulative dose of 0.122 0.046 mg mch . statistical analysis revealed strongly linearly correlated parameters of airway resistance , but no significant relationship between the test results of body plethysmography and forced spirometry . pd+100 sreff and pd+100 srtot significantly correlated in patients with severe , moderate and mild degree of ahr ( figure 3 ) . for pd-20 fev1 and pd+100 correlation of pd+100 srtot and pd+100 sreff in cumulative mch challenge testing inpatients with mild asthma . correlation of pd+100 sreff and of pd-20 fev1 in cumulative mch challenge tests in patients with mild asthma . coinciding positive or negative results in reff and sreff were found in 95% of the mch tests , sreff and srtot matched in 88% of the patients , 85% matched in sreff and sgeff , but only 47% coinciding responses were obtained in sreff and fev1 . under baseline conditions , the mean values of respiratory parameters of males with mild asthma were in the normal range for sreff ( mean 0.77 0.036 kpa s , range 0.26 - 1.6 ) , fev1 ( 3.51 0.90 l , range 0.95 - 5.55 , or 95% according eccs reference values ) and sgeff ( mean 1.53 0.63 1/kpa s ) ( table 1 ) . the mean baseline values for females corresponded to eccs reference values for fev1 ( 2.45 0.60 l , range 1.23 - 4.16 l , or 95.5% predicted ) and to plethysmographically measured sreff ( 0.835 0.306 kpa s , range 0.12 - 1.65 ) and sgeff ( mean 1.37 0.63 1/kpa s ) . according to pd+100 sreff , 25 patients suffered from severe ahr , in 47 a moderate ahr was found , in 41 patients a mild airway ahr , and in another 41 patients normal responses with an increase in sreff < 2.0 so a total of 113 patients ( 75% ) showed positive mch challenges based on sreff ( figure 1 ) . number of patients with mch responses , classified as severe , moderate , mild , and normal , based on pd+100 for sreff , srtot , reff , rtot , pd-40 for sgeff and sgrtot and pd-20 for fev1 . regarding pd-40 sgeff with a decrease of 40% as the threshold value , 28 patients had severe , 58 patients moderate , and 45 mild ahr , whereas only 19 patients had normal responses , so 88% of the patients had positive test results by sgeff . taking a fall in fev1 > 20% of baseline value ( pd-20 fev1 ) as a criterion , only 6 patients had severe ahr , 23 had moderate responses , 21 had mild responses , and the majority of 104 patients had no response . so , 50 from the 154 patients ( 32% ) had a positive test result with fev1 ( figure 2 ) . accuracy of responses classified as severe , moderate , mild and normal , based on pd+100 for sreff , srtot , reff , rtot , pd-40 for sgeff and sgrtot , and pd-20 for fev1 . the mean mch - dose for diagnosis of severe ahr was 0.026 0.012 mg mch . moderate ahr was diagnosed after step 3 or 4 , at a mean cumulative dose of 0.122 0.046 mg mch . statistical analysis revealed strongly linearly correlated parameters of airway resistance , but no significant relationship between the test results of body plethysmography and forced spirometry . pd+100 sreff and pd+100 srtot significantly correlated in patients with severe , moderate and mild degree of ahr ( figure 3 ) . for pd-20 fev1 and pd+100 correlation of pd+100 srtot and pd+100 sreff in cumulative mch challenge testing inpatients with mild asthma . correlation of pd+100 sreff and of pd-20 fev1 in cumulative mch challenge tests in patients with mild asthma . coinciding positive or negative results in reff and sreff were found in 95% of the mch tests , sreff and srtot matched in 88% of the patients , 85% matched in sreff and sgeff , but only 47% coinciding responses were obtained in sreff and fev1 . the concordance of forced spirometry and tidal breathing recording , studied by body plethysmography , was investigated by bronchial challenge tests in patients with chronic cough or suspected mild asthma . in cumulative mch challenges , we found specific airway resistance ( sreff ) to be the most useful parameter in the detection of ahr . the measurement of specific airway resistance ( sraw ) by body plethysmography is largely independent of patient 's cooperation , facilitating the interpretation of measurements . furthermore , higher mch doses were necessary before fev1 , as a key parameter of forced spirometry , showed a significant decrease above 20% . although most practitioners only measure fev1 , reliance upon this measurement alone can lead to both false negative and false positive test results . goldstein et al reported that sensitivity of a mch challenge determined by fev1 alone was only 60% , but increased to 97% after the assessments of fvc , sgaw , and thoracic gas volume ( tgv ) were added to the analysis . this result is in accordance with the known axial heterogeneity of the response of airways of different caliber to bronchoactive agents . furthermore , if patients not fully inhale to total lung capacity ( tlc ) , a fall in fev1 can occur and cause a false positive result . generally , the sensitivity of a positive mch challenge test for the diagnosis of asthma is 85% . a positive predictive value is more limited , as false positive results may be seen in patients with allergic rhinitis , cystic fibrosis , congestive heart failure , copd , and bronchitis . . however , patients with allergic rhinitis , cystic fibrosis , heart failure , or copd were not included in this study . on the other hand , avoidance of maximal maneuvers enhances reactions of bronchoconstrictor agents . taking deep breaths before bronchial challenge tests diminshes obstructive responses as a consequence . therefore , deep inspirations during forced expiratory maneuvers provide an effective protection against airways obstruction . however , the beneficial effects of deep inspirations are impaired in asthma and during acute exacerbations of asthma . in these clinical settings in chronic obstructive pulmonary disease ( copd ) , bronchodilatory effects of deep inspirations are effectively reduced , which may depend on the degree of parenchymal damage . airway inflammation is a typical feature of asthma and copd , although the composition of inflammatory cells is different . inflammatory processes induce airway remodelling and thus increasing thickness of airway walls and reducing the elasticity of the airways . the result would be a reduced strain transmission from lung parenchyma to the airway walls and diminished effects of stretching the airways . slats et al demonstrated that the bronchodilatory effects of deep inspirations in asthma are related to inflammatory cell counts in bronchial walls and smooth muscle layers , whereas in moderate copd this relationship could not be found . they conclude that the physiologic protection against narrowing of the airways by deep inspirations is impaired in asthma and copd , but depends on different mechanisms . using body plethysmography for the assessment of airway resistance responses , measurements are performed during quiet breathing and are not masked by the bronchodilatory responses of deep inhalations or positive intraluminal pressure in the airways . there was a close correlation between reff and rtot , the two parameters for airway resistance , which are calculated from different approaches , and the pd+100 of both parameters . in addition , specific airway resistance sreff and srtot , including lung volume and changes in lung volume during airway obstruction , closely correlate . since raw and intrathoracic gas volume ( itgv ) both increase during acute airway obstruction , the largest response rates can be recorded in sreff and srtot . a close correlation was also found between specific airway resistance and specific conductance , sgaw , the reciprocal value of specific resistance . in further investigations ahr is an important defining feature of asthma and is a manifestation of reversible airflow obstruction due to smooth muscle contraction . ahr represents an exaggerated constrictor response to a variety of physical , chemical , or environmental stimuli . ahr can be quantified by the dose response to pharmacologic agents such as methacholine or histamine , causing a 20% decrease in fev1 from baseline . while ahr is not specific for asthma , patients with asthma typically demonstrate ahr to much lower doses ( e.g. , 10- to 100-fold ) of these agents than normal or allergic individuals . the single , most typical abnormality in many patients with asthma is ahr . as a result , the assessment of changes in ahr using bronchoprovocation testing may be preferable to the reliance upon subjective changes in symptoms alone . this is particularly relevant when asthma control requires a complex , costly , and potentially toxic therapeutic regimen . indications for bronchoprovocation testing include not only the accurate diagnosis of asthma , but also the assessment of the response to asthma therapy , and , less commonly , the identification of triggers for cases involving environmental or occupational asthma . the measurement of ahr by bronchoprovocation testing is potentially useful for several reasons : failure to show ahr argues against the diagnosis of asthma , ahr may be the sole objective evidence of airway dysfunction , ahr is quantitatively associated with the presence and severity of disease , the occurrence of ahr in an asymptomatic person may help predict the future development of asthma , the degree of ahr in a symptomatic person can have prognostic and potentially therapeutic implications , the periodicity of asthma exists in parallel with changes in the degree of ahr . a false negative fev1 in patients with asthma may have serious consequences for the patient due to undertreatment . thus , recording responses of airway resistance by measurements performed during quiet breathing , which are not masked by the bronchodilatory responses of deep inspirations may increase the diagnostic impact of bronchoprovocation . on the other hand , avoidance of maximal maneuvers enhances reactions of bronchoconstrictor agents . taking deep breaths before bronchial challenge tests diminshes obstructive responses as a consequence . therefore , deep inspirations during forced expiratory maneuvers provide an effective protection against airways obstruction . however , the beneficial effects of deep inspirations are impaired in asthma and during acute exacerbations of asthma . in these clinical settings in chronic obstructive pulmonary disease ( copd ) , bronchodilatory effects of deep inspirations are effectively reduced , which may depend on the degree of parenchymal damage . airway inflammation is a typical feature of asthma and copd , although the composition of inflammatory cells is different . inflammatory processes induce airway remodelling and thus increasing thickness of airway walls and reducing the elasticity of the airways . the result would be a reduced strain transmission from lung parenchyma to the airway walls and diminished effects of stretching the airways . slats et al demonstrated that the bronchodilatory effects of deep inspirations in asthma are related to inflammatory cell counts in bronchial walls and smooth muscle layers , whereas in moderate copd this relationship could not be found . they conclude that the physiologic protection against narrowing of the airways by deep inspirations is impaired in asthma and copd , but depends on different mechanisms . using body plethysmography for the assessment of airway resistance responses , measurements are performed during quiet breathing and are not masked by the bronchodilatory responses of deep inhalations or positive intraluminal pressure in the airways . there was a close correlation between reff and rtot , the two parameters for airway resistance , which are calculated from different approaches , and the pd+100 of both parameters . in addition , specific airway resistance sreff and srtot , including lung volume and changes in lung volume during airway obstruction , closely correlate . since raw and intrathoracic gas volume ( itgv ) both increase during acute airway obstruction , the largest response rates can be recorded in sreff and srtot . a close correlation was also found between specific airway resistance and specific conductance , sgaw , the reciprocal value of specific resistance . in further investigations , pd sgaw should be adjusted to pd+100 sreff . ahr is an important defining feature of asthma and is a manifestation of reversible airflow obstruction due to smooth muscle contraction . ahr represents an exaggerated constrictor response to a variety of physical , chemical , or environmental stimuli . ahr can be quantified by the dose response to pharmacologic agents such as methacholine or histamine , causing a 20% decrease in fev1 from baseline . while ahr is not specific for asthma , patients with asthma typically demonstrate ahr to much lower doses ( e.g. , 10- to 100-fold ) of these agents than normal or allergic individuals . the single , most typical abnormality in many patients with asthma is ahr . as a result , the assessment of changes in ahr using bronchoprovocation testing may be preferable to the reliance upon subjective changes in symptoms alone . this is particularly relevant when asthma control requires a complex , costly , and potentially toxic therapeutic regimen . indications for bronchoprovocation testing include not only the accurate diagnosis of asthma , but also the assessment of the response to asthma therapy , and , less commonly , the identification of triggers for cases involving environmental or occupational asthma . the measurement of ahr by bronchoprovocation testing is potentially useful for several reasons : failure to show ahr argues against the diagnosis of asthma , ahr may be the sole objective evidence of airway dysfunction , ahr is quantitatively associated with the presence and severity of disease , the occurrence of ahr in an asymptomatic person may help predict the future development of asthma , the degree of ahr in a symptomatic person can have prognostic and potentially therapeutic implications , the periodicity of asthma exists in parallel with changes in the degree of ahr . a false negative fev1 in patients with asthma may have serious consequences for the patient due to undertreatment . thus , recording responses of airway resistance by measurements performed during quiet breathing , which are not masked by the bronchodilatory responses of deep inspirations may increase the diagnostic impact of bronchoprovocation . the authors compared forced spirometry and body plethysmography in order to evaluate the concordance of the forced respiratory maneuvers and measurements of airway resistance during normal breathing in mch challenges . pd+100 raw and pd-40 gaw for the following plethysmographically measured parameters rtot , reff , srtot , sreff , sgtot , and sgeff , are closely correlated with cumulative mch - challenge tests . the reason for the different responses might be found in the location of the plethymographically measured airway resistance and the flow reduction measured in forced spirometric maneuvers . from our results , we would recommend sreff and sgaw as the reliable parameters for classification of ahr . additional investigations on healthy subjects and patients with asthma and copd should be performed to compare the sensitivity and specificity of body plethysmography and forced spirometry for mch - challenge tests .

introductionbronchial challenge tests by inhalation of aerosolized methacholine ( mch ) are commonly used in the clinical diagnosis of airway hyperresponsiveness ( ahr ) . while the detection of airway narrowing relies on the patient 's cooperation performing forced spirometry , body plethysmographic measurements of airway resistance are less depending on the patient 's cooperation and do not alter the respiratory tract by maximal maneuvers . hence we compared both methods concerning their clinical value and correlation during mch challenges in patients with asthma.materials and methodscumulative mch challenges test , consisting of up to 5 steps , evaluated with body plethysmography on each step were performed in 155 patients with bronchial asthma . airway responses were recorded at each step of mch application ( master - screen body , cardinal health , hchberg ) . at the baseline test and after crossing the provocation dose ( pd ) threshold in body plethysmography ( pd+100 sreff ) , forced expirations were performed and fev1 , fvc , and fev1 % fvc were measured . using regression analysis of the airway parameters and taking the mch dose as the covariate , we could extrapolate to missing spirometric values and interpolate the estimated mch dose when crossing the pd threshold ( pd-20 fev1 ) between two consecutive measurements . the administered pd+100 mch doses for specific airway resistance , srtot , and sreff were compared with resistance parameters rtot and reff , and to pd-20 of fev1 and fev1 % fvc.resultsregarding sreff we found a mild , moderate , or severe ahr in 114 patients ( 75% ) , but only 50 ( 32% ) according to fev1 . a statistical analysis showed strongly linear correlated parameters of airway resistance , but no significant correlation between the results of body plethysmography and forced spirometryconclusionsusing mch challenges , we found specific airway resistance to be the most sensitive parameter to detect ahr . raw is largely independent of height and gender facilitating the interpretation of measurements carried out longitudinally .

Introduction Materials and Methods Study Population Methacholine Challenges Lung Function Measurements Data Analysis Results Lung Function Parameters of Patients Included in the Study Classification of Airway Hyperresponsiveness Discussion Impact of Deep Inspirations during Forced Expiratory Maneuvers on MCH-Testing Changes in Airway Resistance During Quiet Breathing Clinical Relevance of Non-Specific Challenge Testing Summary and Conclusion Conflicts of interest

airway hyperresponsiveness ( ahr ) is defined as an excessive response to an aerosolized drug provocation that elicits little or no response in a normal person . this feature , at some point in time , appears to distinguish most patients with asthma , and underlies the rationale for bronchoprovocation testing [ 1 - 3 ] . bronchial challenge tests by inhalation of aerosolized methacholine ( mch ) are recommended by both the american thoracic society ( ats ) and the european respiratory society ( ers ) . detection of airway narrowing in anglo - american countries commonly relies on forced spirometry ( fev1 ) . fev1 , however , is largely depending on the subject 's cooperation , his compliance , and motivation . slas et al showed that airways inflammation plays an essential role in responses to deep inhalations in healthy subjects and patients with bronchial asthma causing bronchodilatation . in contrast , for body plethysmographic measurements of specific airway resistance ( sraw ) , or specific conductance ( sgaw ) , which have lately been standardized and recommended by the ' deutsche atemwegsliga ' , only quiet breathing is required with a minimum of cooperation . ats and ers consider both spirometry ( fev1 ) and body plethysmography ( sreff and sgeff ) for bronchoprovocation testing ; comparative studies are , however , lacking . in the present study , we compared both methods in order to evaluate the concordance of forced spirometry and body plethysmography , concerning their clinical value during mch challenge testing in patients with confirmed bronchial asthma cumulative mch challenges test were performed in 155 patients ( 82 males , 73 females ) with a mean age of 50.4 15.9 years ( range 18 - 84 years ) , 170 10 cm height ( range 154 - 194 cm ) and a mean bmi of 28 6.2 kg / m(range 16.8 - 48.9 ) during a clinical assessment of patients with chronic cough or suspected mild asthma referred to a 205-bed - center for pulmonology and thoracic surgery prospectively . the mch challenge procedure had the following design : after measuring baseline values and an initial inhalation of aerosolized isotonic saline , maximal 5 provocation steps ( 0.025 , 0.050 , 0.100 , 0.201 , and 0.401 mg ) were performed . the aps provocation system ( cardinal health , hchberg , germany ) uses the effective and reliable nebulizer from medicaid with an aerodynamic mass median diameter of particles of mmad 3.2 m , which is optimal size for effective sedimentation of the aerosol in the lung . the cumulative inhaled doses after each step of inhalation ( 0.025 , 0.075 , 0.175 , 0.376 , and 0.777 mg mch ) were obtained by taking two breaths with mch boluses at step one and two , 4 breaths at step 3 , 8 breaths at step 4 , and 10 breaths at step 5 . classification of ahr was performed in four steps : severe ( mch dose < 0.05 mg ) , moderate ( 0.05 mg < mch dose < 0.20 mg ) mild ( 0.20 < mch dose < 0.65 mg ) , and normal responsive ( mch dose > 0.65 mg ) . specific airway resistance ( sraw ) , specific conductance ( sgaw ) , airway resistance ( raw ) , conductance ( gaw ) and intrathoraxic gas volume ( itgv ) were recorded by body plethysmography ( masterscreen body , cardinal health , hchberg , germany ) . according to the recommendations of the manufactures classification of body plethysmography based on the current recommendations of criee at al . at rest , after saline inhalation and after each step of mch inhalation body plethysmography was measured 2 minutes after mch inhalation , with determination of reff , sreff , rtot , srtot , sgeff and sgtot , and itgv . at baseline and after crossing the pd threshold in body plethysmography ( pd+100 raw , sraw , ) forced spirometric maneuvers were performed and fev1 , fvc , and fev1 % fvc were determined and the pd-20 fev1 extrapolated . utilizing the regression analysis of the recorded airway resistance parameters taking the metacholine dose as the covariate , we could extrapolate to missing spirometric values and interpolate the estimated metacholine dose when crossing the threshold between two consecutive measurements . quality checking was done by calculating the mean goodness - of - fit for each parameter which yielded the following results : using regression analysis of the recorded airway parameters taking the mch dose as the covariate we could extrapolate to missing values and interpolate the estimated mch dose when crossing the threshold of -20% for fev1 , between two consecutive measurements . the required pd+100 mch doses for airway , specific airway resistance ( rtot , reff , srtot , and sreff ) , and specific conductance ( sgaw ) were compared to pd-20 of fev1 . cumulative mch challenges test were performed in 155 patients ( 82 males , 73 females ) with a mean age of 50.4 15.9 years ( range 18 - 84 years ) , 170 10 cm height ( range 154 - 194 cm ) and a mean bmi of 28 6.2 kg / m(range 16.8 - 48.9 ) during a clinical assessment of patients with chronic cough or suspected mild asthma referred to a 205-bed - center for pulmonology and thoracic surgery prospectively . the mch challenge procedure had the following design : after measuring baseline values and an initial inhalation of aerosolized isotonic saline , maximal 5 provocation steps ( 0.025 , 0.050 , 0.100 , 0.201 , and 0.401 mg ) were performed . the aps provocation system ( cardinal health , hchberg , germany ) uses the effective and reliable nebulizer from medicaid with an aerodynamic mass median diameter of particles of mmad 3.2 m , which is optimal size for effective sedimentation of the aerosol in the lung . the cumulative inhaled doses after each step of inhalation ( 0.025 , 0.075 , 0.175 , 0.376 , and 0.777 mg mch ) were obtained by taking two breaths with mch boluses at step one and two , 4 breaths at step 3 , 8 breaths at step 4 , and 10 breaths at step 5 . classification of ahr was performed in four steps : severe ( mch dose < 0.05 mg ) , moderate ( 0.05 mg < mch dose < 0.20 mg ) mild ( 0.20 < mch dose < 0.65 mg ) , and normal responsive ( mch dose > 0.65 mg ) . specific airway resistance ( sraw ) , specific conductance ( sgaw ) , airway resistance ( raw ) , conductance ( gaw ) and intrathoraxic gas volume ( itgv ) were recorded by body plethysmography ( masterscreen body , cardinal health , hchberg , germany ) . according to the recommendations of the manufactures classification of body plethysmography based on the current recommendations of criee at al . at rest , after saline inhalation and after each step of mch inhalation body plethysmography was measured 2 minutes after mch inhalation , with determination of reff , sreff , rtot , srtot , sgeff and sgtot , and itgv . at baseline and after crossing the pd threshold in body plethysmography ( pd+100 raw , sraw , ) forced spirometric maneuvers were performed and fev1 , fvc , and fev1 % fvc were determined and the pd-20 fev1 extrapolated . utilizing the regression analysis of the recorded airway resistance parameters taking the metacholine dose as the covariate , we could extrapolate to missing spirometric values and interpolate the estimated metacholine dose when crossing the threshold between two consecutive measurements . quality checking was done by calculating the mean goodness - of - fit for each parameter which yielded the following results : using regression analysis of the recorded airway parameters taking the mch dose as the covariate we could extrapolate to missing values and interpolate the estimated mch dose when crossing the threshold of -20% for fev1 , between two consecutive measurements . the required pd+100 mch doses for airway , specific airway resistance ( rtot , reff , srtot , and sreff ) , and specific conductance ( sgaw ) were compared to pd-20 of fev1 . under baseline conditions , the mean values of respiratory parameters of males with mild asthma were in the normal range for sreff ( mean 0.77 0.036 kpa s , range 0.26 - 1.6 ) , fev1 ( 3.51 0.90 l , range 0.95 - 5.55 , or 95% according eccs reference values ) and sgeff ( mean 1.53 0.63 1/kpa s ) ( table 1 ) . baseline respiratory parameters of patients with mild asthma included in the study . according to pd+100 sreff , 25 patients suffered from severe ahr , in 47 a moderate ahr was found , in 41 patients a mild airway ahr , and in another 41 patients normal responses with an increase in sreff < 2.0 kpa / l s or < 100% . so a total of 113 patients ( 75% ) showed positive mch challenges based on sreff ( figure 1 ) . number of patients with mch responses , classified as severe , moderate , mild , and normal , based on pd+100 for sreff , srtot , reff , rtot , pd-40 for sgeff and sgrtot and pd-20 for fev1 . regarding pd-40 sgeff with a decrease of 40% as the threshold value , 28 patients had severe , 58 patients moderate , and 45 mild ahr , whereas only 19 patients had normal responses , so 88% of the patients had positive test results by sgeff . taking a fall in fev1 > 20% of baseline value ( pd-20 fev1 ) as a criterion , only 6 patients had severe ahr , 23 had moderate responses , 21 had mild responses , and the majority of 104 patients had no response . so , 50 from the 154 patients ( 32% ) had a positive test result with fev1 ( figure 2 ) . accuracy of responses classified as severe , moderate , mild and normal , based on pd+100 for sreff , srtot , reff , rtot , pd-40 for sgeff and sgrtot , and pd-20 for fev1 . statistical analysis revealed strongly linearly correlated parameters of airway resistance , but no significant relationship between the test results of body plethysmography and forced spirometry . pd+100 sreff and pd+100 srtot significantly correlated in patients with severe , moderate and mild degree of ahr ( figure 3 ) . for pd-20 fev1 and pd+100 correlation of pd+100 srtot and pd+100 sreff in cumulative mch challenge testing inpatients with mild asthma . correlation of pd+100 sreff and of pd-20 fev1 in cumulative mch challenge tests in patients with mild asthma . coinciding positive or negative results in reff and sreff were found in 95% of the mch tests , sreff and srtot matched in 88% of the patients , 85% matched in sreff and sgeff , but only 47% coinciding responses were obtained in sreff and fev1 . under baseline conditions , the mean values of respiratory parameters of males with mild asthma were in the normal range for sreff ( mean 0.77 0.036 kpa s , range 0.26 - 1.6 ) , fev1 ( 3.51 0.90 l , range 0.95 - 5.55 , or 95% according eccs reference values ) and sgeff ( mean 1.53 0.63 1/kpa s ) ( table 1 ) . according to pd+100 sreff , 25 patients suffered from severe ahr , in 47 a moderate ahr was found , in 41 patients a mild airway ahr , and in another 41 patients normal responses with an increase in sreff < 2.0 so a total of 113 patients ( 75% ) showed positive mch challenges based on sreff ( figure 1 ) . number of patients with mch responses , classified as severe , moderate , mild , and normal , based on pd+100 for sreff , srtot , reff , rtot , pd-40 for sgeff and sgrtot and pd-20 for fev1 . regarding pd-40 sgeff with a decrease of 40% as the threshold value , 28 patients had severe , 58 patients moderate , and 45 mild ahr , whereas only 19 patients had normal responses , so 88% of the patients had positive test results by sgeff . taking a fall in fev1 > 20% of baseline value ( pd-20 fev1 ) as a criterion , only 6 patients had severe ahr , 23 had moderate responses , 21 had mild responses , and the majority of 104 patients had no response . so , 50 from the 154 patients ( 32% ) had a positive test result with fev1 ( figure 2 ) . accuracy of responses classified as severe , moderate , mild and normal , based on pd+100 for sreff , srtot , reff , rtot , pd-40 for sgeff and sgrtot , and pd-20 for fev1 . statistical analysis revealed strongly linearly correlated parameters of airway resistance , but no significant relationship between the test results of body plethysmography and forced spirometry . pd+100 sreff and pd+100 srtot significantly correlated in patients with severe , moderate and mild degree of ahr ( figure 3 ) . correlation of pd+100 sreff and of pd-20 fev1 in cumulative mch challenge tests in patients with mild asthma . coinciding positive or negative results in reff and sreff were found in 95% of the mch tests , sreff and srtot matched in 88% of the patients , 85% matched in sreff and sgeff , but only 47% coinciding responses were obtained in sreff and fev1 . the concordance of forced spirometry and tidal breathing recording , studied by body plethysmography , was investigated by bronchial challenge tests in patients with chronic cough or suspected mild asthma . in cumulative mch challenges , we found specific airway resistance ( sreff ) to be the most useful parameter in the detection of ahr . the measurement of specific airway resistance ( sraw ) by body plethysmography is largely independent of patient 's cooperation , facilitating the interpretation of measurements . furthermore , higher mch doses were necessary before fev1 , as a key parameter of forced spirometry , showed a significant decrease above 20% . goldstein et al reported that sensitivity of a mch challenge determined by fev1 alone was only 60% , but increased to 97% after the assessments of fvc , sgaw , and thoracic gas volume ( tgv ) were added to the analysis . using body plethysmography for the assessment of airway resistance responses , measurements are performed during quiet breathing and are not masked by the bronchodilatory responses of deep inhalations or positive intraluminal pressure in the airways . there was a close correlation between reff and rtot , the two parameters for airway resistance , which are calculated from different approaches , and the pd+100 of both parameters . in addition , specific airway resistance sreff and srtot , including lung volume and changes in lung volume during airway obstruction , closely correlate . indications for bronchoprovocation testing include not only the accurate diagnosis of asthma , but also the assessment of the response to asthma therapy , and , less commonly , the identification of triggers for cases involving environmental or occupational asthma . the measurement of ahr by bronchoprovocation testing is potentially useful for several reasons : failure to show ahr argues against the diagnosis of asthma , ahr may be the sole objective evidence of airway dysfunction , ahr is quantitatively associated with the presence and severity of disease , the occurrence of ahr in an asymptomatic person may help predict the future development of asthma , the degree of ahr in a symptomatic person can have prognostic and potentially therapeutic implications , the periodicity of asthma exists in parallel with changes in the degree of ahr . a false negative fev1 in patients with asthma may have serious consequences for the patient due to undertreatment . using body plethysmography for the assessment of airway resistance responses , measurements are performed during quiet breathing and are not masked by the bronchodilatory responses of deep inhalations or positive intraluminal pressure in the airways . there was a close correlation between reff and rtot , the two parameters for airway resistance , which are calculated from different approaches , and the pd+100 of both parameters . indications for bronchoprovocation testing include not only the accurate diagnosis of asthma , but also the assessment of the response to asthma therapy , and , less commonly , the identification of triggers for cases involving environmental or occupational asthma . the measurement of ahr by bronchoprovocation testing is potentially useful for several reasons : failure to show ahr argues against the diagnosis of asthma , ahr may be the sole objective evidence of airway dysfunction , ahr is quantitatively associated with the presence and severity of disease , the occurrence of ahr in an asymptomatic person may help predict the future development of asthma , the degree of ahr in a symptomatic person can have prognostic and potentially therapeutic implications , the periodicity of asthma exists in parallel with changes in the degree of ahr . a false negative fev1 in patients with asthma may have serious consequences for the patient due to undertreatment . the authors compared forced spirometry and body plethysmography in order to evaluate the concordance of the forced respiratory maneuvers and measurements of airway resistance during normal breathing in mch challenges . pd+100 raw and pd-40 gaw for the following plethysmographically measured parameters rtot , reff , srtot , sreff , sgtot , and sgeff , are closely correlated with cumulative mch - challenge tests . additional investigations on healthy subjects and patients with asthma and copd should be performed to compare the sensitivity and specificity of body plethysmography and forced spirometry for mch - challenge tests .

malaria is a major public health problem in tropical and subtropical countries including india and its dynamics vary from place to place . among all anopheline vectors , anopheles culicifacies ( diptera : culicidae ) is a principal malaria vector in rural , periurban and tribal settings ( mishra et al . culicifacies has a wide distribution that extends from ethiopia , yemen and iran in the west via afghanistan , pakistan , india , bangladesh , myanmar and thailand , to laos and vietnam and combodia in the east ( harrison 1980 , van bortel et al . it is found in nepal and southern china , and in the south in sri lanka . culicifacies is a complex of 5 isomorphic types which are designated as species a and b ( green and miles 1980 ) , species c ( subbarao et al . 1983 ) , species d ( subbarao et al . 1989 , vasantha et al . 1991 ) and species e ( kar et al . 1999 ) with varying biological characteristics such as feeding preference , biting activity , and susceptibility to commonly used insecticides in public health programs ( joshi et al . madhya pradesh is situated in the central part of india with an area of 308 thousand km of which forest cover 76,429 km ( about 25% of the total land area ) . madhya pradesh ( population 72.6 million ) along with other states like orrisa ( population 42 million ) , jharkhand ( population 33 million ) , and chhattisgarh ( population 25.5 million ) contributes for more than 60% of reported ( confirmed ) malaria cases in india . according to national vector borne diseases control program ( nvbdcp ) epidemiological data for 2010 from predominantly these tribal states with a total population of 173.1 million ( out of a total of the country population i. e. , 1.21 billion ) represent 14.3 % population show persistent malaria transmission with high api ( annual parasite incidence ) , slide positivity rate ( spr ) and very high pf% ( sharma 2012 ) . madhya pradesh alone account for 6% of the total population of the country but contributes to 8.6% of the total malaria cases . malaria is complex in madhya pradesh because of vast tracts of forest with tribal settlement ( 20% of state population ) ( singh et al . 2004 , anon 2007 ) . the magnitude of the problem can be accessed from an estimate made in 1987 , that 54 million individuals of various ethnic origins residing in forested area of india and accounting for 8% of the total population contributed 30% of total malaria cases , 60% of total falciparum cases and 50% of malaria deaths in the country ( sharma 1996 ) . the reasons for such a high diseases prevalence in madhya pradesh is mainly due to locations of the villages in the deep forest and is characterized by rocky undulation interspersed with ravines and foothills . these streams flow continuously and provide ample breeding sites covered with dense aquatic vegetation for production of number of anophelines particularly an . therefore malaria control in these areas requires specific approaches and control strategies which includes the proper surveillance for distribution of an . culicifacies members and their identification ( pattanayak et al . 1994 ) . since sibling species a , b , c , d and e of an . culicifacies are morphologically indistinguishable at any stage of life and due to practical difficulties associated with classical cytotaxonomic method for the identification of members of the complex , a molecular method using an allele - specific polymerase chain reaction ( as - pcr ) assay targeted to the d3 domain of 28s ribosomal dna was used to distinguish these sibling species ( singh et al . firstly in d3-pcr the species complex is distinguished in two groups i.e. , a and d in one group and species b , c and e in the second group . in second tier involves ad - pcr assay which distinguishes species a from species d , whereas the bce - pcr assay distinguishes species b , c and e with each other . with combination of these two tier pcr assays it is possible to identify individual mosquito of the an . culicifacies species in various districts of madhya pradesh , india . in the earlier reports from this region species a , b , c and d were identified but species e reported from southern parts of india only was not reported from this area . noticeably in our study we encountered species e from some of the districts in co - habitation with species b which indicates that all the five members of an . the adult anopheline mosquito species were collected from different districts of madhya pradesh ie , mandla ( dungaria village ) , jabalpur ( barela village ) , chindwara ( chakarpat and chikhla villages ) , hoshangabad ( dhadav and padav villages ) , narsinghpur ( chinki village ) and khandwa ( chighdhalia ) ( fig . 1 and table 1 ) . these sites were selected on the basis as they represent the tribal belt along the streams of narmada river and also show high incidence of malaria ( singh 2004 , 2006 , 2009 , sharma 2012 ) . the collections were made during the transmission period i.e. february march and august september in the morning period between 0600 h to 0800 h using mouth aspirator and battery operated torch . the fed anophelines were captured at various collection sites including human dwellings , cattle sheds , mixed dwelling and random collection sites . the fed mosquitoes were captured so that f1 generation of these mosquitoes can be utilized for further use after egg lay . all adult mosquitoes were brought to the lab for their identification by using standard keys ( christopher 1933 , wattal and kalra 1961 , das et al . each representative sample was pinned as a voucher specimen and kept in laboratory as a reference collection . from these collection culicifacies female were separated and allowed for egg laying and the adult emerged from them are used for further standardization and identification of mosquito sibling species using allele specific polymerase chain reaction ( as - pcr ) ( goswami et al . map showing different collection sites in the study list of collection sites and result using as - pcr assay of an . culicifacies mosquito the dna extraction was done by using method as described in our previous publication ( sharma et al . each single adult mosquito was homogenized in the micro centrifuge tube by adding 100 l lysis buffer . the homogenate was immediately kept on ice for 10 minutes and followed by heat treatment at 65 c for 30 minutes . subsequently , 30 l 5 m potassium acetate was added and immediately transferred to ice for one hour followed by centrifugation at 13,000 rpm for 15 minutes at 10 c . to the supernatant obtained , a double volume of absolute chilled ethanol was added for precipitation of dna and kept tubes at 20 c for overnight . after centrifugation at 13,000 rpm for 15 minutes at 10 c , the precipitated dna was washed in 70% ethanol twice . the dna pallet was allowed to air dry and finally dissolved in 50 l te buffer . the targeted region , d3 domain of 28 s rdna , was amplified by pcr using universal primers , d3a and d3b designed for platyhelminth ( litvaitis et al . another set of allele specific primers namely aca and acb which are specific to species a / d and species b / c / e respectively were selected for design of multiplex as - pcr . the sequences for the primers used were given in the table 2 with their annealing temperatures . culicifacies sibling species the amplification was performed in a total of 15 l of reaction mixture consisting of tris . hcl 10 mm ph 9.0 , kcl 50 mm mg cl2 2 mm , dntp 0.2 mm 10 pmoles of primer 0.5 u of taq dna polymerase ( mbi fermentas ) and 10ng of genomic dna . reactions were performed in a ( biorad pcr system icycler ) thermal cycler . the pcr condition consisted of initial denaturation step for 5 min at 95 c followed by 35 cycles of 30 sec at 95 c , 30 sec at 55 c , and 60 sec at 72 c . a total of seven primers of which three primers adf , adr , and df were used in the ad - pcr assay differentiating sibling species a from d , and the other four set of primers bcef , bcr , cr and er were used in the bce - pcr assay for the dedifferentiating species b , c and e from each other ( table 2 ) . optimized condition for ad - pcr assay includes 35 cycles of the initial denaturation temperature at 95 c for 40 s , annealing at 50 c for 40 s , and extension 68 c for 40 s , followed by a final extension at 72 c for 10 min . the pcr reaction was comprised of adf , adr and df primers each at 25 pmol , 200mol / l of each of the dntp , 1.5 mmol/ l mgcl2 , 20 mmol / l ( nh4 ) so4 , 75 mmol/ l tris - hcl ph 9.0 and 0.625 unit of taq dna polymerase . whereas the condition for bce - pcr assay are similar as described for the ad - pcr assay except for the primer concentration of 25 pmol bcef primer , 12 pmol bcr primer , 25 pmol er primer and 30 pmol cr primer respectively . the adult anopheline mosquito species were collected from different districts of madhya pradesh ie , mandla ( dungaria village ) , jabalpur ( barela village ) , chindwara ( chakarpat and chikhla villages ) , hoshangabad ( dhadav and padav villages ) , narsinghpur ( chinki village ) and khandwa ( chighdhalia ) ( fig . 1 and table 1 ) . these sites were selected on the basis as they represent the tribal belt along the streams of narmada river and also show high incidence of malaria ( singh 2004 , 2006 , 2009 , sharma 2012 ) . the collections were made during the transmission period i.e. february march and august september in the morning period between 0600 h to 0800 h using mouth aspirator and battery operated torch . the fed anophelines were captured at various collection sites including human dwellings , cattle sheds , mixed dwelling and random collection sites . the fed mosquitoes were captured so that f1 generation of these mosquitoes can be utilized for further use after egg lay . all adult mosquitoes were brought to the lab for their identification by using standard keys ( christopher 1933 , wattal and kalra 1961 , das et al . each representative sample was pinned as a voucher specimen and kept in laboratory as a reference collection . from these collection culicifacies female were separated and allowed for egg laying and the adult emerged from them are used for further standardization and identification of mosquito sibling species using allele specific polymerase chain reaction ( as - pcr ) ( goswami et al . map showing different collection sites in the study list of collection sites and result using as - pcr assay of an . the dna extraction was done by using method as described in our previous publication ( sharma et al . each single adult mosquito was homogenized in the micro centrifuge tube by adding 100 l lysis buffer . the homogenate was immediately kept on ice for 10 minutes and followed by heat treatment at 65 c for 30 minutes . subsequently , 30 l 5 m potassium acetate was added and immediately transferred to ice for one hour followed by centrifugation at 13,000 rpm for 15 minutes at 10 c . to the supernatant obtained , a double volume of absolute chilled ethanol was added for precipitation of dna and kept tubes at 20 c for overnight . after centrifugation at 13,000 rpm for 15 minutes at 10 c , the precipitated dna was washed in 70% ethanol twice . the dna pallet was allowed to air dry and finally dissolved in 50 l te buffer . the targeted region , d3 domain of 28 s rdna , was amplified by pcr using universal primers , d3a and d3b designed for platyhelminth ( litvaitis et al . another set of allele specific primers namely aca and acb which are specific to species a / d and species b / c / e respectively were selected for design of multiplex as - pcr . the sequences for the primers used were given in the table 2 with their annealing temperatures . culicifacies sibling species the amplification was performed in a total of 15 l of reaction mixture consisting of tris . hcl 10 mm ph 9.0 , kcl 50 mm mg cl2 2 mm , dntp 0.2 mm 10 pmoles of primer 0.5 u of taq dna polymerase ( mbi fermentas ) and 10ng of genomic dna . reactions were performed in a ( biorad pcr system icycler ) thermal cycler . the pcr condition consisted of initial denaturation step for 5 min at 95 c followed by 35 cycles of 30 sec at 95 c , 30 sec at 55 c , and 60 sec at 72 c . a total of seven primers of which three primers adf , adr , and df were used in the ad - pcr assay differentiating sibling species a from d , and the other four set of primers bcef , bcr , cr and er were used in the bce - pcr assay for the dedifferentiating species b , c and e from each other ( table 2 ) . optimized condition for ad - pcr assay includes 35 cycles of the initial denaturation temperature at 95 c for 40 s , annealing at 50 c for 40 s , and extension 68 c for 40 s , followed by a final extension at 72 c for 10 min . the pcr reaction was comprised of adf , adr and df primers each at 25 pmol , 200mol / l of each of the dntp , 1.5 mmol/ l mgcl2 , 20 mmol / l ( nh4 ) so4 , 75 mmol/ l tris - hcl ph 9.0 and 0.625 unit of taq dna polymerase . whereas the condition for bce - pcr assay are similar as described for the ad - pcr assay except for the primer concentration of 25 pmol bcef primer , 12 pmol bcr primer , 25 pmol er primer and 30 pmol cr primer respectively . the targeted region , d3 domain of 28 s rdna , was amplified by pcr using universal primers , d3a and d3b designed for platyhelminth ( litvaitis et al . another set of allele specific primers namely aca and acb which are specific to species a / d and species b / c / e respectively were selected for design of multiplex as - pcr . the sequences for the primers used were given in the table 2 with their annealing temperatures . culicifacies sibling species the amplification was performed in a total of 15 l of reaction mixture consisting of tris . hcl 10 mm ph 9.0 , kcl 50 mm mg cl2 2 mm , dntp 0.2 mm 10 pmoles of primer 0.5 u of taq dna polymerase ( mbi fermentas ) and 10ng of genomic dna . reactions were performed in a ( biorad pcr system icycler ) thermal cycler . the pcr condition consisted of initial denaturation step for 5 min at 95 c followed by 35 cycles of 30 sec at 95 c , 30 sec at 55 c , and 60 sec at 72 c . a total of seven primers of which three primers adf , adr , and df were used in the ad - pcr assay differentiating sibling species a from d , and the other four set of primers bcef , bcr , cr and er were used in the bce - pcr assay for the dedifferentiating species b , c and e from each other ( table 2 ) . optimized condition for ad - pcr assay includes 35 cycles of the initial denaturation temperature at 95 c for 40 s , annealing at 50 c for 40 s , and extension 68 c for 40 s , followed by a final extension at 72 c for 10 min . the pcr reaction was comprised of adf , adr and df primers each at 25 pmol , 200mol / l of each of the dntp , 1.5 mmol/ l mgcl2 , 20 mmol / l ( nh4 ) so4 , 75 mmol/ l tris - hcl ph 9.0 and 0.625 unit of taq dna polymerase . whereas the condition for bce - pcr assay are similar as described for the ad - pcr assay except for the primer concentration of 25 pmol bcef primer , 12 pmol bcr primer , 25 pmol er primer and 30 pmol cr primer respectively . during the collection period a large number of anopheles mosquitoes were collected from the various collection sites ( fig . 1 and table 1 ) and per man per hour ( pmh ) count estimate was also made from each site . culicifacies during the study period was found to be in the range of 8120 pmh , with a high density during august - september ( 90120 pmh ) and to a low density in february march ( 850 pmh ) . the as - pcr assay using different primers , the a / d specific primer ( aca ) in conjunction with d3b produces 313 bp amplification product and b / c / e - specific primer ( acb ) forms 133 bp product with d3a . additionally , the external primers d3a and d3b form common product in all the samples with 382bp products in species a and d whereas 385bp in species b / c / e serving as positive control ( fig . 2 ) . for further distinguishing the sibling species in a / d and b / c / e individually , a total of seven primers of which three primers adf , adr , and df were used in the ad - pcr assay differentiating sibling species a from d , and the other four set of primers bcef , bcr , cr and er were used in the bce - pcr assay differentiating sibling species b , c and e from each other . in ad - pcr , the sibling species a and d produced the bands of 359 bp for d species and 359 bp and 166 bp for sibling species a. on the other hand in bce - pcr , the products are 248 bp for b , 248 bp and 95 bp for c and 248 and 178 for sibling species e respectively ( fig . d3-pcr showing the different bands to differentiate a / d and b / c / e sibling species of an . culicifacies allele specific multiplex ad - bce - pcr showing different bands for each an . culicifacies collected from different districts from madhya pradesh ie , mandla , jabalpur , chindwara , hoshangabad , narsinghpur and khandwa respectively . the collected mosquitoes were subjected to two tier pcr assays . from the d3-pcr only narsinghpur and khandwa samples were identified in a / d group whereas all the other districts samples were identified in and identified in b / c / e group ( fig . results obtained from ad - bce pcr showed the presence of sibling species b from jabalpur , chindwara and hoshangabad , c from hoshangabad only , d from narsinghpur and khandwa and sibling species e from mandla , chindwara and hoshangabad respectively ( fig . the accurate identification and distribution pattern of anopheline mosquitoes is necessary for planning effective vector control strategies and for a better understanding of their potential role in malaria transmission . wattal and kalra in 1961 described 32 species of female anophelines in different regions of india . they divided india in six regions and included madhya pradesh in hyderabad region where about 28 species were described from this region ( wattal and kalra 1961 ) . in our collection we have also encountered about 11 species of anopheles from the study area which includes an . but in the study area malaria is mainly transmitted by two efficient vectors i.e. , an . culicifacies being a major vector of malaria in india is responsible for approximately 65% of total malaria cases ( sharma 1998 ) . in india culicifacies have been found among these species b was found throughout the country whereas species e was reported only from the southern parts of india . they may be different in the feeding pattern an important characteristics that influence vectorial capacity . distinct difference were observed in laboratory studies with reference to insemination rate , fecundity , longevity , oviposition , gonotropic cycle , egg hatching , larval mortality rates and adult emergence time have been observed in different species of an . attempts to find morphological markers for the members of species complex have not been successful so far except the variation in spermatheca of sibling species a and b ( das 1990 ) but this difference still need to be reconfirmed by other techniques . the classical technique of cytotaxonomic is difficult and has limited use as this requires semi gravid females only and moreover it requires highly skilled personnel . with the advent of dna based technology we are now able to differentiate members of an . the dna based technology includes pcr assay which are simple and sensitive at the same time they are applicable to all stages and either sexes of mosquitoes ( goswami et al . 2006 ) . as reported by sharma ( 2012 ) in his review that the epidemiological indices of malaria in madhya pradesh revealed a very dismal picture of malaria . an international team of experts reported a very high incidence of malaria in pregnancy ( mip ) . for example in madhya pradesh ( rural ) 183,0001.5 million per year contract malaria in pregnancy , and result in 73,000629,000 lost foetus and 1,500 to 12,600 maternal deaths . plausible estimate of 220,000 mip cases per year ( 136,000305,000 ) , 95,800 lost fetus ( 56,800147,600 ) and 1,000 maternal deaths ( 6501,600 ) ( diamond et al . culicifacies mosquito species were collected from different malaria endemic district of madhya pradesh by using as - pcr we were able to identify four species b , c , d and e from these areas . species e was not reported earlier from these areas as this species is prevalent only in southern peninsular part of india . species e is highly anthropophagic and possesses high sporozoite rate up to 20% and also known as vector in southern india and srilanka ( kar et al . we encountered species e from mandla , chindwara and hoshangabad districts respectively which have high disease prevalence and represent the tribal belts ( singh 2004 , 2006 , 2009 , sharma 2012 ) . noticeably this species e was found sympatric with a non vector species b from chindwara and hoshangabad district , but in jabalpur district only species b and in mandla only species e were identified . topographically the villages under study from mandla and jabalpur are very close to each other . although species b is a poor vector of malaria in india but through examination of mitotic y chromosome morphology ( kar et al . 1999 ) , that what was reported as b on the sri lanka island is really a sympatric mixture of b and e ( surendran et al . moreover these two sibling species in sri lanka differ in longevity and in their susceptibility to malaria parasite infection and common insecticides ( surendran et al . it is evident from the literature that species e can not be differentiated from species b because they have homosequential polytene chromosome arrangements . species e requires mitotic chromosome examination of male progeny and/or vectorial potential needs to be established for distinction from species b ( kar et al . after screening several enzyme systems , electrophoretic variation at the lactate dehydrogenase ( ldh ) locus was useful . it could group species a and d in one category and species b and c in another category ( adak et al . species e showed the same ldhs allele as in species b and c ( kar et al . e , the paradox that species b females from northern india are not vectors , but homosequential females from rameshwaram island are vectors is resolved . similar studies are needed to be carried out to see the vectorial capacity and disease transmission in the studied districts of madhya pradesh . as in mainland areas close to rameshwaram , species e were found , populations in other parts of tamilnadu state where species b has been identified also should be examined ( kar et al . culicifacies populations identified as species b in sri lanka also should be examined immediately for y - chromosome variations and correlated with malaria infection ( surendran et al . 1997 ) has reported acrocentric and submetacentric y - chromosomes within species b but no epidemiological or dissection data is available that indicates that species b is a vector . thus , there is an urgent need to develop suitable markers that can differentiate species b and e and also to see the other biological characteristics of these two species in such cohabiting areas to conclude that which species is responsible for disease transmission . moreover new areas should be explored for the presence of species e as it is a potent vector of malaria . hence there is an urgent need for nationwide surveillance and identification of vector sibling species distribution once again so that modified species pattern in these areas could be established . apart from confirming their identity , distribution pattern and their differential malaria vector status , it will be important to determine the susceptibility of these sibling species to insecticides in each part of the states/ country as this affects the efficiency of vector control operations in the malaria control programs in india . in conclusion we can say that as we encountered a new sibling species e of an . existence of such modified distribution of sibling species may exist in other areas also which necessitate for reconsidering the sibling species distribution in newer area . knowledge of proper identification and distribution pattern of sibling species may further help us in development of vector control strategies .

background : anopheles culicifacies is an important vector of malaria in southeast asia , contributing to almost 70% of malaria cases in india . it exists as a complex of five morphologically indistinguishable species a , b , c , d and e with varied geographical distribution patterns . in india , 8% of the total population of madhya pradesh ( central india ) contributes about 30% of total malaria cases , 60% of total falciparum cases and 50% of malaria deaths . an . culicifacies is the major malaria vector in this state . vector control mainly relies on the proper identification and distribution of vector species exists in a particular area . the present study was carried out to identify the distribution of an . culicifacies sibling species in certain endemic district of central india , madhya pradesh.methods:the an . culicifacies mosquitoes collected from the study districts were identified morphologically . the genomic dna was isolated from the mosquitoes and subjected to allele specific pcr targeting d3 domain of 28s ribosomal dna.results:the mean prevalence of an . culicifacies during the study period was in the range of 8120 per man per hour ( pmh ) . from the study areas species b was identified from jabalpur , chindwara and hoshangabad , species c from hoshangabad only , species d from narsinghpur and khandwa and sibling species e from mandla , chindwara and hoshangabad respectively.conclusion:this is the first report to detect species e from madhya pradesh region which necessitate for reconsideration of species distribution of each an . culicifacies sibling species that would enable to develop required vector control strategies .

Introduction Materials and Methods Collection of Mosquitoes DNA Isolation Allele Specific PCR (AS-PCR) D3-PCR AD-PCR and BCE-PCR Results Discussion Conclusion

among all anopheline vectors , anopheles culicifacies ( diptera : culicidae ) is a principal malaria vector in rural , periurban and tribal settings ( mishra et al . culicifacies has a wide distribution that extends from ethiopia , yemen and iran in the west via afghanistan , pakistan , india , bangladesh , myanmar and thailand , to laos and vietnam and combodia in the east ( harrison 1980 , van bortel et al . it is found in nepal and southern china , and in the south in sri lanka . culicifacies is a complex of 5 isomorphic types which are designated as species a and b ( green and miles 1980 ) , species c ( subbarao et al . 1983 ) , species d ( subbarao et al . madhya pradesh is situated in the central part of india with an area of 308 thousand km of which forest cover 76,429 km ( about 25% of the total land area ) . madhya pradesh ( population 72.6 million ) along with other states like orrisa ( population 42 million ) , jharkhand ( population 33 million ) , and chhattisgarh ( population 25.5 million ) contributes for more than 60% of reported ( confirmed ) malaria cases in india . according to national vector borne diseases control program ( nvbdcp ) epidemiological data for 2010 from predominantly these tribal states with a total population of 173.1 million ( out of a total of the country population i. e. , 1.21 billion ) represent 14.3 % population show persistent malaria transmission with high api ( annual parasite incidence ) , slide positivity rate ( spr ) and very high pf% ( sharma 2012 ) . madhya pradesh alone account for 6% of the total population of the country but contributes to 8.6% of the total malaria cases . the magnitude of the problem can be accessed from an estimate made in 1987 , that 54 million individuals of various ethnic origins residing in forested area of india and accounting for 8% of the total population contributed 30% of total malaria cases , 60% of total falciparum cases and 50% of malaria deaths in the country ( sharma 1996 ) . the reasons for such a high diseases prevalence in madhya pradesh is mainly due to locations of the villages in the deep forest and is characterized by rocky undulation interspersed with ravines and foothills . therefore malaria control in these areas requires specific approaches and control strategies which includes the proper surveillance for distribution of an . since sibling species a , b , c , d and e of an . culicifacies are morphologically indistinguishable at any stage of life and due to practical difficulties associated with classical cytotaxonomic method for the identification of members of the complex , a molecular method using an allele - specific polymerase chain reaction ( as - pcr ) assay targeted to the d3 domain of 28s ribosomal dna was used to distinguish these sibling species ( singh et al . , a and d in one group and species b , c and e in the second group . in second tier involves ad - pcr assay which distinguishes species a from species d , whereas the bce - pcr assay distinguishes species b , c and e with each other . with combination of these two tier pcr assays it is possible to identify individual mosquito of the an . culicifacies species in various districts of madhya pradesh , india . in the earlier reports from this region species a , b , c and d were identified but species e reported from southern parts of india only was not reported from this area . noticeably in our study we encountered species e from some of the districts in co - habitation with species b which indicates that all the five members of an . the adult anopheline mosquito species were collected from different districts of madhya pradesh ie , mandla ( dungaria village ) , jabalpur ( barela village ) , chindwara ( chakarpat and chikhla villages ) , hoshangabad ( dhadav and padav villages ) , narsinghpur ( chinki village ) and khandwa ( chighdhalia ) ( fig . these sites were selected on the basis as they represent the tribal belt along the streams of narmada river and also show high incidence of malaria ( singh 2004 , 2006 , 2009 , sharma 2012 ) . february march and august september in the morning period between 0600 h to 0800 h using mouth aspirator and battery operated torch . from these collection culicifacies female were separated and allowed for egg laying and the adult emerged from them are used for further standardization and identification of mosquito sibling species using allele specific polymerase chain reaction ( as - pcr ) ( goswami et al . map showing different collection sites in the study list of collection sites and result using as - pcr assay of an . each single adult mosquito was homogenized in the micro centrifuge tube by adding 100 l lysis buffer . after centrifugation at 13,000 rpm for 15 minutes at 10 c , the precipitated dna was washed in 70% ethanol twice . the targeted region , d3 domain of 28 s rdna , was amplified by pcr using universal primers , d3a and d3b designed for platyhelminth ( litvaitis et al . another set of allele specific primers namely aca and acb which are specific to species a / d and species b / c / e respectively were selected for design of multiplex as - pcr . the sequences for the primers used were given in the table 2 with their annealing temperatures . culicifacies sibling species the amplification was performed in a total of 15 l of reaction mixture consisting of tris . a total of seven primers of which three primers adf , adr , and df were used in the ad - pcr assay differentiating sibling species a from d , and the other four set of primers bcef , bcr , cr and er were used in the bce - pcr assay for the dedifferentiating species b , c and e from each other ( table 2 ) . optimized condition for ad - pcr assay includes 35 cycles of the initial denaturation temperature at 95 c for 40 s , annealing at 50 c for 40 s , and extension 68 c for 40 s , followed by a final extension at 72 c for 10 min . the pcr reaction was comprised of adf , adr and df primers each at 25 pmol , 200mol / l of each of the dntp , 1.5 mmol/ l mgcl2 , 20 mmol / l ( nh4 ) so4 , 75 mmol/ l tris - hcl ph 9.0 and 0.625 unit of taq dna polymerase . the adult anopheline mosquito species were collected from different districts of madhya pradesh ie , mandla ( dungaria village ) , jabalpur ( barela village ) , chindwara ( chakarpat and chikhla villages ) , hoshangabad ( dhadav and padav villages ) , narsinghpur ( chinki village ) and khandwa ( chighdhalia ) ( fig . these sites were selected on the basis as they represent the tribal belt along the streams of narmada river and also show high incidence of malaria ( singh 2004 , 2006 , 2009 , sharma 2012 ) . the collections were made during the transmission period i.e. each representative sample was pinned as a voucher specimen and kept in laboratory as a reference collection . from these collection culicifacies female were separated and allowed for egg laying and the adult emerged from them are used for further standardization and identification of mosquito sibling species using allele specific polymerase chain reaction ( as - pcr ) ( goswami et al . map showing different collection sites in the study list of collection sites and result using as - pcr assay of an . after centrifugation at 13,000 rpm for 15 minutes at 10 c , the precipitated dna was washed in 70% ethanol twice . the targeted region , d3 domain of 28 s rdna , was amplified by pcr using universal primers , d3a and d3b designed for platyhelminth ( litvaitis et al . another set of allele specific primers namely aca and acb which are specific to species a / d and species b / c / e respectively were selected for design of multiplex as - pcr . culicifacies sibling species the amplification was performed in a total of 15 l of reaction mixture consisting of tris . hcl 10 mm ph 9.0 , kcl 50 mm mg cl2 2 mm , dntp 0.2 mm 10 pmoles of primer 0.5 u of taq dna polymerase ( mbi fermentas ) and 10ng of genomic dna . reactions were performed in a ( biorad pcr system icycler ) thermal cycler . the pcr condition consisted of initial denaturation step for 5 min at 95 c followed by 35 cycles of 30 sec at 95 c , 30 sec at 55 c , and 60 sec at 72 c . a total of seven primers of which three primers adf , adr , and df were used in the ad - pcr assay differentiating sibling species a from d , and the other four set of primers bcef , bcr , cr and er were used in the bce - pcr assay for the dedifferentiating species b , c and e from each other ( table 2 ) . optimized condition for ad - pcr assay includes 35 cycles of the initial denaturation temperature at 95 c for 40 s , annealing at 50 c for 40 s , and extension 68 c for 40 s , followed by a final extension at 72 c for 10 min . the pcr reaction was comprised of adf , adr and df primers each at 25 pmol , 200mol / l of each of the dntp , 1.5 mmol/ l mgcl2 , 20 mmol / l ( nh4 ) so4 , 75 mmol/ l tris - hcl ph 9.0 and 0.625 unit of taq dna polymerase . the targeted region , d3 domain of 28 s rdna , was amplified by pcr using universal primers , d3a and d3b designed for platyhelminth ( litvaitis et al . another set of allele specific primers namely aca and acb which are specific to species a / d and species b / c / e respectively were selected for design of multiplex as - pcr . the sequences for the primers used were given in the table 2 with their annealing temperatures . culicifacies sibling species the amplification was performed in a total of 15 l of reaction mixture consisting of tris . hcl 10 mm ph 9.0 , kcl 50 mm mg cl2 2 mm , dntp 0.2 mm 10 pmoles of primer 0.5 u of taq dna polymerase ( mbi fermentas ) and 10ng of genomic dna . a total of seven primers of which three primers adf , adr , and df were used in the ad - pcr assay differentiating sibling species a from d , and the other four set of primers bcef , bcr , cr and er were used in the bce - pcr assay for the dedifferentiating species b , c and e from each other ( table 2 ) . the pcr reaction was comprised of adf , adr and df primers each at 25 pmol , 200mol / l of each of the dntp , 1.5 mmol/ l mgcl2 , 20 mmol / l ( nh4 ) so4 , 75 mmol/ l tris - hcl ph 9.0 and 0.625 unit of taq dna polymerase . during the collection period a large number of anopheles mosquitoes were collected from the various collection sites ( fig . 1 and table 1 ) and per man per hour ( pmh ) count estimate was also made from each site . culicifacies during the study period was found to be in the range of 8120 pmh , with a high density during august - september ( 90120 pmh ) and to a low density in february march ( 850 pmh ) . additionally , the external primers d3a and d3b form common product in all the samples with 382bp products in species a and d whereas 385bp in species b / c / e serving as positive control ( fig . for further distinguishing the sibling species in a / d and b / c / e individually , a total of seven primers of which three primers adf , adr , and df were used in the ad - pcr assay differentiating sibling species a from d , and the other four set of primers bcef , bcr , cr and er were used in the bce - pcr assay differentiating sibling species b , c and e from each other . in ad - pcr , the sibling species a and d produced the bands of 359 bp for d species and 359 bp and 166 bp for sibling species a. on the other hand in bce - pcr , the products are 248 bp for b , 248 bp and 95 bp for c and 248 and 178 for sibling species e respectively ( fig . d3-pcr showing the different bands to differentiate a / d and b / c / e sibling species of an . culicifacies allele specific multiplex ad - bce - pcr showing different bands for each an . culicifacies collected from different districts from madhya pradesh ie , mandla , jabalpur , chindwara , hoshangabad , narsinghpur and khandwa respectively . from the d3-pcr only narsinghpur and khandwa samples were identified in a / d group whereas all the other districts samples were identified in and identified in b / c / e group ( fig . results obtained from ad - bce pcr showed the presence of sibling species b from jabalpur , chindwara and hoshangabad , c from hoshangabad only , d from narsinghpur and khandwa and sibling species e from mandla , chindwara and hoshangabad respectively ( fig . the accurate identification and distribution pattern of anopheline mosquitoes is necessary for planning effective vector control strategies and for a better understanding of their potential role in malaria transmission . they divided india in six regions and included madhya pradesh in hyderabad region where about 28 species were described from this region ( wattal and kalra 1961 ) . in our collection we have also encountered about 11 species of anopheles from the study area which includes an . but in the study area malaria is mainly transmitted by two efficient vectors i.e. culicifacies being a major vector of malaria in india is responsible for approximately 65% of total malaria cases ( sharma 1998 ) . in india culicifacies have been found among these species b was found throughout the country whereas species e was reported only from the southern parts of india . they may be different in the feeding pattern an important characteristics that influence vectorial capacity . distinct difference were observed in laboratory studies with reference to insemination rate , fecundity , longevity , oviposition , gonotropic cycle , egg hatching , larval mortality rates and adult emergence time have been observed in different species of an . attempts to find morphological markers for the members of species complex have not been successful so far except the variation in spermatheca of sibling species a and b ( das 1990 ) but this difference still need to be reconfirmed by other techniques . with the advent of dna based technology we are now able to differentiate members of an . as reported by sharma ( 2012 ) in his review that the epidemiological indices of malaria in madhya pradesh revealed a very dismal picture of malaria . an international team of experts reported a very high incidence of malaria in pregnancy ( mip ) . for example in madhya pradesh ( rural ) 183,0001.5 million per year contract malaria in pregnancy , and result in 73,000629,000 lost foetus and 1,500 to 12,600 maternal deaths . culicifacies mosquito species were collected from different malaria endemic district of madhya pradesh by using as - pcr we were able to identify four species b , c , d and e from these areas . species e is highly anthropophagic and possesses high sporozoite rate up to 20% and also known as vector in southern india and srilanka ( kar et al . we encountered species e from mandla , chindwara and hoshangabad districts respectively which have high disease prevalence and represent the tribal belts ( singh 2004 , 2006 , 2009 , sharma 2012 ) . noticeably this species e was found sympatric with a non vector species b from chindwara and hoshangabad district , but in jabalpur district only species b and in mandla only species e were identified . although species b is a poor vector of malaria in india but through examination of mitotic y chromosome morphology ( kar et al . 1999 ) , that what was reported as b on the sri lanka island is really a sympatric mixture of b and e ( surendran et al . moreover these two sibling species in sri lanka differ in longevity and in their susceptibility to malaria parasite infection and common insecticides ( surendran et al . it is evident from the literature that species e can not be differentiated from species b because they have homosequential polytene chromosome arrangements . species e requires mitotic chromosome examination of male progeny and/or vectorial potential needs to be established for distinction from species b ( kar et al . it could group species a and d in one category and species b and c in another category ( adak et al . species e showed the same ldhs allele as in species b and c ( kar et al . e , the paradox that species b females from northern india are not vectors , but homosequential females from rameshwaram island are vectors is resolved . similar studies are needed to be carried out to see the vectorial capacity and disease transmission in the studied districts of madhya pradesh . as in mainland areas close to rameshwaram , species e were found , populations in other parts of tamilnadu state where species b has been identified also should be examined ( kar et al . thus , there is an urgent need to develop suitable markers that can differentiate species b and e and also to see the other biological characteristics of these two species in such cohabiting areas to conclude that which species is responsible for disease transmission . moreover new areas should be explored for the presence of species e as it is a potent vector of malaria . hence there is an urgent need for nationwide surveillance and identification of vector sibling species distribution once again so that modified species pattern in these areas could be established . apart from confirming their identity , distribution pattern and their differential malaria vector status , it will be important to determine the susceptibility of these sibling species to insecticides in each part of the states/ country as this affects the efficiency of vector control operations in the malaria control programs in india . in conclusion we can say that as we encountered a new sibling species e of an . existence of such modified distribution of sibling species may exist in other areas also which necessitate for reconsidering the sibling species distribution in newer area . knowledge of proper identification and distribution pattern of sibling species may further help us in development of vector control strategies .

reactive oxygen species ( ros ) , chemically reactive molecules containing oxygen , are formed by natural byproducts of the normal metabolism of oxygen and by environmental factors such as ultraviolet radiation , pollutants , and drugs . however , imbalance between ros production and removal leads to oxidative stress . in the liver , in addition , the excessive generation of ros , with the reduction of antioxidant defense activities , has been known to be closely related with the induction and progression of hepatic cell death . water dropwort(oenanthe javanica ( o. javanica ) ) is an aquatic perennial herb with a distinctive aroma and taste and is one of the medicinal plants , which has been used to treat jaundice , hypertension , fever , and abdominal pain . o. javanica has high antioxidant properties via scavenging ros and has been known to protect primary cultured rat cortical cells from glutamate - induced neurotoxicity . in addition , o. javanica has anti - proliferative effects on hepg2 cells , not normal chang liver cells . furthermore , it has been well - known that o. javanica has protective effects against various liver injuries : o. javanica displays hepatoprotective effects against tert - butyl hydroperoxide - induced damage in hepg2 cells , carbon tetrachloride - induced liver injury , alcohol toxication , and hepatitis b virus . recently , we reported that o. javanica extract ( oje ) significantly increased immunoreactivities of superoxide dismutase ( sod1 ) , manganese superoxide dismutase ( sod2 ) , catalase ( cat ) , and glutathione peroxidase ( gpx ) in the rat kidney . however , no studies regarding the effect of o. javanica on the normal liver have been reported yet , although some studies have showed hepatoprotective effects of o. javanica in various animal models . in the present study , therefore , we examined the effect of oje on antioxidant enzymes , such as copper , zinc - sod1 , sod2 , cat , and gpx , in the rat liver using immunohistochemistry and western blot analysis . male sprague - dawley rats were obtained from the experimental animal center , kangwon national university , chuncheon , south korea . the rats were used at 12 weeks of age ( body weight , 300320 g ) . they were housed in a conventional state under adequate temperature ( 23c ) and humidity ( 60% ) control with a 12-h light/12-h dark cycle , and free access to food and water . the procedures for animal handling and care adhered to guidelines that are in compliance with the current international laws and policies ( guide for the care and use of laboratory animals , the national academies press , 8 ed . , 2011 ) , and they were approved by the institutional animal care and use committee at kangwon national university . all efforts were also made to minimize animal suffering , as well as , the number of the animals used . as previously described , o. javanica was collected by professor jong dai kim in kangwon province ( south korea ) , in march 2013 and kept in a deep freezer ( 70c ) . o. javanica was extracted with 10 volume ( v / w ) of 70% ethanol at 70c for 4 h , and extraction was repeated three times . 2 ) , concentrated with a vacuum evaporator , and completely dried with a freeze - drier . the extraction yield was 14.5% . the animals were divided into three groups : ( 1 ) normal - group ( n = 14 ) , which is served as a negative control and received a normal composition pellets diet and tap water ad libitum ; ( 2 ) ascorbic acid ( aa)-group ( n = 14 ) , which is served as a positive control ; aa is known as a representative antioxidant chemical , which is mixed by 0.5% of pellets weight ( w / w ) [ table 1 ] ; and ( 3 ) oje - group ( n = 14 ) ; oje was mixed by 0.5% of pellets weight ( w / w ) . each group was designated to receive different amounts composition of the pellets for 28 days . composition of experimental diets ( % ) for histological analysis , the animals ( n = 7 in each group ) were anesthetized with sodium pentobarbital and perfused transcardially with 0.1 mol / l phosphate - buffered saline ( pbs , ph 7.4 ) followed by 4% paraformaldehyde in 0.1 m phosphate buffer ( pb , ph 7.4 ) at 28 days after aa or oje diet . the liver was removed and postfixed in the 10% buffered formalin . the sections were cut at a 6-m thickness with a microtome ( leica , wetzlar , germany ) . to examine the pathological changes of the liver in each group using hematoxylin and eosin ( h & e ) staining , the sections were stained and dehydrated by immersing in serial ethanol baths , and they were then mounted with canada balsam ( kanto chemical , tokyo , japan ) . immunohistochemistry was carried out with sod1 , sod2 , cat , and gpx in order to examine changes in the antioxidants in the liver according to the method of our previous study . in brief , liver sections were sequentially treated with 0.3% hydrogen peroxide ( h2o2 ) in pbs for 30 min and 10% normal goat or donkey serum in 0.05 m pbs for 30 min . the sections were next incubated with goat anti - sod1 ( diluted 1:500 , calbiochem , darmstadt , germany ) , goat anti - sod2 ( diluted 1:1000 , calbiochem ) , rabbit anti - cat ( diluted 1:1000 , lab frontier , seoul , korea ) and sheep anti - gpx ( diluted 1:1000 , chemicon international , billerica , ma ) . thereafter , the tissues were exposed to biotinylated goat anti - rabbit igg , goat anti - sheep igg , donkey anti - goat igg , and streptavidin peroxidase complex ( vector , burlingame , ca , usa ) . and they were visualized with 3,3-diaminobenzidine in 0.1 m tris - hcl buffer . after dehydration , a negative control test was carried out using preimmune serum instead of primary antibody in order to establish the specificity of the immunostaining . the negative control resulted in the absence of immunoreactivity in all structures . in order to quantitatively analyze sod1 , sod2 , cat , and gpx immunoreactivity , digital images of the liver to center the central vein were captured with an axiom1 light microscope ( carl zeiss , germany ) equipped with a digital camera ( axiocam , carl zeiss , germany ) connected to a pc monitor . sod1 , sod2 , cat , and gpx immunoreactive cells were counted at 200x magnification . six coronal sections with 150-m interval per animal were selected , and cell counts were obtained by averaging the counts from each animal . to examine the protein levels of antioxidants in the liver , the animals ( n = 7 in each group ) were used for western blot analysis according to the method of our previous study . after sacrificing animals and removing the liver , and the tissues were homogenized in 50 mm pbs ( ph 7.4 ) containing 0.1 mm ethylene glycol bis ( 2-aminoethyl ether)-n , n , n , n tetraacetic acid ( ph 8.0 ) , 0.2% nonidet p-40 , 10 mm ethylenediamine tetraacetic acid ( ph 8.0 ) , 15 mm sodium pyrophosphate , 100 mm -glycerophosphate , 50 mm naf , 150 mm nacl , 2 mm sodium orthovanadate , 1 mm phenylmethylsulfonyl fluoride and 1 mm dithiothreitol ( dtt ) . after centrifugation , the protein level was determined in the supernatants using a micro bca protein assay kit with bovine serum albumin as the standard ( pierce chemical , rockford , il , usa ) . aliquots containing 20 g of total protein were boiled in loading buffer containing 150 mm tris ( ph 6.8 ) , 3 mm dtt , 6% sds , 0.3% bromophenol blue and 30% glycerol . after electrophoresis , the gels were transferred to nitrocellulose transfer membranes ( pall corp . , east hills , ny , usa ) . to reduce background staining , the membranes were incubated with 5% nonfat dry milk in pbs containing 0.1% tween 20 for 45 min , followed by incubation with goat anti - sod1 ( diluted 1:1000 , calbiochem , darmstadt , germany ) , goat anti - sod2 ( diluted 1:1000 , calbiochem ) , rabbit anti - cat ( diluted 1:1000 , labfrontier , seoul , korea ) and sheep anti - gpx ( diluted 1:1000 , chemicon international , billerica , ma ) , peroxidase - conjugated donkey anti - goat igg , goat anti - rabbit igg or goat anti - sheep igg ( santa cruz , usa ) , and an ecl kit ( pierce chemical , rockford , usa ) . the result of western blot analysis was scanned , and densitometric analysis for the quantification of the bands was done using image j 1.46 ( national institutes of health , bethesda , md , usa ) , which was used to count relative optical density ( rod ) : a ratio of the rod was calibrated as % , with normal - group designated as 100% . a multiple - sample comparison was applied to test the differences between groups ( anova and the tukey multiple range test as post - hoc test using the criterion of the least significant differences ) . male sprague - dawley rats were obtained from the experimental animal center , kangwon national university , chuncheon , south korea . the rats were used at 12 weeks of age ( body weight , 300320 g ) . they were housed in a conventional state under adequate temperature ( 23c ) and humidity ( 60% ) control with a 12-h light/12-h dark cycle , and free access to food and water . the procedures for animal handling and care adhered to guidelines that are in compliance with the current international laws and policies ( guide for the care and use of laboratory animals , the national academies press , 8 ed . , 2011 ) , and they were approved by the institutional animal care and use committee at kangwon national university . all efforts were also made to minimize animal suffering , as well as , the number of the animals used . as previously described , o. javanica was collected by professor jong dai kim in kangwon province ( south korea ) , in march 2013 and kept in a deep freezer ( 70c ) . o. javanica was extracted with 10 volume ( v / w ) of 70% ethanol at 70c for 4 h , and extraction was repeated three times . 2 ) , concentrated with a vacuum evaporator , and completely dried with a freeze - drier . the extraction yield was 14.5% . the animals were divided into three groups : ( 1 ) normal - group ( n = 14 ) , which is served as a negative control and received a normal composition pellets diet and tap water ad libitum ; ( 2 ) ascorbic acid ( aa)-group ( n = 14 ) , which is served as a positive control ; aa is known as a representative antioxidant chemical , which is mixed by 0.5% of pellets weight ( w / w ) [ table 1 ] ; and ( 3 ) oje - group ( n = 14 ) ; oje was mixed by 0.5% of pellets weight ( w / w ) . each group was designated to receive different amounts composition of the pellets for 28 days . for histological analysis , the animals ( n = 7 in each group ) were anesthetized with sodium pentobarbital and perfused transcardially with 0.1 mol / l phosphate - buffered saline ( pbs , ph 7.4 ) followed by 4% paraformaldehyde in 0.1 m phosphate buffer ( pb , ph 7.4 ) at 28 days after aa or oje diet . the sections were cut at a 6-m thickness with a microtome ( leica , wetzlar , germany ) . to examine the pathological changes of the liver in each group using hematoxylin and eosin ( h & e ) staining , the sections were mounted on gelatin - coated microscopy slides . the sections were stained and dehydrated by immersing in serial ethanol baths , and they were then mounted with canada balsam ( kanto chemical , tokyo , japan ) . immunohistochemistry was carried out with sod1 , sod2 , cat , and gpx in order to examine changes in the antioxidants in the liver according to the method of our previous study . in brief , liver sections were sequentially treated with 0.3% hydrogen peroxide ( h2o2 ) in pbs for 30 min and 10% normal goat or donkey serum in 0.05 m pbs for 30 min . the sections were next incubated with goat anti - sod1 ( diluted 1:500 , calbiochem , darmstadt , germany ) , goat anti - sod2 ( diluted 1:1000 , calbiochem ) , rabbit anti - cat ( diluted 1:1000 , lab frontier , seoul , korea ) and sheep anti - gpx ( diluted 1:1000 , chemicon international , billerica , ma ) . thereafter , the tissues were exposed to biotinylated goat anti - rabbit igg , goat anti - sheep igg , donkey anti - goat igg , and streptavidin peroxidase complex ( vector , burlingame , ca , usa ) . and they were visualized with 3,3-diaminobenzidine in 0.1 m tris - hcl buffer . after dehydration , the sections were mounted with canada balsam ( kanto , tokyo , japan ) . a negative control test was carried out using preimmune serum instead of primary antibody in order to establish the specificity of the immunostaining . the negative control resulted in the absence of immunoreactivity in all structures . in order to quantitatively analyze sod1 , sod2 , cat , and gpx immunoreactivity , digital images of the liver to center the central vein were captured with an axiom1 light microscope ( carl zeiss , germany ) equipped with a digital camera ( axiocam , carl zeiss , germany ) connected to a pc monitor . sod1 , sod2 , cat , and gpx immunoreactive cells were counted at 200x magnification . six coronal sections with 150-m interval per animal were selected , and cell counts were obtained by averaging the counts from each animal . to examine the protein levels of antioxidants in the liver , the animals ( n = 7 in each group ) were used for western blot analysis according to the method of our previous study . after sacrificing animals and removing the liver , and the tissues were homogenized in 50 mm pbs ( ph 7.4 ) containing 0.1 mm ethylene glycol bis ( 2-aminoethyl ether)-n , n , n , n tetraacetic acid ( ph 8.0 ) , 0.2% nonidet p-40 , 10 mm ethylenediamine tetraacetic acid ( ph 8.0 ) , 15 mm sodium pyrophosphate , 100 mm -glycerophosphate , 50 mm naf , 150 mm nacl , 2 mm sodium orthovanadate , 1 mm phenylmethylsulfonyl fluoride and 1 mm dithiothreitol ( dtt ) . after centrifugation , the protein level was determined in the supernatants using a micro bca protein assay kit with bovine serum albumin as the standard ( pierce chemical , rockford , il , usa ) . aliquots containing 20 g of total protein were boiled in loading buffer containing 150 mm tris ( ph 6.8 ) , 3 mm dtt , 6% sds , 0.3% bromophenol blue and 30% glycerol . after electrophoresis , the gels were transferred to nitrocellulose transfer membranes ( pall corp . , east hills , ny , usa ) . to reduce background staining , the membranes were incubated with 5% nonfat dry milk in pbs containing 0.1% tween 20 for 45 min , followed by incubation with goat anti - sod1 ( diluted 1:1000 , calbiochem , darmstadt , germany ) , goat anti - sod2 ( diluted 1:1000 , calbiochem ) , rabbit anti - cat ( diluted 1:1000 , labfrontier , seoul , korea ) and sheep anti - gpx ( diluted 1:1000 , chemicon international , billerica , ma ) , peroxidase - conjugated donkey anti - goat igg , goat anti - rabbit igg or goat anti - sheep igg ( santa cruz , usa ) , and an ecl kit ( pierce chemical , rockford , usa ) . the result of western blot analysis was scanned , and densitometric analysis for the quantification of the bands was done using image j 1.46 ( national institutes of health , bethesda , md , usa ) , which was used to count relative optical density ( rod ) : a ratio of the rod was calibrated as % , with normal - group designated as 100% . a multiple - sample comparison was applied to test the differences between groups ( anova and the tukey multiple range test as post - hoc test using the criterion of the least significant differences ) . the effect of oje treatment on the liver histology was evaluated using h & e staining . we found that histological findings in the aa- and oje - groups were very similar to those in the normal - group . these results show that aa and oje did not affect normal liver histology compared with that in the normal - group [ figure 1 ] . h and e staining in the rat liver of the normal-(a ) , ascorbic acid -(b ) and oenanthe javanica extract - ( c ) groups . superoxide dismutase 1 superoxide dismutase 1 immunoreactive cells were hardly detected in the normal - group , although sod1 immunoreactivity is shown in noncellular tissue [ figures 2a and 3 ] . sod1 immunoreactive cells were significantly increased in the aa - group compared with those in the normal - group [ figures 2b and 3 ] . in the oje - group , the mean number of sod1 immunoreactive cells was significantly increased by about 190% compared with that in the aa - group [ figures 2c and 3 ] . immunohistochemistry for superoxide dismutase ( sod1 ) ( a - c ) , manganese superoxide dismutase ( sod2 ) ( d - f ) , catalase ( cat ) ( g - i ) and glutathione peroxidase ( gpx ) ( j - l ) in the rat liver of the normal-(left column ) , ascorbic acid - ( middle column ) and oenanthe javanica extract ( oje ) - ( right column ) groups . strong sod1 , sod2 , cat , and gpx immunoreactive cells ( arrows ) are markedly increased in the oje - group . the mean number of superoxide dismutase ( sod1 ) , manganese superoxide dismutase , catalase and glutathione peroxidase immunoreactive cells in the rat liver of the normal- , ascorbic acid ( aa)- and oenanthe javanica extract - groups ( n = 7 per group ; * p < 0.05 , significantly different from the normal - group , p < 0.05 , significantly different from the aa - group ) . manganese superoxide dismutase the pattern of sod2 immunoreactivity in all the experimental groups was similar to that observed in the immunohistochemical data of sod1 [ figure 2d2f ] . sod2 immunoreactive cells were increased in the aa- and oje - groups compared with those in the normal - group [ figures 2e and 3 ] ; especially , the mean number of sod2 immunoreactive cells in the oje - group was significantly increased by about 478% compared with that in the aa - group [ figures 2f and 3 ] . in the normal - group , cat immunoreactivity was hardly detected in the liver [ figures 2 g and 3 ] . however , a few cat immunoreactive cells were observed in the aa - group [ figures 2h and 3 ] . in the oje - group , cat immunoreactive cells were significantly increased by about 685% compared with that in the aa - group [ figures 2i and 3 ] . glutathione peroxidase although sod1 immunoreactivity is shown in noncellular tissue , gpx immunoreactive cells were hardly found in the liver of the normal - group [ figures 2j and 3 ] . however , many gpx immunoreactive cells were easily detected in the aa- and oje - groups [ figures 2k , 2l and 3 ] ; especially , strong gpx immunoreactive cells were significantly increased by about 346% in the oje - group compared with that in the aa - group [ figures 2l and 3 ] . in this study , we examined changes in sod1 , sod2 , cat , and gpx protein levels in the liver using western blot analysis [ figure 4 ] . in the aa - group , the levels of all the antioxidant enzymes were significantly increased compared with that in the normal - group , and the levels of all the antioxidant enzymes in the oje - group were significantly higher than those in the aa - group . western blot analysis of superoxide dismutase , manganese superoxide dismutase , catalase , and glutathione peroxidase in the rat liver of the normal- , ascorbic acid ( aa)- and oenanthe javanica extract -groups . relative optical density ( rod ) as the mean percentage values of the immunoblot band is represented ( n = 7 per group ; * p < 0.05 , significantly different from the normal - group , p < 0.05 , significantly different from the aa - group ) . the effect of oje treatment on the liver histology was evaluated using h & e staining . we found that histological findings in the aa- and oje - groups were very similar to those in the normal - group . these results show that aa and oje did not affect normal liver histology compared with that in the normal - group [ figure 1 ] . h and e staining in the rat liver of the normal-(a ) , ascorbic acid -(b ) and oenanthe javanica extract - ( c ) groups . superoxide dismutase 1 superoxide dismutase 1 immunoreactive cells were hardly detected in the normal - group , although sod1 immunoreactivity is shown in noncellular tissue [ figures 2a and 3 ] . sod1 immunoreactive cells were significantly increased in the aa - group compared with those in the normal - group [ figures 2b and 3 ] . in the oje - group , the mean number of sod1 immunoreactive cells was significantly increased by about 190% compared with that in the aa - group [ figures 2c and 3 ] . immunohistochemistry for superoxide dismutase ( sod1 ) ( a - c ) , manganese superoxide dismutase ( sod2 ) ( d - f ) , catalase ( cat ) ( g - i ) and glutathione peroxidase ( gpx ) ( j - l ) in the rat liver of the normal-(left column ) , ascorbic acid - ( middle column ) and oenanthe javanica extract ( oje ) - ( right column ) groups . strong sod1 , sod2 , cat , and gpx immunoreactive cells ( arrows ) are markedly increased in the oje - group . the mean number of superoxide dismutase ( sod1 ) , manganese superoxide dismutase , catalase and glutathione peroxidase immunoreactive cells in the rat liver of the normal- , ascorbic acid ( aa)- and oenanthe javanica extract - groups ( n = 7 per group ; * p < 0.05 , significantly different from the normal - group , p < 0.05 , significantly different from the aa - group ) . manganese superoxide dismutase the pattern of sod2 immunoreactivity in all the experimental groups was similar to that observed in the immunohistochemical data of sod1 [ figure 2d2f ] . sod2 immunoreactive cells were increased in the aa- and oje - groups compared with those in the normal - group [ figures 2e and 3 ] ; especially , the mean number of sod2 immunoreactive cells in the oje - group was significantly increased by about 478% compared with that in the aa - group [ figures 2f and 3 ] . in the normal - group , cat immunoreactivity was hardly detected in the liver [ figures 2 g and 3 ] . however , a few cat immunoreactive cells were observed in the aa - group [ figures 2h and 3 ] . in the oje - group , cat immunoreactive cells were significantly increased by about 685% compared with that in the aa - group [ figures 2i and 3 ] . glutathione peroxidase although sod1 immunoreactivity is shown in noncellular tissue , gpx immunoreactive cells were hardly found in the liver of the normal - group [ figures 2j and 3 ] . however , many gpx immunoreactive cells were easily detected in the aa- and oje - groups [ figures 2k , 2l and 3 ] ; especially , strong gpx immunoreactive cells were significantly increased by about 346% in the oje - group compared with that in the aa - group [ figures 2l and 3 ] . in this study , we examined changes in sod1 , sod2 , cat , and gpx protein levels in the liver using western blot analysis [ figure 4 ] . in the aa - group , the levels of all the antioxidant enzymes were significantly increased compared with that in the normal - group , and the levels of all the antioxidant enzymes in the oje - group were significantly higher than those in the aa - group . western blot analysis of superoxide dismutase , manganese superoxide dismutase , catalase , and glutathione peroxidase in the rat liver of the normal- , ascorbic acid ( aa)- and oenanthe javanica extract -groups . relative optical density ( rod ) as the mean percentage values of the immunoblot band is represented ( n = 7 per group ; * p < 0.05 , significantly different from the normal - group , p < 0.05 , significantly different from the aa - group ) . it has been widely accepted that sods , cat , and gpx play important roles in protection against oxidative stresses . it is well - known that sod enzymes participate in the protection against harmful oxidative stresses by catalyzing the dismutation of superoxide anions into hydrogen peroxide and oxygen . especially , cat and gpx are important enzymes , which convert hydrogen peroxide into water and oxygen . in this study aa has been well - known to have antioxidant property by scavenging ros and to act as an essential coenzyme in oxidative stress pathways . it was reported that the oral administration of 100 mg / kg aa for 4 weeks could reduce malondialdehyde and ros levels and increase sod and cat activities in the cadmium - treated rat liver . we , in the present study , found that significant increases in the number of sod1 , sod2 , cat , and gpx immunoreactive cells and their protein levels in the aa - group ( positive control - group ) compared with those in the normal - group . many natural products have been known to be hepatoprotective agents through their effects on the antioxidant system . in the present study , we first examined the effect of oje on antioxidant enzymes in the liver , and we found that more significant increases in the number of sod1 , sod2 , cat , and gpx immunoreactive cells and their protein levels were significantly increased in the oje - group compared with those in the aa - group as well as the normal - group . it was reported that oje , which has gallic acid , catechin , chlorogenic acid and caffeic acid , displayed a great potential as an antioxidant functional ingredient in food applications . furthermore , we recently showed that oje administration in the rat markedly increased antioxidant enzymes immunoreactive cells in the kidney . therefore , it can be postulated that oje pretreatment could provide hepatoprotection against oxidative stress - related liver cell damage via increasing antioxidant activity although we did not examine this in the present study . in conclusion , we show that oje treatment significantly induced augments of endogenous enzymatic antioxidants ( sod1 , sod2 , cat , and gpx ) in the rat liver and the findings indicate that oje pretreatment could provide hepatoprotection against some oxidative stresses in the liver .

background : oenanthe javanica ( o. javanica ) has been known to have high antioxidant properties via scavenging reactive oxygen species . we examined the effect of o. javanica extract ( oje ) on antioxidant enzymes in the rat liver.methods:we examined the effect of the oje on copper , zinc - superoxide dismutase ( sod1 ) , manganese superoxide dismutase ( sod2 ) , catalase ( cat ) , and glutathione peroxidase ( gpx ) in the rat liver using immunohistochemistry and western blot analysis . sprague - dawley rats were randomly assigned to three groups ; ( 1 ) normal diet fed group ( normal - group ) , ( 2 ) diet containing ascorbic acid ( aa)-fed group ( aa - group ) as a positive control , ( 3 ) diet containing oje - fed group ( oje - group).results : in this study , no histopathological finding in the rat liver was found in all the experimental groups . numbers of sod1 , sod2 , cat , and gpx immunoreactive cells and their protein levels were significantly increased in the aa - fed group compared with those in the normal - group . on the other hand , in the oje - group , numbers of sod1 , sod2 , cat , and gpx immunoreactive cells in the liver were significantly increased by about 190% , 478% , 685% , and 346% , respectively , compared with those in the aa - group . in addition , protein levels of sod1 , sod2 , cat , and gpx in the oje - group were also significantly much higher than those in the aa-group.conclusion:oje significantly increased expressions of sod1 and sod2 , cat , and gpx in the liver cells of the rat , and these suggests that significant enhancements of endogenous enzymatic antioxidants by oje might be a legitimate strategy for decreasing oxidative stresses in the liver .

I M Experimental animals Preparation of Treatment of Tissue processing for histology Hematoxylin and eosin staining Immunohistochemistry for antioxidant enzymes Western blot analysis for antioxidant enzymes Statistical analysis R Histological finding Immunoreactivity of antioxidant enzymes Protein levels of antioxidant enzymes D

reactive oxygen species ( ros ) , chemically reactive molecules containing oxygen , are formed by natural byproducts of the normal metabolism of oxygen and by environmental factors such as ultraviolet radiation , pollutants , and drugs . in the liver , in addition , the excessive generation of ros , with the reduction of antioxidant defense activities , has been known to be closely related with the induction and progression of hepatic cell death . water dropwort(oenanthe javanica ( o. javanica ) ) is an aquatic perennial herb with a distinctive aroma and taste and is one of the medicinal plants , which has been used to treat jaundice , hypertension , fever , and abdominal pain . o. javanica has high antioxidant properties via scavenging ros and has been known to protect primary cultured rat cortical cells from glutamate - induced neurotoxicity . in addition , o. javanica has anti - proliferative effects on hepg2 cells , not normal chang liver cells . furthermore , it has been well - known that o. javanica has protective effects against various liver injuries : o. javanica displays hepatoprotective effects against tert - butyl hydroperoxide - induced damage in hepg2 cells , carbon tetrachloride - induced liver injury , alcohol toxication , and hepatitis b virus . recently , we reported that o. javanica extract ( oje ) significantly increased immunoreactivities of superoxide dismutase ( sod1 ) , manganese superoxide dismutase ( sod2 ) , catalase ( cat ) , and glutathione peroxidase ( gpx ) in the rat kidney . however , no studies regarding the effect of o. javanica on the normal liver have been reported yet , although some studies have showed hepatoprotective effects of o. javanica in various animal models . in the present study , therefore , we examined the effect of oje on antioxidant enzymes , such as copper , zinc - sod1 , sod2 , cat , and gpx , in the rat liver using immunohistochemistry and western blot analysis . male sprague - dawley rats were obtained from the experimental animal center , kangwon national university , chuncheon , south korea . as previously described , o. javanica was collected by professor jong dai kim in kangwon province ( south korea ) , in march 2013 and kept in a deep freezer ( 70c ) . 2 ) , concentrated with a vacuum evaporator , and completely dried with a freeze - drier . the animals were divided into three groups : ( 1 ) normal - group ( n = 14 ) , which is served as a negative control and received a normal composition pellets diet and tap water ad libitum ; ( 2 ) ascorbic acid ( aa)-group ( n = 14 ) , which is served as a positive control ; aa is known as a representative antioxidant chemical , which is mixed by 0.5% of pellets weight ( w / w ) [ table 1 ] ; and ( 3 ) oje - group ( n = 14 ) ; oje was mixed by 0.5% of pellets weight ( w / w ) . the liver was removed and postfixed in the 10% buffered formalin . to examine the pathological changes of the liver in each group using hematoxylin and eosin ( h & e ) staining , the sections were stained and dehydrated by immersing in serial ethanol baths , and they were then mounted with canada balsam ( kanto chemical , tokyo , japan ) . immunohistochemistry was carried out with sod1 , sod2 , cat , and gpx in order to examine changes in the antioxidants in the liver according to the method of our previous study . in order to quantitatively analyze sod1 , sod2 , cat , and gpx immunoreactivity , digital images of the liver to center the central vein were captured with an axiom1 light microscope ( carl zeiss , germany ) equipped with a digital camera ( axiocam , carl zeiss , germany ) connected to a pc monitor . sod1 , sod2 , cat , and gpx immunoreactive cells were counted at 200x magnification . to examine the protein levels of antioxidants in the liver , the animals ( n = 7 in each group ) were used for western blot analysis according to the method of our previous study . after sacrificing animals and removing the liver , and the tissues were homogenized in 50 mm pbs ( ph 7.4 ) containing 0.1 mm ethylene glycol bis ( 2-aminoethyl ether)-n , n , n , n tetraacetic acid ( ph 8.0 ) , 0.2% nonidet p-40 , 10 mm ethylenediamine tetraacetic acid ( ph 8.0 ) , 15 mm sodium pyrophosphate , 100 mm -glycerophosphate , 50 mm naf , 150 mm nacl , 2 mm sodium orthovanadate , 1 mm phenylmethylsulfonyl fluoride and 1 mm dithiothreitol ( dtt ) . the result of western blot analysis was scanned , and densitometric analysis for the quantification of the bands was done using image j 1.46 ( national institutes of health , bethesda , md , usa ) , which was used to count relative optical density ( rod ) : a ratio of the rod was calibrated as % , with normal - group designated as 100% . male sprague - dawley rats were obtained from the experimental animal center , kangwon national university , chuncheon , south korea . as previously described , o. javanica was collected by professor jong dai kim in kangwon province ( south korea ) , in march 2013 and kept in a deep freezer ( 70c ) . 2 ) , concentrated with a vacuum evaporator , and completely dried with a freeze - drier . the animals were divided into three groups : ( 1 ) normal - group ( n = 14 ) , which is served as a negative control and received a normal composition pellets diet and tap water ad libitum ; ( 2 ) ascorbic acid ( aa)-group ( n = 14 ) , which is served as a positive control ; aa is known as a representative antioxidant chemical , which is mixed by 0.5% of pellets weight ( w / w ) [ table 1 ] ; and ( 3 ) oje - group ( n = 14 ) ; oje was mixed by 0.5% of pellets weight ( w / w ) . immunohistochemistry was carried out with sod1 , sod2 , cat , and gpx in order to examine changes in the antioxidants in the liver according to the method of our previous study . in order to quantitatively analyze sod1 , sod2 , cat , and gpx immunoreactivity , digital images of the liver to center the central vein were captured with an axiom1 light microscope ( carl zeiss , germany ) equipped with a digital camera ( axiocam , carl zeiss , germany ) connected to a pc monitor . sod1 , sod2 , cat , and gpx immunoreactive cells were counted at 200x magnification . to examine the protein levels of antioxidants in the liver , the animals ( n = 7 in each group ) were used for western blot analysis according to the method of our previous study . after sacrificing animals and removing the liver , and the tissues were homogenized in 50 mm pbs ( ph 7.4 ) containing 0.1 mm ethylene glycol bis ( 2-aminoethyl ether)-n , n , n , n tetraacetic acid ( ph 8.0 ) , 0.2% nonidet p-40 , 10 mm ethylenediamine tetraacetic acid ( ph 8.0 ) , 15 mm sodium pyrophosphate , 100 mm -glycerophosphate , 50 mm naf , 150 mm nacl , 2 mm sodium orthovanadate , 1 mm phenylmethylsulfonyl fluoride and 1 mm dithiothreitol ( dtt ) . the result of western blot analysis was scanned , and densitometric analysis for the quantification of the bands was done using image j 1.46 ( national institutes of health , bethesda , md , usa ) , which was used to count relative optical density ( rod ) : a ratio of the rod was calibrated as % , with normal - group designated as 100% . the effect of oje treatment on the liver histology was evaluated using h & e staining . we found that histological findings in the aa- and oje - groups were very similar to those in the normal - group . these results show that aa and oje did not affect normal liver histology compared with that in the normal - group [ figure 1 ] . h and e staining in the rat liver of the normal-(a ) , ascorbic acid -(b ) and oenanthe javanica extract - ( c ) groups . superoxide dismutase 1 superoxide dismutase 1 immunoreactive cells were hardly detected in the normal - group , although sod1 immunoreactivity is shown in noncellular tissue [ figures 2a and 3 ] . sod1 immunoreactive cells were significantly increased in the aa - group compared with those in the normal - group [ figures 2b and 3 ] . in the oje - group , the mean number of sod1 immunoreactive cells was significantly increased by about 190% compared with that in the aa - group [ figures 2c and 3 ] . immunohistochemistry for superoxide dismutase ( sod1 ) ( a - c ) , manganese superoxide dismutase ( sod2 ) ( d - f ) , catalase ( cat ) ( g - i ) and glutathione peroxidase ( gpx ) ( j - l ) in the rat liver of the normal-(left column ) , ascorbic acid - ( middle column ) and oenanthe javanica extract ( oje ) - ( right column ) groups . strong sod1 , sod2 , cat , and gpx immunoreactive cells ( arrows ) are markedly increased in the oje - group . the mean number of superoxide dismutase ( sod1 ) , manganese superoxide dismutase , catalase and glutathione peroxidase immunoreactive cells in the rat liver of the normal- , ascorbic acid ( aa)- and oenanthe javanica extract - groups ( n = 7 per group ; * p < 0.05 , significantly different from the normal - group , p < 0.05 , significantly different from the aa - group ) . manganese superoxide dismutase the pattern of sod2 immunoreactivity in all the experimental groups was similar to that observed in the immunohistochemical data of sod1 [ figure 2d2f ] . sod2 immunoreactive cells were increased in the aa- and oje - groups compared with those in the normal - group [ figures 2e and 3 ] ; especially , the mean number of sod2 immunoreactive cells in the oje - group was significantly increased by about 478% compared with that in the aa - group [ figures 2f and 3 ] . in the normal - group , cat immunoreactivity was hardly detected in the liver [ figures 2 g and 3 ] . however , a few cat immunoreactive cells were observed in the aa - group [ figures 2h and 3 ] . in the oje - group , cat immunoreactive cells were significantly increased by about 685% compared with that in the aa - group [ figures 2i and 3 ] . glutathione peroxidase although sod1 immunoreactivity is shown in noncellular tissue , gpx immunoreactive cells were hardly found in the liver of the normal - group [ figures 2j and 3 ] . however , many gpx immunoreactive cells were easily detected in the aa- and oje - groups [ figures 2k , 2l and 3 ] ; especially , strong gpx immunoreactive cells were significantly increased by about 346% in the oje - group compared with that in the aa - group [ figures 2l and 3 ] . in this study , we examined changes in sod1 , sod2 , cat , and gpx protein levels in the liver using western blot analysis [ figure 4 ] . in the aa - group , the levels of all the antioxidant enzymes were significantly increased compared with that in the normal - group , and the levels of all the antioxidant enzymes in the oje - group were significantly higher than those in the aa - group . western blot analysis of superoxide dismutase , manganese superoxide dismutase , catalase , and glutathione peroxidase in the rat liver of the normal- , ascorbic acid ( aa)- and oenanthe javanica extract -groups . relative optical density ( rod ) as the mean percentage values of the immunoblot band is represented ( n = 7 per group ; * p < 0.05 , significantly different from the normal - group , p < 0.05 , significantly different from the aa - group ) . the effect of oje treatment on the liver histology was evaluated using h & e staining . we found that histological findings in the aa- and oje - groups were very similar to those in the normal - group . these results show that aa and oje did not affect normal liver histology compared with that in the normal - group [ figure 1 ] . h and e staining in the rat liver of the normal-(a ) , ascorbic acid -(b ) and oenanthe javanica extract - ( c ) groups . superoxide dismutase 1 superoxide dismutase 1 immunoreactive cells were hardly detected in the normal - group , although sod1 immunoreactivity is shown in noncellular tissue [ figures 2a and 3 ] . sod1 immunoreactive cells were significantly increased in the aa - group compared with those in the normal - group [ figures 2b and 3 ] . in the oje - group , the mean number of sod1 immunoreactive cells was significantly increased by about 190% compared with that in the aa - group [ figures 2c and 3 ] . immunohistochemistry for superoxide dismutase ( sod1 ) ( a - c ) , manganese superoxide dismutase ( sod2 ) ( d - f ) , catalase ( cat ) ( g - i ) and glutathione peroxidase ( gpx ) ( j - l ) in the rat liver of the normal-(left column ) , ascorbic acid - ( middle column ) and oenanthe javanica extract ( oje ) - ( right column ) groups . strong sod1 , sod2 , cat , and gpx immunoreactive cells ( arrows ) are markedly increased in the oje - group . the mean number of superoxide dismutase ( sod1 ) , manganese superoxide dismutase , catalase and glutathione peroxidase immunoreactive cells in the rat liver of the normal- , ascorbic acid ( aa)- and oenanthe javanica extract - groups ( n = 7 per group ; * p < 0.05 , significantly different from the normal - group , p < 0.05 , significantly different from the aa - group ) . manganese superoxide dismutase the pattern of sod2 immunoreactivity in all the experimental groups was similar to that observed in the immunohistochemical data of sod1 [ figure 2d2f ] . sod2 immunoreactive cells were increased in the aa- and oje - groups compared with those in the normal - group [ figures 2e and 3 ] ; especially , the mean number of sod2 immunoreactive cells in the oje - group was significantly increased by about 478% compared with that in the aa - group [ figures 2f and 3 ] . in the normal - group , cat immunoreactivity was hardly detected in the liver [ figures 2 g and 3 ] . however , a few cat immunoreactive cells were observed in the aa - group [ figures 2h and 3 ] . in the oje - group , cat immunoreactive cells were significantly increased by about 685% compared with that in the aa - group [ figures 2i and 3 ] . glutathione peroxidase although sod1 immunoreactivity is shown in noncellular tissue , gpx immunoreactive cells were hardly found in the liver of the normal - group [ figures 2j and 3 ] . however , many gpx immunoreactive cells were easily detected in the aa- and oje - groups [ figures 2k , 2l and 3 ] ; especially , strong gpx immunoreactive cells were significantly increased by about 346% in the oje - group compared with that in the aa - group [ figures 2l and 3 ] . in this study , we examined changes in sod1 , sod2 , cat , and gpx protein levels in the liver using western blot analysis [ figure 4 ] . in the aa - group , the levels of all the antioxidant enzymes were significantly increased compared with that in the normal - group , and the levels of all the antioxidant enzymes in the oje - group were significantly higher than those in the aa - group . western blot analysis of superoxide dismutase , manganese superoxide dismutase , catalase , and glutathione peroxidase in the rat liver of the normal- , ascorbic acid ( aa)- and oenanthe javanica extract -groups . relative optical density ( rod ) as the mean percentage values of the immunoblot band is represented ( n = 7 per group ; * p < 0.05 , significantly different from the normal - group , p < 0.05 , significantly different from the aa - group ) . it has been widely accepted that sods , cat , and gpx play important roles in protection against oxidative stresses . in this study aa has been well - known to have antioxidant property by scavenging ros and to act as an essential coenzyme in oxidative stress pathways . we , in the present study , found that significant increases in the number of sod1 , sod2 , cat , and gpx immunoreactive cells and their protein levels in the aa - group ( positive control - group ) compared with those in the normal - group . many natural products have been known to be hepatoprotective agents through their effects on the antioxidant system . in the present study , we first examined the effect of oje on antioxidant enzymes in the liver , and we found that more significant increases in the number of sod1 , sod2 , cat , and gpx immunoreactive cells and their protein levels were significantly increased in the oje - group compared with those in the aa - group as well as the normal - group . furthermore , we recently showed that oje administration in the rat markedly increased antioxidant enzymes immunoreactive cells in the kidney . in conclusion , we show that oje treatment significantly induced augments of endogenous enzymatic antioxidants ( sod1 , sod2 , cat , and gpx ) in the rat liver and the findings indicate that oje pretreatment could provide hepatoprotection against some oxidative stresses in the liver .

the initial surgical treatment for osteochondral defect ( ocd ) of the talus is usually arthroscopic debridement via curettage . however , many patients do not achieve adequate pain relief or restoration of mobility with debridement alone [ 34 , 36 ] . in addition , even among those patients for whom debridement appears to be effective , symptoms often recur over time . for these patients , standard treatments include osteoarticular autograft ( or allograft ) transfer ( oats ) [ 18 , 19 ] , or autologous chondrocyte implantation ( aci ) [ 2 , 3 , 14 , 17 , 25 ] . these open procedures are more invasive and often more expensive than arthroscopy and can result in considerable morbidity . in addition , debridement does not repair the underlying bone defect and may thus allow continued progression of the lesion . researchers have sought treatment alternatives for ocd that could stabilise the interior of the lesion and prevent further collapse [ 1 , 2 , 7 ] . this report introduces a new arthroscopic procedure in which a volume - filling , osteoconductive material containing a diffusible growth factor is inserted into the ocd lesion to stimulate repair of the patient s own subchondral bone . this growth factor , recombinant human platelet - derived growth factor ( rhpdgf ) , has been found to promote healing ; regeneration ; and repair of bone , ligament , periodontal cementum , and other tissues [ 9 , 2224 , 2633 , 35 ] . in this new procedure , arthroscopic debridement to stable , viable bone is followed by arthroscopic placement of rhpdgf mixed with an osteoconductive matrix of beta - tricalcium phosphate ( -tcp ) . the defect is then sealed with fibrin glue , and the patient is kept non - weight bearing for 6 weeks prior to being remobilised . the primary objective of this case - series - based study was to evaluate the initial safety , efficacy , and clinical utility of rhpdgf/-tcp as an arthroscopic treatment following debridement for ocd of the talus , over the course of an initial 6-month follow - up . the hypothesis was that the procedure would result in improved pain and functional scores for patients with and ocd . the clinical relevance of this study is that , if successful , the procedure could provide a less invasive option with improved healing compared to standard techniques . this single - site , single - surgeon , prospective study , conducted with enrolment from february 2012 until november 2012 , was designed as a case - series - based clinical study . patients diagnosed with a talar ocd were recruited for the study from the clinical practice of the authors . inclusion criteria included signed reb - approved informed consent form ( icf ) prior to enrolment ; diagnosis of an isolated ocd larger than 5 mm long , 3 mm wide , and 5 mm deep , confirmed by mri ; independent , ambulatory and can comply with all post - operative evaluations and visits ; 21 years of age or older ; skeletally mature ; stable ankle joint on history and similar ligament stability with the opposite ankle ; < 15 of hindfoot valgus and 5 of hindfoot varus ; ocd is chronic and not secondary to acute trauma within the previous 6 months ; if history of fracture , no residual deformity of the tibia , fibula , or syndesmosis ; no prior fusions of the hindfoot ( subtalar and talonavicular joints ) ; body mass index ( bmi ) 35 ; american society of anesthesiologists ( asa ) physical status classification of 1 or 2 ; and has exhausted non - operative treatment . exclusion criteria included > 15 of hindfoot valgus or 5 of hindfoot varus ; defect > 30 mm length , 25 mm width , or 20 mm depth in size on mri assessment ; allergy to yeast - derived products ; has metallic or electronically , magnetically , or mechanically activated implants that would contraindicate mri scans of the foot ; history of malignancy anywhere in the body ; physically or mentally compromised and unable or unlikely to remain compliant to follow - up ; history of drug / alcohol abuse within the 12 months prior to screening for study entry ; pregnant , or able to become pregnant but not practicing a medically accepted form of birth control ; current acute infection in area surrounding surgical site ; history of anaphylaxis ; condition is bilateral and surgery is to be scheduled over the course of the study ; requires concomitant osteotomy of tibia , fibula , or calcaneus for hindfoot deformity , or requires concomitant hindfoot fusion for hindfoot arthritis or deformity ; undergoing any concomitant surgery that may invalidate outcome scores for this study ; ocd of the tibia in isolation or in combination with the talar lesion ; nicotine addiction or using medication or substances containing nicotine ; cocaine abuse or use of cocaine derivatives ; undergoing revision debridement of an ocd . diagnosis of ocd was confirmed via baseline magnetic resonance imaging ( mri ) scans of the lesion site prior to enrolment . location and size of defect ( width , depth , and length ) , were determined via ankle arthroscopy and recorded prior to treatment . photographs of defect before and after debridement as well as any associated pathology ( osteophytes , synovitis ) were also obtained . patient assessments were completed preoperatively and at 2 , 6 , 12 , and 24 weeks post - operatively . clinical outcomes were recorded preoperatively and at each follow - up visit using the visual analogue scale ( vas ) for pain , the ankle osteoarthritis scale ( aos ) , the short - form-36 ( sf-36 ) health survey , and ap and lateral x - rays . all adverse events affecting the ankles were also recorded at each study visit ( see appendix ) . follow - up mri was conducted at 12 and 24 weeks , in order to evaluate bone marrow oedema . in addition , ct scans at 2 and 24 weeks were compared in order to determine healing status at the base of the ocd and fill of the graft . all scans were measured using inteleviewer software tools ( intelerad , calgary , ab , canada ) , which enable automated , quantitative segmentation and calculation of two - dimensional area and three - dimensional volume measurements ( with read - outs in 1-mm increments ) , as well as image registration for pre- and post - treatment comparisons of patient lesions . the mri scans were evaluated with sagittal and coronal sequences cut with the greatest amount of oedema ( t2-weighted signal on fat suppressed images ) being measured for square surface area of involvement . the oedema signal was measured preoperatively and at each follow - up period . the ct scan was evaluated for size of defect preoperatively and for fill with bone graft at both time periods . the ct was measured on one 0.6-mm slice on the sagittal view and one 0.6-mm slice on the coronal view . all procedures were performed by a single surgeon having advanced skills in arthroscopy and foot and ankle surgery . patients were treated with arthroscopic debridement of the ocd to stable , viable bone via abrasive osteochondroplasty without microfracture or drilling . ( microfracture was not used because the subchondral bone was breached and softened in all cases ) . debridement was followed by arthroscopic placement of a preparation containing rhpdgf and -tcp ( see below ) . routine ankle arthroscopy was performed using anterior medial , anterior lateral , and medial portals to access and debride the joint . an augment bone graft preparation ( biomimetic inc . , memphis , tn ) containing -tcp and rhpdgf was then prepared using standard techniques and placed into the joint via the arthroscopic portal . following mixing , the final consistency of the graft material is similar to that of granular wet sand . when placed near a viable host bone , it acts as scaffold for new bone growth ; it subsequently undergoes remodelling and is finally replaced by host bone . all materials used in the components have been previously approved by health canada for the treatment of foot and ankle fusion procedures and by the fda for the treatment of periodontal bone defects . the foot was dorsiflexed while the fibrin glue set , then the ankle kept dorsiflexed until the wounds were closed and the limb placed in a below - the - knee slab in dorsiflexion for 2 weeks post - surgery . if all appeared stable at 2 weeks , subjects began joint range - of - motion exercises in a walker boot and remained non - weight bearing until the 6-week follow - up visit . the primary outcome measures were the vas for pain and the ankle osteoarthritis scale ( aos ) . the vas for pain is a unidimensional , self - reported measure of pain intensity [ 16 , 20 , 21 ] , which has been widely used in diverse adult populations , including those with rheumatic disease [ 12 , 13 ] . the aos is a reliable , validated , patient - reported , ankle - specific functional outcome instrument . ( the score ranges from 0 to 100 , with a lower score indicating more normal function ) . secondary outcome measures included the physical component summary ( pcs ) and mental component summary ( mcs ) subscales of the sf-36 health survey questionnaire , and mri and ct scans of the affected ankles . the sf-36 is a generic , patient - reported measure of general health status [ 4 , 6 ] that has been shown to have acceptable criterion validity in patients with end - stage ankle arthritis . this study was approved by the institutional review board of the providence health care research institute at the university of british columbia ( reb # h03 - 50062 ) and was therefore performed in accordance with the ethical standards laid down in the 1964 declaration of helsinki and its later amendments . all patients provided informed consent for study enrolment and for the surgical procedure prior to questionnaire administration . treatment safety and efficacy were assessed by comparing the preoperative baseline scores on the primary and secondary outcome measures with the scores recorded during follow - up evaluations at 6 , 12 , and 24 weeks , using student s t test . ninety - five per cent confidence intervals were used to assess variability using aos scores and mean area of oedema ( mm ) via mri . diagnosis of ocd was confirmed via baseline magnetic resonance imaging ( mri ) scans of the lesion site prior to enrolment . location and size of defect ( width , depth , and length ) , were determined via ankle arthroscopy and recorded prior to treatment . photographs of defect before and after debridement as well as any associated pathology ( osteophytes , synovitis ) were also obtained . patient assessments were completed preoperatively and at 2 , 6 , 12 , and 24 weeks post - operatively . clinical outcomes were recorded preoperatively and at each follow - up visit using the visual analogue scale ( vas ) for pain , the ankle osteoarthritis scale ( aos ) , the short - form-36 ( sf-36 ) health survey , and ap and lateral x - rays . all adverse events affecting the ankles were also recorded at each study visit ( see appendix ) . follow - up mri was conducted at 12 and 24 weeks , in order to evaluate bone marrow oedema . in addition , ct scans at 2 and 24 weeks were compared in order to determine healing status at the base of the ocd and fill of the graft . all scans were measured using inteleviewer software tools ( intelerad , calgary , ab , canada ) , which enable automated , quantitative segmentation and calculation of two - dimensional area and three - dimensional volume measurements ( with read - outs in 1-mm increments ) , as well as image registration for pre- and post - treatment comparisons of patient lesions . the mri scans were evaluated with sagittal and coronal sequences cut with the greatest amount of oedema ( t2-weighted signal on fat suppressed images ) being measured for square surface area of involvement . the ct scan was evaluated for size of defect preoperatively and for fill with bone graft at both time periods . the ct was measured on one 0.6-mm slice on the sagittal view and one 0.6-mm slice on the coronal view . all procedures were performed by a single surgeon having advanced skills in arthroscopy and foot and ankle surgery . patients were treated with arthroscopic debridement of the ocd to stable , viable bone via abrasive osteochondroplasty without microfracture or drilling . ( microfracture was not used because the subchondral bone was breached and softened in all cases ) . debridement was followed by arthroscopic placement of a preparation containing rhpdgf and -tcp ( see below ) . routine ankle arthroscopy was performed using anterior medial , anterior lateral , and medial portals to access and debride the joint . an augment bone graft preparation ( biomimetic inc . , memphis , tn ) containing -tcp and rhpdgf was then prepared using standard techniques and placed into the joint via the arthroscopic portal . following mixing , the final consistency of the graft material is similar to that of granular wet sand . when placed near a viable host bone , it acts as scaffold for new bone growth ; it subsequently undergoes remodelling and is finally replaced by host bone . all materials used in the components have been previously approved by health canada for the treatment of foot and ankle fusion procedures and by the fda for the treatment of periodontal bone defects . the foot was dorsiflexed while the fibrin glue set , then the ankle kept dorsiflexed until the wounds were closed and the limb placed in a below - the - knee slab in dorsiflexion for 2 weeks post - surgery . if all appeared stable at 2 weeks , subjects began joint range - of - motion exercises in a walker boot and remained non - weight bearing until the 6-week follow - up visit . the primary outcome measures were the vas for pain and the ankle osteoarthritis scale ( aos ) . the vas for pain is a unidimensional , self - reported measure of pain intensity [ 16 , 20 , 21 ] , which has been widely used in diverse adult populations , including those with rheumatic disease [ 12 , 13 ] . the aos is a reliable , validated , patient - reported , ankle - specific functional outcome instrument . ( the score ranges from 0 to 100 , with a lower score indicating more normal function ) . secondary outcome measures included the physical component summary ( pcs ) and mental component summary ( mcs ) subscales of the sf-36 health survey questionnaire , and mri and ct scans of the affected ankles . the sf-36 is a generic , patient - reported measure of general health status [ 4 , 6 ] that has been shown to have acceptable criterion validity in patients with end - stage ankle arthritis . this study was approved by the institutional review board of the providence health care research institute at the university of british columbia ( reb # h03 - 50062 ) and was therefore performed in accordance with the ethical standards laid down in the 1964 declaration of helsinki and its later amendments . all patients provided informed consent for study enrolment and for the surgical procedure prior to questionnaire administration . treatment safety and efficacy were assessed by comparing the preoperative baseline scores on the primary and secondary outcome measures with the scores recorded during follow - up evaluations at 6 , 12 , and 24 weeks , using student s t test . ninety - five per cent confidence intervals were used to assess variability using aos scores and mean area of oedema ( mm ) via mri . thirty patients with verified ocd were initially assessed for inclusion in this pilot study . of these , 25 patients were excluded due to ankle instability or other associated pathology , or were from the practice of another surgeon . five patients with six ocd lesions of the talus met all inclusion and exclusion criteria , and underwent arthroscopic debridement followed by arthroscopic filling of the defect with rhpdgf - bb/-tcp matrix . none of these patients withdrew from the study before the final follow - up visit at 24 weeks ; all patients were seen for follow - up at the correct intervals and had complete data . mean patient age was 52 8.5 years , and mean bmi was 26.3 5.0 . three of the patients were female and two were male . in one patient , the ocd occurred in the left ankle ; in the remainder , it occurred in the right ankle . one patient had two lesions on the same ankle ( one on the medial and one on the lateral side of the talus ) . four patients had undergone a prior debridement , and three had undergone more than one prior debridement . four lesions were shoulder lesions and two were not.table 1dimensions of ocd lesions from arthroscopic assessmentaveragestandard deviationlength ( mm)143width ( mm)92depth ( mm)112surface area ( mm)10337volume ( mm)285139 surface area was calculated as an ellipse volume was calculated as a hemi - ellipsoid dimensions of ocd lesions from arthroscopic assessment surface area was calculated as an ellipse volume was calculated as a hemi - ellipsoid mean scores for pain and disability at baseline and at 24 weeks post - operative follow - up ( as measured by the vas , sf-36 , and aos scales ) are listed in table 2 . changes in these scores are illustrated in figs . 1 , 2 and 3.table 2mean outcome scores , pain , and disabilityvas pain scaleaossf-36non - wbwbpaindisabilitytotalpcsmcspreoperative baseline3.44.137.238.237.735.153.2week 241.12.125.927.626.742.953.5 vas visual analogue scale , aos ankle osteoarthritis scale , sf-36 short - form ( 36 ) health survey , wb weight bearing , pcs physical component summary , mcs mental component summaryfig . 1average scores on the vas pain questionnaire , from preoperative baseline ( preop ) to final follow - up ( week 24 ) . 2average scores on the mental and physical components of the sf-36 health survey questionnaire , from preoperative baseline ( preop ) to final follow - up ( week 24 ) . 3average scores on the total ankle osteoarthritis scale ( aos ) and on the pain and disability components , from preoperative baseline ( preop ) to final follow - up ( week 24 ) . b post - operative sagittal view showing reduction in the oedema signal . c preoperative coronal view showing bone marrow oedema . d post - operative coronal view showing reduced marrow signal mean outcome scores , pain , and disability vas visual analogue scale , aos ankle osteoarthritis scale , sf-36 short - form ( 36 ) health survey , wb weight bearing , pcs physical component summary , mcs mental component summary average scores on the vas pain questionnaire , from preoperative baseline ( preop ) to final follow - up ( week 24 ) . a lower score indicates decreased pain average scores on the mental and physical components of the sf-36 health survey questionnaire , from preoperative baseline ( preop ) to final follow - up ( week 24 ) . increased scores indicate improvement average scores on the total ankle osteoarthritis scale ( aos ) and on the pain and disability components , from preoperative baseline ( preop ) to final follow - up ( week 24 ) . decreased scores indicate improvement magnetic resonance imaging ( mri ) scans . a preoperative sagittal view showing bone marrow oedema . b post - operative sagittal view showing reduction in the oedema signal . c preoperative coronal view showing bone marrow oedema . d post - operative coronal view showing reduced marrow signal no major adverse events related to the graft were observed . over the course of the study , there were no wound infections , wound breakdowns , reoperations , loss of motion , symptoms of loose body / impingement , or synovitis . ( any additional adverse events affecting the ankle are listed by patient in the appendix ) . evaluation of bone marrow oedema as visualised by mri , preoperatively and at 24 weeks post - operatively , is shown in table 3 and fig . 4 ( note that retrograde drilling was not possible in this joint , as only minimal cartilage remained).table 3mean area of oedema ( mm ) via mriparameterpreop24 weekscoronal oedema95 ( ci 20171)71 ( ci 0208)sagittal oedema140 ( ci 50229)37 ( ci 093)average118 ( ci 45190)54 ( ci 0150)table 4mean area of ocd lesions and percentage filled by graft , as visualised via ct scanparameter2 weeks24 weekscoronal view ( mm)6958sagittal view ( mm)8976amount of defect filled by graft ( coronal view ) ( % ) 85.254amount of defect filled by graft ( sagittal view ) ( % ) 84.661 mean area of oedema ( mm ) via mri mean area of ocd lesions and percentage filled by graft , as visualised via ct scan incorporation of the graft as visualised by ct scan , at 2- and 24-week follow - ups , is shown in table 4 and fig . 5.fig . 5post - operative computed tomography ( ct ) scans . a coronal view at 2 weeks showing fill of the osteochondral defect . b coronal view at final scan ( 24 weeks ) showing healing of the bone in the base of the osteochondral defect and resorption of periarticular tricalcium phosphate granules post - operative computed tomography ( ct ) scans . a coronal view at 2 weeks showing fill of the osteochondral defect . b coronal view at final scan ( 24 weeks ) showing healing of the bone in the base of the osteochondral defect and resorption of periarticular tricalcium phosphate granules the most important finding of the present study was that a new arthroscopic procedure yielded improved pain and functional scores for patients with ocd , and enhanced healing of these lesions ( as confirmed by mri and ct scans ) . no short - term complications were observed despite close monitoring of the patients . this prospective , single - centre , case - series - based , proof - of - concept study evaluated the initial safety , efficacy , and clinical utility of rhpdgf/-tcp as an arthroscopic treatment following debridement for ocd of the talus in five patients . results at 6-month follow - up indicate that the method is both safe and effective , at least initially . mean non - weight bearing vas pain scores decreased by 68 % , mean weight bearing vas pain scores decreased by 49 % ( vas ) , and mean scores on the aos disability component decreased by 28 % ( table 2 ; figs . 1 , 3 ) . mri scans confirmed a reduction in subchondral oedema over time in all patients , with the mean area decreasing by 25 % on the coronal view and by 74 % on the sagittal view from preoperative baseline to final follow - up scans at 24 weeks ( table 3 ; fig . no heterotopic ossification was evident on follow - up ct scans , and the graft was incorporated in all five cases ( table 4 ; fig . 5 ) . in addition , no major adverse events related to the graft were observed , and none of the patients required reoperation for their ocd . with procedures such as oats , not only is there increased risk of knee donor - site morbidity , but the force of impact during graft insertion can damage both the talus and the chondrocytes in the graft . thus , gentler insertion of the non - cellular preparation used in our procedure confers the added advantage of reduced damage to ankle and graft , as well as the knee . in a large , multi - centre , prospective , randomised , controlled , clinical trial , digiovanni et al . compared the safety and efficacy of pdgf/-tcp versus autogenous bone grafts ( autografts ) in patients requiring hindfoot or ankle fusion . the study found that the pdgf treatment resulted in comparable fusion rates , less pain , and fewer side effects compared to treatment with autograft . due to its role as a mitogenic and chemotactic factor for fibroblasts [ 30 , 32 ] , osteoblasts [ 2224 , 26 , 29 ] , chondrocytes [ 28 , 33 ] , and tenoblasts [ 9 , 27 , 35 ] , pdgf also holds considerable promise in restoring bone integrity in lesions such as ocds . healing and restoration of the subchondral bone may thus make our method more effective in the long term than cartilage grafts , oats , or aci . in addition , this new procedure offers an alternative that should prove to be less invasive and more economical than either oats or aci . especially promising was the fact that four of the five patients had undergone previous debridements that required revision , but no revisions or additional procedures were required following the rhpdgf/-tcp treatment . limitations included small sample size , a short follow - up period , lack of a concurrent control group , and potential selection bias due to patient recruitment from a single clinical centre . in addition , the improved area of marrow oedema observed at final follow - up could be due in part to a period of relatively limited mobility . a larger , multi - centre , randomised , controlled trial with a minimum 2-year follow - up period is needed and is planned in order to address these limitations . the clinical relevance of this study is that it demonstrates that a new technique using a human growth factor for the treatment of ocd can be safe , well tolerated , and clinically useful . if the results of the present study are confirmed in a larger trial , the potential benefits include lower cost , less post - operative pain , fewer wound complications , and shorter recovery time due to its less invasive nature ; no non - unions of osteotomies ; considerable reductions in patient - reported pain several months after surgery ; and better long - term outcomes , compared with more invasive procedures such as oats or aci , which are currently the standard of care . in addition , there is no need for grafts ( and therefore no donor - site morbidity ) , and this mixture of growth factor and osteoconductive matrix may help to stabilise the lesion after debridement , preventing further progression . in conclusion , the use of rhpdgf in an osteoconductive matrix shows promise as a clinically useful alternative for the treatment of ocd lesions of the talus following arthroscopic debridement . an expanded series or randomised , see tables 5 , 6 , and 7.table 5adverse events as reported by patientspatient idadverse event descriptiononset date001fell using crutches , stuck splint at study ankle12 march 2012slipped on stairs , muscles sore at ankle11 may 2012development of stress fracture at calcaneus of study foot ( osteoporosis - z - score-3.7 , severely osteoporotic)noted during may 2012small clot with superficial phlebitis right anklenoted during 17 may 2012 appointmentsevered acl right leg ( study side)noted during unscheduled visit 18 july 2012fell off bike , landed on right side ( knee area)early august 2012occasional catching and locking symptoms study anklenoted during 24 august 2012 visitmedial pain , study anklenoted during 24 august 2012 visitdropped 1-lb weight on study anklenoted during 24 august 2012 visit002right hip replacement6 march 2013continued pain left study ankle . patient had injection for thisnoted during unscheduled visit 4 april 2013003cast causing pain removed and replacednoted during unscheduled visit 23 october 2012sharp , acute pain right study ankle5 november 2012shooting pains medial ankle , decreased sensation throughout entire dorsum of footnoted during 15 november 2012 visitpatient notes colour difference right study ankle , swelling in foot , lump on bottom of foot ( noticed during december 2012 ) , sore big toe with pain radiating to foot , numbnessnoted during unscheduled visit 21 december 2012severe oa left knee referred to specialistreferral 27 december 2012after short walk , sharp pain in ankle6 january 2013corneal surgery for displaced lens10 january 2013004graft not contained as well as hopednoted during 7 june 2012 visitstiff anklenoted during 12 july 2012ankle becomes swollen after running any distancenoted during 15 november 2012 visit005slipped in elevator , caught himself24 june 2012patient notes occasional numb / tingling sensationnoted during 25 june 2012 visitresidual material back of jointnoted during 25 june 2012 visitdropped brick on ankle16 august 2012withdrawal symptoms from discontinuation of tramadol insomnia , loss of appetitenoted during 30 august 2012 visittable 6adverse event definitions : severity criteriadegreedescriptionmild ( grade 1)symptom(s ) barely noticeable to subject or does not make subject uncomfortable ; does not influence performance or functioning ; prescription drug not ordinarily needed for relief of symptom(s ) , but may be given because of the nature of subjectmoderate ( grade 2)symptom(s ) of a sufficient severity to make subject uncomfortable ; performance of daily activity is influenced ; subject is able to continue in study ; treatment of symptom(s ) may be neededsevere ( grade 3)cause severe discomfort ; symptoms cause incapacitation or significant impact on subject s daily life ; severity may cause cessation of treatment with study device ; treatment for symptom(s ) may be given and/or subject hospitalisedlife - threatening ( grade 4)extreme limitation in activity ; significant assistance required ; significant medical intervention / therapy required ; hospitalisation or hospice care requiredtable 7adverse event definitions : relationship to procedurerelationshipdescriptionnot relatedany reaction that does not follow a reasonable temporal sequence from administration of the study device and that is likely to have been produced by the subject s clinical state or other modes of therapy administered to the subjectunlikelyany reaction that does not follow a reasonable temporal sequence from administration of the study device or that is likely to have been produced by the subject s clinical state or other modes of therapy administered to the subjectlikelya reaction that follows a reasonable temporal sequence from administration of the study device or that follows a known response pattern to the suspended device and that could not be reasonably explained by the known characteristics of the subject s clinical state or other modes of therapy administered to the subjectdefinitea reaction that follows a reasonable temporal sequence from administration of the study device and that follows a known response pattern to the suspected device and that recurs with rechallenge , and/or is improved by removing the device adverse events as reported by patients adverse event definitions : severity criteria adverse event definitions : relationship to procedure none of the authors have received financial support , including pharmaceutical company support , for authorship , or promotion of this study .

purpose an arthroscopic procedure for the treatment of osteochondral defects using platelet - derived growth factor ( pdgf ) carried out in a matrix of tricalcium phosphate was developed . this prospective , case - series - based study was designed to evaluate the safety and clinical utility of this procedure.methodspatients with an isolated osteochondral defect larger than 5 mm long , 3 mm wide , and 5 mm deep and smaller than 30 mm long , 25 mm wide , or 20 mm deep were considered for enrolment . only patients with chronic lesions were enroled . arthroscopic debridement was followed by the placement of recombinant human pdgf in a matrix of tricalcium phosphate . the ankle osteoarthritis scale ( aos ) , visual analogue scale ( vas ) for pain , and sf-36 questionnaires were administered at 0 , 2 , 6 , 12 , and 24 weeks . magnetic resonance imaging ( mri ) and computed tomography ( ct ) scans were taken before and after surgery.resultsfive patients were ultimately enroled in this proof - of - concept trial . all outcome measures demonstrated marked improvement from baseline to final follow - up : the mean weight bearing vas pain score improved by 49 % , and the mean aos functional score improved by 28 % . bone healing was seen on ct , and reduction in oedema signal was seen on mri.conclusionthis new procedure may offer a promising alternative for the treatment of osteochondral defects . further high - quality studies are needed to confirm these results and to analyse the long - term effects of the procedure . the clinical relevance of this study is that the procedure may provide a less invasive option with improved bone healing compared to standard techniques .level of evidenceiv .

Level of evidence Introduction Materials and methods Data collection Surgical techniques Outcome measures Statistical analysis Results Discussion Conclusion Appendix: adverse events Conflict of interest

the initial surgical treatment for osteochondral defect ( ocd ) of the talus is usually arthroscopic debridement via curettage . for these patients , standard treatments include osteoarticular autograft ( or allograft ) transfer ( oats ) [ 18 , 19 ] , or autologous chondrocyte implantation ( aci ) [ 2 , 3 , 14 , 17 , 25 ] . researchers have sought treatment alternatives for ocd that could stabilise the interior of the lesion and prevent further collapse [ 1 , 2 , 7 ] . this report introduces a new arthroscopic procedure in which a volume - filling , osteoconductive material containing a diffusible growth factor is inserted into the ocd lesion to stimulate repair of the patient s own subchondral bone . this growth factor , recombinant human platelet - derived growth factor ( rhpdgf ) , has been found to promote healing ; regeneration ; and repair of bone , ligament , periodontal cementum , and other tissues [ 9 , 2224 , 2633 , 35 ] . in this new procedure , arthroscopic debridement to stable , viable bone is followed by arthroscopic placement of rhpdgf mixed with an osteoconductive matrix of beta - tricalcium phosphate ( -tcp ) . the defect is then sealed with fibrin glue , and the patient is kept non - weight bearing for 6 weeks prior to being remobilised . the primary objective of this case - series - based study was to evaluate the initial safety , efficacy , and clinical utility of rhpdgf/-tcp as an arthroscopic treatment following debridement for ocd of the talus , over the course of an initial 6-month follow - up . the hypothesis was that the procedure would result in improved pain and functional scores for patients with and ocd . the clinical relevance of this study is that , if successful , the procedure could provide a less invasive option with improved healing compared to standard techniques . this single - site , single - surgeon , prospective study , conducted with enrolment from february 2012 until november 2012 , was designed as a case - series - based clinical study . inclusion criteria included signed reb - approved informed consent form ( icf ) prior to enrolment ; diagnosis of an isolated ocd larger than 5 mm long , 3 mm wide , and 5 mm deep , confirmed by mri ; independent , ambulatory and can comply with all post - operative evaluations and visits ; 21 years of age or older ; skeletally mature ; stable ankle joint on history and similar ligament stability with the opposite ankle ; < 15 of hindfoot valgus and 5 of hindfoot varus ; ocd is chronic and not secondary to acute trauma within the previous 6 months ; if history of fracture , no residual deformity of the tibia , fibula , or syndesmosis ; no prior fusions of the hindfoot ( subtalar and talonavicular joints ) ; body mass index ( bmi ) 35 ; american society of anesthesiologists ( asa ) physical status classification of 1 or 2 ; and has exhausted non - operative treatment . exclusion criteria included > 15 of hindfoot valgus or 5 of hindfoot varus ; defect > 30 mm length , 25 mm width , or 20 mm depth in size on mri assessment ; allergy to yeast - derived products ; has metallic or electronically , magnetically , or mechanically activated implants that would contraindicate mri scans of the foot ; history of malignancy anywhere in the body ; physically or mentally compromised and unable or unlikely to remain compliant to follow - up ; history of drug / alcohol abuse within the 12 months prior to screening for study entry ; pregnant , or able to become pregnant but not practicing a medically accepted form of birth control ; current acute infection in area surrounding surgical site ; history of anaphylaxis ; condition is bilateral and surgery is to be scheduled over the course of the study ; requires concomitant osteotomy of tibia , fibula , or calcaneus for hindfoot deformity , or requires concomitant hindfoot fusion for hindfoot arthritis or deformity ; undergoing any concomitant surgery that may invalidate outcome scores for this study ; ocd of the tibia in isolation or in combination with the talar lesion ; nicotine addiction or using medication or substances containing nicotine ; cocaine abuse or use of cocaine derivatives ; undergoing revision debridement of an ocd . diagnosis of ocd was confirmed via baseline magnetic resonance imaging ( mri ) scans of the lesion site prior to enrolment . patient assessments were completed preoperatively and at 2 , 6 , 12 , and 24 weeks post - operatively . clinical outcomes were recorded preoperatively and at each follow - up visit using the visual analogue scale ( vas ) for pain , the ankle osteoarthritis scale ( aos ) , the short - form-36 ( sf-36 ) health survey , and ap and lateral x - rays . follow - up mri was conducted at 12 and 24 weeks , in order to evaluate bone marrow oedema . the oedema signal was measured preoperatively and at each follow - up period . debridement was followed by arthroscopic placement of a preparation containing rhpdgf and -tcp ( see below ) . all materials used in the components have been previously approved by health canada for the treatment of foot and ankle fusion procedures and by the fda for the treatment of periodontal bone defects . if all appeared stable at 2 weeks , subjects began joint range - of - motion exercises in a walker boot and remained non - weight bearing until the 6-week follow - up visit . the primary outcome measures were the vas for pain and the ankle osteoarthritis scale ( aos ) . secondary outcome measures included the physical component summary ( pcs ) and mental component summary ( mcs ) subscales of the sf-36 health survey questionnaire , and mri and ct scans of the affected ankles . this study was approved by the institutional review board of the providence health care research institute at the university of british columbia ( reb # h03 - 50062 ) and was therefore performed in accordance with the ethical standards laid down in the 1964 declaration of helsinki and its later amendments . treatment safety and efficacy were assessed by comparing the preoperative baseline scores on the primary and secondary outcome measures with the scores recorded during follow - up evaluations at 6 , 12 , and 24 weeks , using student s t test . diagnosis of ocd was confirmed via baseline magnetic resonance imaging ( mri ) scans of the lesion site prior to enrolment . patient assessments were completed preoperatively and at 2 , 6 , 12 , and 24 weeks post - operatively . clinical outcomes were recorded preoperatively and at each follow - up visit using the visual analogue scale ( vas ) for pain , the ankle osteoarthritis scale ( aos ) , the short - form-36 ( sf-36 ) health survey , and ap and lateral x - rays . follow - up mri was conducted at 12 and 24 weeks , in order to evaluate bone marrow oedema . in addition , ct scans at 2 and 24 weeks were compared in order to determine healing status at the base of the ocd and fill of the graft . patients were treated with arthroscopic debridement of the ocd to stable , viable bone via abrasive osteochondroplasty without microfracture or drilling . debridement was followed by arthroscopic placement of a preparation containing rhpdgf and -tcp ( see below ) . all materials used in the components have been previously approved by health canada for the treatment of foot and ankle fusion procedures and by the fda for the treatment of periodontal bone defects . if all appeared stable at 2 weeks , subjects began joint range - of - motion exercises in a walker boot and remained non - weight bearing until the 6-week follow - up visit . the primary outcome measures were the vas for pain and the ankle osteoarthritis scale ( aos ) . secondary outcome measures included the physical component summary ( pcs ) and mental component summary ( mcs ) subscales of the sf-36 health survey questionnaire , and mri and ct scans of the affected ankles . this study was approved by the institutional review board of the providence health care research institute at the university of british columbia ( reb # h03 - 50062 ) and was therefore performed in accordance with the ethical standards laid down in the 1964 declaration of helsinki and its later amendments . treatment safety and efficacy were assessed by comparing the preoperative baseline scores on the primary and secondary outcome measures with the scores recorded during follow - up evaluations at 6 , 12 , and 24 weeks , using student s t test . five patients with six ocd lesions of the talus met all inclusion and exclusion criteria , and underwent arthroscopic debridement followed by arthroscopic filling of the defect with rhpdgf - bb/-tcp matrix . none of these patients withdrew from the study before the final follow - up visit at 24 weeks ; all patients were seen for follow - up at the correct intervals and had complete data . four lesions were shoulder lesions and two were not.table 1dimensions of ocd lesions from arthroscopic assessmentaveragestandard deviationlength ( mm)143width ( mm)92depth ( mm)112surface area ( mm)10337volume ( mm)285139 surface area was calculated as an ellipse volume was calculated as a hemi - ellipsoid dimensions of ocd lesions from arthroscopic assessment surface area was calculated as an ellipse volume was calculated as a hemi - ellipsoid mean scores for pain and disability at baseline and at 24 weeks post - operative follow - up ( as measured by the vas , sf-36 , and aos scales ) are listed in table 2 . 1 , 2 and 3.table 2mean outcome scores , pain , and disabilityvas pain scaleaossf-36non - wbwbpaindisabilitytotalpcsmcspreoperative baseline3.44.137.238.237.735.153.2week 241.12.125.927.626.742.953.5 vas visual analogue scale , aos ankle osteoarthritis scale , sf-36 short - form ( 36 ) health survey , wb weight bearing , pcs physical component summary , mcs mental component summaryfig . 1average scores on the vas pain questionnaire , from preoperative baseline ( preop ) to final follow - up ( week 24 ) . 2average scores on the mental and physical components of the sf-36 health survey questionnaire , from preoperative baseline ( preop ) to final follow - up ( week 24 ) . 3average scores on the total ankle osteoarthritis scale ( aos ) and on the pain and disability components , from preoperative baseline ( preop ) to final follow - up ( week 24 ) . d post - operative coronal view showing reduced marrow signal mean outcome scores , pain , and disability vas visual analogue scale , aos ankle osteoarthritis scale , sf-36 short - form ( 36 ) health survey , wb weight bearing , pcs physical component summary , mcs mental component summary average scores on the vas pain questionnaire , from preoperative baseline ( preop ) to final follow - up ( week 24 ) . a lower score indicates decreased pain average scores on the mental and physical components of the sf-36 health survey questionnaire , from preoperative baseline ( preop ) to final follow - up ( week 24 ) . increased scores indicate improvement average scores on the total ankle osteoarthritis scale ( aos ) and on the pain and disability components , from preoperative baseline ( preop ) to final follow - up ( week 24 ) . decreased scores indicate improvement magnetic resonance imaging ( mri ) scans . 5post - operative computed tomography ( ct ) scans . b coronal view at final scan ( 24 weeks ) showing healing of the bone in the base of the osteochondral defect and resorption of periarticular tricalcium phosphate granules post - operative computed tomography ( ct ) scans . b coronal view at final scan ( 24 weeks ) showing healing of the bone in the base of the osteochondral defect and resorption of periarticular tricalcium phosphate granules the most important finding of the present study was that a new arthroscopic procedure yielded improved pain and functional scores for patients with ocd , and enhanced healing of these lesions ( as confirmed by mri and ct scans ) . this prospective , single - centre , case - series - based , proof - of - concept study evaluated the initial safety , efficacy , and clinical utility of rhpdgf/-tcp as an arthroscopic treatment following debridement for ocd of the talus in five patients . results at 6-month follow - up indicate that the method is both safe and effective , at least initially . mean non - weight bearing vas pain scores decreased by 68 % , mean weight bearing vas pain scores decreased by 49 % ( vas ) , and mean scores on the aos disability component decreased by 28 % ( table 2 ; figs . mri scans confirmed a reduction in subchondral oedema over time in all patients , with the mean area decreasing by 25 % on the coronal view and by 74 % on the sagittal view from preoperative baseline to final follow - up scans at 24 weeks ( table 3 ; fig . no heterotopic ossification was evident on follow - up ct scans , and the graft was incorporated in all five cases ( table 4 ; fig . the study found that the pdgf treatment resulted in comparable fusion rates , less pain , and fewer side effects compared to treatment with autograft . healing and restoration of the subchondral bone may thus make our method more effective in the long term than cartilage grafts , oats , or aci . limitations included small sample size , a short follow - up period , lack of a concurrent control group , and potential selection bias due to patient recruitment from a single clinical centre . the clinical relevance of this study is that it demonstrates that a new technique using a human growth factor for the treatment of ocd can be safe , well tolerated , and clinically useful . if the results of the present study are confirmed in a larger trial , the potential benefits include lower cost , less post - operative pain , fewer wound complications , and shorter recovery time due to its less invasive nature ; no non - unions of osteotomies ; considerable reductions in patient - reported pain several months after surgery ; and better long - term outcomes , compared with more invasive procedures such as oats or aci , which are currently the standard of care . in conclusion , the use of rhpdgf in an osteoconductive matrix shows promise as a clinically useful alternative for the treatment of ocd lesions of the talus following arthroscopic debridement . an expanded series or randomised , see tables 5 , 6 , and 7.table 5adverse events as reported by patientspatient idadverse event descriptiononset date001fell using crutches , stuck splint at study ankle12 march 2012slipped on stairs , muscles sore at ankle11 may 2012development of stress fracture at calcaneus of study foot ( osteoporosis - z - score-3.7 , severely osteoporotic)noted during may 2012small clot with superficial phlebitis right anklenoted during 17 may 2012 appointmentsevered acl right leg ( study side)noted during unscheduled visit 18 july 2012fell off bike , landed on right side ( knee area)early august 2012occasional catching and locking symptoms study anklenoted during 24 august 2012 visitmedial pain , study anklenoted during 24 august 2012 visitdropped 1-lb weight on study anklenoted during 24 august 2012 visit002right hip replacement6 march 2013continued pain left study ankle . patient had injection for thisnoted during unscheduled visit 4 april 2013003cast causing pain removed and replacednoted during unscheduled visit 23 october 2012sharp , acute pain right study ankle5 november 2012shooting pains medial ankle , decreased sensation throughout entire dorsum of footnoted during 15 november 2012 visitpatient notes colour difference right study ankle , swelling in foot , lump on bottom of foot ( noticed during december 2012 ) , sore big toe with pain radiating to foot , numbnessnoted during unscheduled visit 21 december 2012severe oa left knee referred to specialistreferral 27 december 2012after short walk , sharp pain in ankle6 january 2013corneal surgery for displaced lens10 january 2013004graft not contained as well as hopednoted during 7 june 2012 visitstiff anklenoted during 12 july 2012ankle becomes swollen after running any distancenoted during 15 november 2012 visit005slipped in elevator , caught himself24 june 2012patient notes occasional numb / tingling sensationnoted during 25 june 2012 visitresidual material back of jointnoted during 25 june 2012 visitdropped brick on ankle16 august 2012withdrawal symptoms from discontinuation of tramadol insomnia , loss of appetitenoted during 30 august 2012 visittable 6adverse event definitions : severity criteriadegreedescriptionmild ( grade 1)symptom(s ) barely noticeable to subject or does not make subject uncomfortable ; does not influence performance or functioning ; prescription drug not ordinarily needed for relief of symptom(s ) , but may be given because of the nature of subjectmoderate ( grade 2)symptom(s ) of a sufficient severity to make subject uncomfortable ; performance of daily activity is influenced ; subject is able to continue in study ; treatment of symptom(s ) may be neededsevere ( grade 3)cause severe discomfort ; symptoms cause incapacitation or significant impact on subject s daily life ; severity may cause cessation of treatment with study device ; treatment for symptom(s ) may be given and/or subject hospitalisedlife - threatening ( grade 4)extreme limitation in activity ; significant assistance required ; significant medical intervention / therapy required ; hospitalisation or hospice care requiredtable 7adverse event definitions : relationship to procedurerelationshipdescriptionnot relatedany reaction that does not follow a reasonable temporal sequence from administration of the study device and that is likely to have been produced by the subject s clinical state or other modes of therapy administered to the subjectunlikelyany reaction that does not follow a reasonable temporal sequence from administration of the study device or that is likely to have been produced by the subject s clinical state or other modes of therapy administered to the subjectlikelya reaction that follows a reasonable temporal sequence from administration of the study device or that follows a known response pattern to the suspended device and that could not be reasonably explained by the known characteristics of the subject s clinical state or other modes of therapy administered to the subjectdefinitea reaction that follows a reasonable temporal sequence from administration of the study device and that follows a known response pattern to the suspected device and that recurs with rechallenge , and/or is improved by removing the device adverse events as reported by patients adverse event definitions : severity criteria adverse event definitions : relationship to procedure none of the authors have received financial support , including pharmaceutical company support , for authorship , or promotion of this study .

ubiquitination is a posttranslational modification ( ptm ) that involves the attachment of a ubiquitin molecule ( ~9 kda ) to a target protein . it is now well accepted that most of the cellular processes required for the maintenance of the cell homeostasis are regulated by the ubiquitin - proteasome system ( ups ) , including the regulation of innate immune signalling . ubiquitination is mediated by a set of three enzymes , a ubiquitin - activating enzyme ( e1 ) , a ubiquitin - conjugating enzyme ( e2 ) , and a ubiquitin ligase ( e3 ) . ubiquitin ( ub ) is attached as a monomer or as polyubiquitin ( poly - ub ) chains . this attachment occurs between a lysine group in the target protein and the carboxy - terminal glycine of ub . the formation of ub chains , however , occurs by formation of a bond between the carboxy - terminal glycine of ub and one of the seven lysines ( k6 , k11 , k27 , k29 , k33 , k48 , and k63 ) or the methionine ( m1 ) present in the acceptor ub molecule allowing the generation of a wide variety of poly - ub chains . each poly - ub chain type will influence the fate of the target protein differently . for instance , k48-conjugated ub chains are considered a signal for protein degradation at the proteasome while k63 and m1 chains play important roles in signalling pathways . ubiquitination is a reversible process , and its reversibility is mediated by a family of proteases called deubiquitinases ( deubiquitinating enzymes , dubs ) . keeping the balance between the addition and removal of ubiquitin moieties is crucial in maintaining cellular homeostasis and any disturbances in this balance can have adverse consequences for the cell . there are four thiol protease families , the ubiquitin - specific proteases ( usp ) , ubiquitin c - terminal hydrolases ( uch ) , ovarian tumour domain containing proteases ( otu ) , and machado joseph disease ( mjd)/josephin domain dubs , and one zinc - metalloprotease group , the jab1/mpn / mov34 metalloenzyme family . the main functions of dubs are ( i ) generation / release of free ubiquitin from ub precursors ( de novo ub synthesis ) , ( ii ) subtle editing of poly - ub chains , and ( iii ) removal of the poly - ub chains from substrates prior to degradation by proteasome - bound dubs . dubs , similarly to other proteases , are tightly regulated to avoid aberrant function that could be therefore detrimental to the cell . this is achieved by a combination of different layers of regulation at transcriptional and nontranscriptional levels . as one of the best examples of transcriptional regulation is a20 ( tnfaip3 ) , which is a member of the otu family of dubs . a20 expression levels are highly upregulated in a proinflammatory environment ( i.e. , in response to tlr4 activation ) , reflecting its important role as a negative regulator in the inflammatory response , as we will discuss below . there are other dubs , which are regulated in response to cytokines , including dub1 , dub2 , usp17 ( dub3 ) , and otud-6b . dub1 is specifically induced by il-3 , il-5 , and gm - csf , while dub2 is stimulated by il-2 . usp17 ( dub3 ) is involved in the regulation of cell growth and survival and it is regulated by the cytokines il-4 and il-6 . ovarian tumour domain containing 6b ( otud-6b ) is a dub , whose expression in b lymphocytes is induced by secretion of il-3 , il-4 , il-13 , or gm - csf . with prolonged stimulation , these cytokines have an opposite effect and instead lead to a decrease in otud-6b expression . a higher expression of otud-6b was associated with inhibition of cell growth , an increase in apoptosis , and arrest of cells in g1 phase . dubs can acquire specificity due to recruitment factors that guide them towards a specific substrate . one example is usp10 that requires the protein mcpip-1 ( monocyte chemotactic protein induced protein 1 ) to interact with and deubiquitinate its substrate nemo inhibiting the nf-b signalling cascade . in other cases binding of for instance , usp7 , whose catalytic triad exists in an inactive configuration , changes towards an active one upon ubiquitin binding suggesting that usp7 catalytic domain is only fully active when a ubiquitin molecule is correctly bound . the presence of dubs in molecular complexes is a common way to modulate their activity . this mechanism is essential for usp1 , an inefficient enzyme alone , but its activity highly increases when bound to the wd40-repeat protein uaf-1 due to conformational changes that increase its catalytic activity . usp1 is involved in dna damage response , mainly in the fanconi anemia ( fa ) pathway where it mediates the deubiquitination of fancd2 and fanci , a crucial step for the correct function of the fa pathway [ 9 , 10 ] . additionally dub activity can be further adjusted by posttranslational modifications such as phosphorylation or ubiquitination . for instance , phosphorylation of cyld at ser418 or usp7 at ser18 led to an increase in the activities of these two dubs . in the case of cyld , this modification can be induced by lps ( lipopolysaccharide ) or tnf- ( tumour necrosis factor ) treatment and it can suppress its deubiquitinating activity on tnf receptor - associated factor 2 ( traf2 ) . furthermore , this phosphorylation also occurs in dendritic cells ( dcs ) treated with lps / lex , which leads to a diminished activity of cyld but not to its complete loss . this effect can be reversed by an inhibition of dc - sign signalling and also by depletion of ikk . one example is the production of reactive oxygen species ( ros ) generated during mitochondrial oxidative metabolism as well as in cellular responses to cytokines or bacterial invasion , which can inhibit cellular dub activity by oxidation of the catalytic cysteine residue [ 14 , 15 ] . to summarize , more than one regulatory mechanism can apply to certain dubs , which highlights the importance of a fine and multifaceted control of dub expression and activity . innate immunity is triggered in response to either pamps , which are derived from microbial pathogens , or damps such as atp , cholesterol , or monosodium urate crystals . these danger signals are recognized by pattern recognition receptors either at the cell membranes by toll - like receptors ( tlrs ) or at the cytosol by receptors such as the nod - like receptors ( nlrs ) . activation of these prr receptors results in a variety of immune signalling cascades which lead to the induction of immune mediators and proinflammatory cytokines , such as tnf or il-1 , capable of triggering appropriate immune responses . these cytokines lead to the recruitment of immune cells to the site of infection or tissue damage , which initiates an inflammatory response . tlr- and nlr - mediated signalling is heavily controlled by the ubiquitin system , which plays an essential role in maintaining the appropriate regulation of these cellular pathways . although dubs can be involved in many other inflammatory aspects , here we will discuss how dubs contribute to tlr and nlr - induced pathways , focusing on the activation of two very important and related processes , nf-b pathway and inflammasome activation . tlrs are transmembrane glycoproteins , which play a key role in the immune response against microbes . ten human tlrs have been identified to date and they either localize to the cell surface ( tlrs 1 , 2 , 4 , 5 , 6 , and 10 ) or have endosomal localization ( tlrs 3 , 7 , 8 , and 9 ) . there are two distinguishable pathways of tlr signalling , one via the myd88 ( myeloid differentiation primary - response protein 88 ) and the second one via trif ( tir domain containing adaptor protein inducing interferon / ) and apart from tlr3 , most other tlrs are associated with the myd88 pathway . ubiquitination is critically involved in optimal tlr - triggered myd88 and trif signalling ( figure 1 ) . tlr3 engagement induces the recruitment of trif and modification of traf3 with k63 poly - ub , which consequently recruits the tbk1 ( traf family member - associated nf-b activator - binding kinase)/ikk kinase complex . in contrast , tlr4 or tlr2 activation leads to the assembly of the myd88 signalling complex , recruiting traf6 , ciap1 , and ciap2 . these ubiquitin ligases mediate k48-linked poly - ub of traf3 , and traf3 is consequently degraded by the proteasome . traf6 ubiquitin ligase activity is essential for the synthesis of k63-linked poly - ub chains , which act as a scaffold to recruit other proteins required for signalling . traf6 k63-linked poly - ub chains recruit both the tak1 and ikk complexes through their respective ubiquitin - binding subunits , tab2/3 and nemo . this occurs with the help of the lubac ubiquitin ligase complex , which leads to the linear ubiquitination of nemo required for the recruitment of the ikk complex ( ikk and ikk ) . as a result , tak1 phosphorylates ikk , which in turn phosphorylates ib and subsequently undergoes ubiquitination and proteasomal degradation this allows nf-b to translocate to the nucleus from cytosol and regulate the transcription of a variety of target genes ( figure 1 ) . deubiquitination also plays a key role in tlr signalling pathways by reversing the effect of ubiquitination and controlling the intensity of the immune response ( figure 1 ) . several dubs have been identified to participate in the tlr signalling , the most studied and best characterized being a20 ( tnfaip3 ) and cyld . a20 contains an otu domain , which has dub activity specific towards several nf-b signalling factors , such as traf6 , ripk1 , or nemo , which consequently leads to suppressed nf-b activation . a20 is an unusual dub because it encodes seven zinc - finger ( znf ) motifs , which confer e3 ubiquitin ligase activity on a20 . this allows a20 to perform an editing function : in addition to removing k63-linked polyubiquitin chains from substrates such as ripk1 , a20 can introduce k48-polyubiquitin chains in the same substrate tagging it for a proteasomal degradation . in addition to this , a20 can also regulate nf-b independently of its enzymatic activity . a20 can bind polyubiquitin chains through its znf domain allowing the interaction of ubiquitinated nemo with a20 . this ubiquitin - induced recruitment of a20 to nemo is sufficient to block ikk phosphorylation by its upstream kinase tak1 preventing nf-b activation . in contrast cyld is a tumour suppressor , whose loss leads to familial cylindromatosis , a skin tumour hereditary disorder , but that also controls nf-b activation . cyld achieves this by specifically cleaving k63-linked poly - ub chains and linear poly - ub chains from ripk1 , traf2 , and nemo and similarly to a20 negatively regulates nf-b signalling . usp7 was first identified as a herpesvirus associated protein , hence its alternative name hausp ( herpesvirus associated usp ) . . it can regulate nf-b transcriptional activity in the nucleus , by deubiquitinating nf-b and preventing its degradation , hence increasing its transcriptional activity . but usp7 can also act as a negative cytosolic regulator by deubiquitinating nemo and consequently decreasing proteasomal degradation of ib. this in turn retains nf-b in the cytoplasm and further suppresses nf-b activity . these two reported and opposing roles suggest that usp7 can perform different functions roles , depending on substrate recognition or cellular localization , highlighting the tight activity control of this protease . as previously mentioned , usp10 is required for mediated inhibition of nf-b activation . by mediating usp10-dependent deubiquitination of nemo traf family member - associated nf-b activator ( tank ) interacts with both mcpip1 and usp10 , which leads to decrease in traf6 ubiquitination and the termination of the nf-b activation in response to tlr activation . in accordance with this usp18 is responsible for counteracting isg15 conjugation and it is an important negative regulator of the ifn responses , thereby playing important roles in viral responses . however , we now know that usp18 also mediates and regulates tlr - induced nf-b activation by cleavage of k63-polyubiquitin chains , but not k48 chains , of tak1 and nemo . in addition to the dubs described here there are several others implicated in the downregulation of the nf-b pathway upon tlr activation , although these are not well characterized . these include the usp family members usp2a , usp4 , usp15 , usp21 , and usp31 and the member of the jamm family mysm1 and their substrates have been summarized in table 1 . the nlr family presents a characteristic tripartite domain architecture with a variable c - terminus , a middle nacht domain , and a leucine rich repeat ( lrr ) n - terminus . the c - terminal lrr domain is involved in the ligand binding or activator sensing while the n - terminal domain performs effector functions by interacting with other proteins . nlrs are classified into four subfamilies according to their n - terminal domains : the acidic transactivation domain ( nlra ) , the baculoviral inhibitory repeat - like domain ( nlrb ) that includes nod1 and nod2 , the caspase activation and recruitment domain ( card ; nlrc ) , and the pyrin domain ( nlrp ) . nlrs can recognize a wide variety of ligands including pathogens , endogenous molecules , or environmental factors . their functions can vary and they are divided into four steps : inflammasome formation , signalling transduction , transcription activation , and autophagy . similarly to tlrs , nlr activation is also tightly regulated and ptms play an important role here . although ubiquitination in nlr signalling is well accepted , the role of dubs in these pathways is just emerging . nod1 and nod2 receptors are important bacterial sensors , which recognize peptidoglycan ( pgn ) . nod1 senses the ie - dap dipeptide , which is found in pgn of all gram - negative and certain gram - positive bacteria , while nod2 recognizes mdp ( muramyl dipeptide ) , the minimal bioactive peptidoglycan motif common to all bacteria ( figure 2(a ) ) . upon encountering with these ligands , nod1 and nod2 form oligomeric complexes , leading to the activation of nf-b and mapk . iaps ( ciap1 , ciap2 , and xiap ) are central regulators of nod1 and nod2 signalling . upon oligomerization ripk2 this allows the recruitment of tak1/tab2/tab3 complex and lubac , which can also mediate the linear ubiquitination of ripk2 , and further contributes to the nf-b and mapk pathway activation by ubiquitination of nemo [ 30 , 31 ] . ubiquitin can directly bind to the card domain of nod1 or nod2 and compete with ripk2 for its association with these receptors , suggesting that ubiquitin might play a negative regulatory role [ 32 , 33 ] ( figure 2(b ) ) . a20 also plays a regulatory role in nod2 signalling by deubiquitinating ripk2 to control the extent of the inflammatory signals . a20-deficient cells present an amplified response to mdp , including increased ripk2 ubiquitination and nf-b signalling . one of the dubs , which is relatively poorly characterized but which has been shown to play key functions in nod2 signalling , is otulin . this protein specifically deconjugates linear ( m1 ) poly - ub chains assembled by lubac and in this way it modulates linear ubiquitination of lubac 's substrates and provides fine - tuning of the initial activation of nf-b . by deubiquitinating ripk2 because lubac continuously ubiquitinates itself and other substrates , otulin plays an important role to avoid accumulation of met1-ub chains and overactivation of this pathway ( table 1 , figure 2(b ) ) . the inflammasome is a molecular complex , which consists of a sensor molecule ( nlr , e.g. , nlrp1 , nlrp3 , nlrc4 , or nlrp6 ) , an adaptor protein ( asc , apoptosis - associated speck - like protein containing a card domain ) , and an effector molecule ( caspase-1 ) . the main function of the effector molecule is to induce the cleavage and activation of the proinflammatory cytokines , il-1 and il-18 . these proinflammatory proteins are synthesized as precursor molecules and require caspase-1 activation within the inflammasome in order to be released and cleaved and perform their biological activity . first , an nf-b mediated initial step leads to increased expression of nlrp3 and pro - il-1. then an activating signal triggers rapid activation of caspase-1 . caspase-1 activation can be achieved by several k - releasing molecules , including nigericin , crystals , or extracellular atp through the activation of the atp - gated p2x7 receptor ( p2x7r ) . after the inflammasome is fully activated , it can lead to pyroptotic cell death , which can be distinguished from other cell death types by pore formation in the plasma membrane followed by osmotic cell lysis and finally the release of il-1 and il-18 . given the important role of ubiquitin in signalling cascades derived from tlr and nlr activation , it is not surprising to find that assembly and activation of an inflammasome is also regulated by the ubiquitin system . ubiquitination can regulate canonical inflammasome activation by modulation of three major components : nlr , asc , and caspase-1 . this can be exemplified by march7 , which promotes ubiquitination of nlrp3 , and this causes its degradation upon dopamine stimulation as a mean to control inflammasome activation . another example is scffbxl2 , whose activity is impaired upon lps priming preventing nlrp3 ubiquitination and its consequent degradation ( figure 3 ) . for instance , trim30 can negatively regulate nlrp3 inflammasome by modulating the levels of ros species in the cell . trim30 macrophages produce higher levels of ros and potentiate nlrp3 inflammasome activation ; however the mechanisms by which trim30 controls this remain unknown . however , trim33 is essential for cytosolic rna - induced nlrp3 inflammasome activation . trim33 ubiquitinates dhx33 , a cytosolic dsdna sensor for nlrp3 , allowing dhx33-nlrp3 interactions and consequent inflammasome activation . similarly to the nod2 receptor activation , ciap e3s are also involved in the inflammasome activation . attenuation of ciap activities , either by their deletion or by inhibition , triggers nlrp3 and caspase-1 activation as well as rip3 kinase - dependent il-1 processing and secretion . on the other hand , ciap1 and ciap2 can attach k63-linked poly - ub chains to caspase-1 , thereby facilitating caspase-1 activation and il-1 release . caspase-1 ubiquitination also occurs in response to the nlrp1 activator anthrax lethal toxin [ 43 , 44 ] although the type of ubiquitin chains and whether this is a requirement for caspase-1 activation still remain unclear . in addition to nlr and caspase-1 , ubiquitin - mediated inflammasome activation can be also promoted by modification of the adaptor protein asc . in this situation , k63 poly - ub modification of asc allows for its interaction with the autophagic adaptor p62 and delivery of asc to the autophagosome . traf3 ubiquitin ligase ubiquitinates asc , and abolishment of the target lysine ( k174 ) prevents inflammasome activation and il-1 release in response to viral infection . also , traf6-mediated asc ubiquitination has been recently reported in response to far - infrared and proposed to constitute a mechanism , which dampens inflammasome activation in repair processes . interestingly asc has been identified as a substrate of hoil-1l , a member of linear ubiquitination complex lubac , and hoil deficient macrophages present an impaired inflammasome response . in line with this , macrophages deficient in sharpin , which is a different member of the lubac complex , are not able to mount an optimal inflammasome response . all this evidence reveals that ubiquitination is an essential modification for the control of the inflammasome activation . it is then logical to assume that dubs are important players of these regulatory mechanisms . this was first suggested by juliana et al . , who showed that nlrp3 is ubiquitinated in resting macrophages and that , upon cell activation with priming ( lps ) and activating signals ( atp , nigericin , and msu crystals ) , these ubiquitin chains are removed by dubs , allowing activation of the complex . this report was quickly followed by two other studies supporting these results [ 51 , 52 ] , and it was py et al . who identified brcc3 as the first dub to be directly involved in inflammasome activation . these reports showed that inhibition of dub activity with the dub inhibitors bap-15 , wp1130 , pr-619 , and g5 blocks nlrp3 but not nlrc4 or aim2 mediated il-1 release and pyroptosis ( figure 3 ; table 1 ) . moreover , a recent report has demonstrated that histone deacetylase 6 ( hdac6 ) negatively regulates nlrp3 inflammasome activation . hdac6 interacts with nlrp3 's ubiquitin - binding domain and treatment with the dub inhibitor pr-619 results in an increased interaction of nlrp3 with hdac6 . the authors suggest this is due to an increased ubiquitination of nlrp3 and the consequent inhibition of nlrp3-dependent caspase-1 activation . the ability of these dub inhibitors to block inflammasome activation could explain the inhibitory effect of the compound bay 11 - 7082 on nlrp3 inflammasome independently of its nf-b inhibitory activity since this compound can inhibit components of the ubiquitin system , including dubs [ 55 , 56 ] . the other dub , which has been directly implicated in the inflammasome activation , is a20 . in contrast to brcc3 , a20 acts as a negative regulator of nlrp3 and suppresses inflammasome activation by restricting ubiquitination of il-1 and nlrp3 activation [ 57 , 58 ] . given the fine - tuning and the layers of regulation required for both the inflammasome and dub activation , it is quite likely to think that different dubs might perform opposing functions pertaining to the inflammasome activation . whether dubs regulate the ubiquitination state of asc or caspase-1 involved in the inflammasome assembly still remains unknown . during pathogenesis , deubiquitinating enzymes are regulated both by microorganisms and by a host cell . pathogens can exploit the host ubiquitin system by expressing their own ubiquitin - specific enzymes , and the host cell can up- or downregulate expression and/or activity of host dubs . first , an example of a pathogen - encoded deubiquitinase disturbing the host innate immune pathways is salmonella 's avra , which is a dub that facilitates inhibition of the nf-b pathway . also , depletion of avra in salmonella leads to significantly increased secretion of cytokine il-6 in the host cell , which is dependent on nf-b pathway [ 6063 ] . as a second example , chlamydia trachomatis encodes two dubs , chladub1 and chladub2 , which are specific for ubiquitin but they also harbour deneddylating activity . chladub1 binds and stabilizes ib , most likely via its deubiquitination , and finally this can lead to an inhibition of nf-b activation . since several known bacterial dubs directly target important functions in the host immune system , development of selective inhibitors for pathogenic dubs could be exploited as a therapeutic approach in the treatment of infections . bacterial infection can induce inflammasome activation in the host cell and deubiquitination has been implicated in this process . salmonella typhimurium infection leads to changes in the activity of several host dubs , such as usp4 , usp5 , uchl3 , and uchl5 , and increased activity of uchl5 was found to contribute to the inflammasome activation during this infection . additionally , enteropathogenic escherichia coli protein nlea associates with and interrupts deubiquitination of nlrp3 , thereby repressing inflammasome activation . dubs are genetically altered in many human cancers ( i.e. , cyld , a20 , or usp6 ) or contribute to the stability of oncogenes or tumour suppressors ( i.e. , usp7 , usp8 , or brcc3 ) . here we will highlight dubs with potential implications in immune disease although the scope for other dubs contributing to disease is very high . although many of the studies mentioned in this review have been performed in vitro in cell culture models , the involvement of dubs in inflammatory responses has been also studied by using animal models , highlighting the relevance of these proteases in a relevant tissue and immune context ( table 1 ) . mutations in the cyld gene lead to a subtype of the benign cancer predisposition syndrome of skin appendages also known as brooke - spiegler syndrome , although inactivation or downregulation of cyld is also observed in a variety of other cancers , including melanoma , and breast , colon , lung , breast , cervical , and , recently , prostate cancer . as previously mentioned cyld can bind to nemo and nf-b that have been identified as its substrates . it is possible that the negative regulation of nf-b mediated by cyld contributes to its tumour suppression function given the increasingly recognized role for nf-b in cancer advancement . cyld deactivation could provide specific advantage to tumour cells by enhanced nf-b signalling [ 6971 ] . they show increased basal and induced nf-b activation and can develop autoimmune symptoms and colonic inflammation with features of human inflammatory bowel disease , and their inflammatory responses in response to pathogenic infection are potentiated . multiple mutations in the a20 gene have been identified ; however no inheritable syndrome has so far been linked with a20 abnormalities . a20 mutations are strongly linked to autoimmunity , lymphomas , and asthma [ 74 , 75 ] , highlighting important differences to cyld despite both targeting nf-b . this might be explained by different chain preference , k48 and k11 for a20 compared to the k63 and m1 chain preference showed by cyld . a20 mice fail to regulate nf-b responses , develop severe inflammation and are hypersensitive to lps or tnf leading to premature death . cell specific ablation of a20 has revealed important knowledge about the contribution of a20 to disease pathogenesis and generated very useful mouse models for several conditions like rheumatoid arthritis , lupus erythematosus , or inflammatory bowel disease . usp18 has been thoroughly studied in the context of viral responses , since it regulates protein isgylation in response to viral infection . however liu et al . also demonstrated that usp18 deficient mice are resistant to experimental autoimmune encephalomyelitis ( eae ) . this study proposes that usp18 regulates tak1-tab interaction and is hence necessary for th17 differentiation and autoimmune response . dubs can contribute to disease not only by mutations , but also by an altered expression or activity . an example of this is usp7 , whose increased activity mediates the deubiquitination and destabilization of a number of critical tumour suppressors , including p53 or pten , and is by inference an oncogenic prosurvival protein . the interrelationship between p53 , usp7 , and mdm2 ubiquitin ligase is quite unique and complex . usp7 can deubiquitinate and stabilize p53 , but interestingly it can also deubiquitinate and stabilize mdm2 indirectly leading to p53 destabilization and its degradation by the proteasome . usp7 also interacts and stabilizes the icp0 ubiquitin e3 ligase of herpes simplex virus ( hsv ) , which is required for the effective initiation of the lytic cycle , facilitating lytic viral growth . usp7 can also interact with other viral proteins , such as the ebna1 protein of the epstein - barr virus ( ebv ) and the viral interferon regulatory factor 1 ( virf1 ) of a kaposi sarcoma herpesvirus protein . in addition , and as mentioned before , usp7 plays a role by regulating nf-b signalling [ 24 , 25 ] . unfortunately usp7 mice are embryonically lethal explaining the lack of in vivo studies to further characterize the role of usp7 in immune responses and associated pathologies . given the importance of dubs in inflammatory and other pathological responses , it is certainly easy to think of dubs as potential therapeutic targets , whose modulation could be beneficial for inflammatory conditions . however , up to date there are no dub targeting compounds that have been approved for clinical use , either in the inflammatory or in cancer context . the identification and success of inhibitors that target other elements of the ubiquitin system suggest that altering inflammation by targeting the ubiquitin system , including dubs , could be a viable approach to develop novel anti - inflammatory treatments . an example of successful development of ups inhibitors has been achieved with the proteasomal inhibitors bortezomib or carfilzomib , which have been effected in multiple myeloma treatment . another compound , mln4924 ( nedd8-e1 enzyme inhibitor ) , has reached phase i clinical trials and smac mimetics , which promote proteasomal degradation of ciaps , have recently proved to work in cancer patients through phase i clinical trials . dubs present the advantage of being druggable targets since they have a catalytic domain , and unlike other ups members , such as the e3 ubiquitin ligase family with approx . given the clear evidence of the contribution of dubs to disease there is a considerable effort put into the development of compounds that modulate dub activity . intensive research is being channelled to develop selective dub inhibitors , which could be applied to such diseases like cancer , neurological and inflammatory disorders , or infectious disease . despite these intensive efforts and great advances in the dub field although no dub - selective compound has yet reached clinical trials , the field is moving fast and in the right direction . mission therapeutics is developing new dub inhibitors that present good oral bioavailability and low ec50s in cell viability assays . proteostasis therapeutics in collaboration with biogen is developing very promising usp14 inhibitor series , while genentech and almac might be developing a new therapeutic generation of usp7 inhibitors [ 86 , 87 ] . this is due to two main challenges : first not all dubs work in the same manner hence different strategies need to be followed to develop these compounds and second we do not completely understand how these enzymes function and/or are regulated . in addition , many of the studies , which address dub functions , have been developed in in vitro systems using either isolated proteins or cell lines that are not relevant to function or disease . this might not reflect the reality of dub behaviour in a tissue - specific context and more work has to be developed using in vivo mice models and primary human cells . to achieve this , new and more powerful tools are required , including in - cell based assays to discriminate selective dub function and cytotoxicity and the development of inducible mouse models , which would allow for the study of tissue - specific dub functions . it is fundamental that basic research and drug development teams work in close collaboration to allow the success of these compounds [ 86 , 87 ] . based on our actual knowledge on dubs thinking that not all dubs will be good therapeutic targets is likely , since some of them might share more than one substrate , which play opposing roles in different tissues or be essential to maintain homeostasis and health . for instance , targeting usp7 in the oncology context would be a good therapeutic strategy ; however we need to very carefully consider the possible effects of inhibiting usp7 on the inflammatory response to the tumour . similarly , we could argue that potentiating a20 function in an inflammatory context would be a plausible treatment ; however more detailed studies in the consequences of this approach are required . the presence of dubs in pathogens causative of disease , such as virus , bacteria , or parasites , has also highlighted the possibility of developing dub inhibitors , which specifically target the pathogen and not the host . in the following years new knowledge emerging from ongoing research will allow scientists to discern those that constitute good targets and offer promising new alternatives to existing therapeutics . immune responses are strongly regulated by the addition and removal of ubiquitin molecules , and although the roles of e3 ubiquitin ligases in these signalling pathways are well established , it is still unclear how dubs contribute to prr signalling . the advances in this field due to novel tools and approaches including advanced mass spectroscopic techniques , ubiquitin linkage - specific antibodies , and structural and biochemical studies will provide new insights into the regulatory mechanism of immune signalling molecules by dubs and vice versa . since the involvement of dubs in several inflammatory conditions is clear , development of potent and selective dub - specific inhibitors or agonists could provide new therapeutics to treat these conditions . for instance , given the high regulation of nod1/2 by ubiquitin and the contribution of nod mutations to inflammatory diseases such as inflammatory bowel disease ( ibd ) or crohn 's disease , it is possible that dubs could be used as a target in nod - associated inflammatory conditions . similarly to the kinase research area 20 years ago the dub field is in its infancy . there are many challenges that remain to be solved to further advance our understanding of dub function , specificity , and activity and to develop compounds that inhibit this activity . however , the field is advancing quickly , and hopefully new highly selective dub inhibitors will be developed very soon .

inflammation is a protective response of the organism to tissue injury or infection . it occurs when the immune system recognizes pathogen - associated molecular patterns ( pamps ) or damage - associated molecular pattern ( damps ) through the activation of pattern recognition receptors . this initiates a variety of signalling events that conclude in the upregulation of proinflammatory molecules , which initiate an appropriate immune response . this response is tightly regulated since any aberrant activation of immune responses would have severe pathological consequences such as sepsis or chronic inflammatory and autoimmune diseases . accumulative evidence shows that the ubiquitin system , and in particular ubiquitin - specific isopeptidases also known as deubiquitinases ( dubs ) , plays crucial roles in the control of these immune pathways . in this review we will give an up - to - date overview on the role of dubs in the nf-b pathway and inflammasome activation , two intrinsically related events triggered by activation of the membrane tlrs as well as the cytosolic nod and nlr receptors . modulation of dub activity by small molecules has been proposed as a way to control dysregulation or overactivation of these key players of the inflammatory response . we will also discuss the advances and challenges of a potential use of dubs as therapeutic targets in inflammatory pathologies .

1. Introduction 2. Mechanisms of Regulation of DUBs 3. Deubiquitination in TLR- and NLR-Mediated Immune Signalling 4. Pathogen Manipulation of DUBs to Control PRR Signalling 5. Deubiquitinases and Inflammatory Disease 6. Modulating DUB Activity as a Novel Inflammatory Therapeutic Approach 7. Concluding Remarks

ubiquitination is a posttranslational modification ( ptm ) that involves the attachment of a ubiquitin molecule ( ~9 kda ) to a target protein . it is now well accepted that most of the cellular processes required for the maintenance of the cell homeostasis are regulated by the ubiquitin - proteasome system ( ups ) , including the regulation of innate immune signalling . ubiquitination is mediated by a set of three enzymes , a ubiquitin - activating enzyme ( e1 ) , a ubiquitin - conjugating enzyme ( e2 ) , and a ubiquitin ligase ( e3 ) . the formation of ub chains , however , occurs by formation of a bond between the carboxy - terminal glycine of ub and one of the seven lysines ( k6 , k11 , k27 , k29 , k33 , k48 , and k63 ) or the methionine ( m1 ) present in the acceptor ub molecule allowing the generation of a wide variety of poly - ub chains . ubiquitination is a reversible process , and its reversibility is mediated by a family of proteases called deubiquitinases ( deubiquitinating enzymes , dubs ) . there are four thiol protease families , the ubiquitin - specific proteases ( usp ) , ubiquitin c - terminal hydrolases ( uch ) , ovarian tumour domain containing proteases ( otu ) , and machado joseph disease ( mjd)/josephin domain dubs , and one zinc - metalloprotease group , the jab1/mpn / mov34 metalloenzyme family . the main functions of dubs are ( i ) generation / release of free ubiquitin from ub precursors ( de novo ub synthesis ) , ( ii ) subtle editing of poly - ub chains , and ( iii ) removal of the poly - ub chains from substrates prior to degradation by proteasome - bound dubs . as one of the best examples of transcriptional regulation is a20 ( tnfaip3 ) , which is a member of the otu family of dubs . , in response to tlr4 activation ) , reflecting its important role as a negative regulator in the inflammatory response , as we will discuss below . there are other dubs , which are regulated in response to cytokines , including dub1 , dub2 , usp17 ( dub3 ) , and otud-6b . the presence of dubs in molecular complexes is a common way to modulate their activity . usp1 is involved in dna damage response , mainly in the fanconi anemia ( fa ) pathway where it mediates the deubiquitination of fancd2 and fanci , a crucial step for the correct function of the fa pathway [ 9 , 10 ] . additionally dub activity can be further adjusted by posttranslational modifications such as phosphorylation or ubiquitination . for instance , phosphorylation of cyld at ser418 or usp7 at ser18 led to an increase in the activities of these two dubs . in the case of cyld , this modification can be induced by lps ( lipopolysaccharide ) or tnf- ( tumour necrosis factor ) treatment and it can suppress its deubiquitinating activity on tnf receptor - associated factor 2 ( traf2 ) . one example is the production of reactive oxygen species ( ros ) generated during mitochondrial oxidative metabolism as well as in cellular responses to cytokines or bacterial invasion , which can inhibit cellular dub activity by oxidation of the catalytic cysteine residue [ 14 , 15 ] . to summarize , more than one regulatory mechanism can apply to certain dubs , which highlights the importance of a fine and multifaceted control of dub expression and activity . innate immunity is triggered in response to either pamps , which are derived from microbial pathogens , or damps such as atp , cholesterol , or monosodium urate crystals . these danger signals are recognized by pattern recognition receptors either at the cell membranes by toll - like receptors ( tlrs ) or at the cytosol by receptors such as the nod - like receptors ( nlrs ) . activation of these prr receptors results in a variety of immune signalling cascades which lead to the induction of immune mediators and proinflammatory cytokines , such as tnf or il-1 , capable of triggering appropriate immune responses . these cytokines lead to the recruitment of immune cells to the site of infection or tissue damage , which initiates an inflammatory response . tlr- and nlr - mediated signalling is heavily controlled by the ubiquitin system , which plays an essential role in maintaining the appropriate regulation of these cellular pathways . although dubs can be involved in many other inflammatory aspects , here we will discuss how dubs contribute to tlr and nlr - induced pathways , focusing on the activation of two very important and related processes , nf-b pathway and inflammasome activation . tlrs are transmembrane glycoproteins , which play a key role in the immune response against microbes . ten human tlrs have been identified to date and they either localize to the cell surface ( tlrs 1 , 2 , 4 , 5 , 6 , and 10 ) or have endosomal localization ( tlrs 3 , 7 , 8 , and 9 ) . tlr3 engagement induces the recruitment of trif and modification of traf3 with k63 poly - ub , which consequently recruits the tbk1 ( traf family member - associated nf-b activator - binding kinase)/ikk kinase complex . in contrast , tlr4 or tlr2 activation leads to the assembly of the myd88 signalling complex , recruiting traf6 , ciap1 , and ciap2 . this occurs with the help of the lubac ubiquitin ligase complex , which leads to the linear ubiquitination of nemo required for the recruitment of the ikk complex ( ikk and ikk ) . as a result , tak1 phosphorylates ikk , which in turn phosphorylates ib and subsequently undergoes ubiquitination and proteasomal degradation this allows nf-b to translocate to the nucleus from cytosol and regulate the transcription of a variety of target genes ( figure 1 ) . deubiquitination also plays a key role in tlr signalling pathways by reversing the effect of ubiquitination and controlling the intensity of the immune response ( figure 1 ) . a20 contains an otu domain , which has dub activity specific towards several nf-b signalling factors , such as traf6 , ripk1 , or nemo , which consequently leads to suppressed nf-b activation . this allows a20 to perform an editing function : in addition to removing k63-linked polyubiquitin chains from substrates such as ripk1 , a20 can introduce k48-polyubiquitin chains in the same substrate tagging it for a proteasomal degradation . by mediating usp10-dependent deubiquitination of nemo traf family member - associated nf-b activator ( tank ) interacts with both mcpip1 and usp10 , which leads to decrease in traf6 ubiquitination and the termination of the nf-b activation in response to tlr activation . in addition to the dubs described here there are several others implicated in the downregulation of the nf-b pathway upon tlr activation , although these are not well characterized . nlrs are classified into four subfamilies according to their n - terminal domains : the acidic transactivation domain ( nlra ) , the baculoviral inhibitory repeat - like domain ( nlrb ) that includes nod1 and nod2 , the caspase activation and recruitment domain ( card ; nlrc ) , and the pyrin domain ( nlrp ) . nlrs can recognize a wide variety of ligands including pathogens , endogenous molecules , or environmental factors . although ubiquitination in nlr signalling is well accepted , the role of dubs in these pathways is just emerging . upon oligomerization ripk2 this allows the recruitment of tak1/tab2/tab3 complex and lubac , which can also mediate the linear ubiquitination of ripk2 , and further contributes to the nf-b and mapk pathway activation by ubiquitination of nemo [ 30 , 31 ] . a20 also plays a regulatory role in nod2 signalling by deubiquitinating ripk2 to control the extent of the inflammatory signals . one of the dubs , which is relatively poorly characterized but which has been shown to play key functions in nod2 signalling , is otulin . this protein specifically deconjugates linear ( m1 ) poly - ub chains assembled by lubac and in this way it modulates linear ubiquitination of lubac 's substrates and provides fine - tuning of the initial activation of nf-b . the inflammasome is a molecular complex , which consists of a sensor molecule ( nlr , e.g. , nlrp1 , nlrp3 , nlrc4 , or nlrp6 ) , an adaptor protein ( asc , apoptosis - associated speck - like protein containing a card domain ) , and an effector molecule ( caspase-1 ) . the main function of the effector molecule is to induce the cleavage and activation of the proinflammatory cytokines , il-1 and il-18 . caspase-1 activation can be achieved by several k - releasing molecules , including nigericin , crystals , or extracellular atp through the activation of the atp - gated p2x7 receptor ( p2x7r ) . after the inflammasome is fully activated , it can lead to pyroptotic cell death , which can be distinguished from other cell death types by pore formation in the plasma membrane followed by osmotic cell lysis and finally the release of il-1 and il-18 . given the important role of ubiquitin in signalling cascades derived from tlr and nlr activation , it is not surprising to find that assembly and activation of an inflammasome is also regulated by the ubiquitin system . ubiquitination can regulate canonical inflammasome activation by modulation of three major components : nlr , asc , and caspase-1 . this can be exemplified by march7 , which promotes ubiquitination of nlrp3 , and this causes its degradation upon dopamine stimulation as a mean to control inflammasome activation . similarly to the nod2 receptor activation , ciap e3s are also involved in the inflammasome activation . attenuation of ciap activities , either by their deletion or by inhibition , triggers nlrp3 and caspase-1 activation as well as rip3 kinase - dependent il-1 processing and secretion . in addition to nlr and caspase-1 , ubiquitin - mediated inflammasome activation can be also promoted by modification of the adaptor protein asc . traf3 ubiquitin ligase ubiquitinates asc , and abolishment of the target lysine ( k174 ) prevents inflammasome activation and il-1 release in response to viral infection . also , traf6-mediated asc ubiquitination has been recently reported in response to far - infrared and proposed to constitute a mechanism , which dampens inflammasome activation in repair processes . interestingly asc has been identified as a substrate of hoil-1l , a member of linear ubiquitination complex lubac , and hoil deficient macrophages present an impaired inflammasome response . in line with this , macrophages deficient in sharpin , which is a different member of the lubac complex , are not able to mount an optimal inflammasome response . all this evidence reveals that ubiquitination is an essential modification for the control of the inflammasome activation . it is then logical to assume that dubs are important players of these regulatory mechanisms . , who showed that nlrp3 is ubiquitinated in resting macrophages and that , upon cell activation with priming ( lps ) and activating signals ( atp , nigericin , and msu crystals ) , these ubiquitin chains are removed by dubs , allowing activation of the complex . who identified brcc3 as the first dub to be directly involved in inflammasome activation . these reports showed that inhibition of dub activity with the dub inhibitors bap-15 , wp1130 , pr-619 , and g5 blocks nlrp3 but not nlrc4 or aim2 mediated il-1 release and pyroptosis ( figure 3 ; table 1 ) . the ability of these dub inhibitors to block inflammasome activation could explain the inhibitory effect of the compound bay 11 - 7082 on nlrp3 inflammasome independently of its nf-b inhibitory activity since this compound can inhibit components of the ubiquitin system , including dubs [ 55 , 56 ] . the other dub , which has been directly implicated in the inflammasome activation , is a20 . in contrast to brcc3 , a20 acts as a negative regulator of nlrp3 and suppresses inflammasome activation by restricting ubiquitination of il-1 and nlrp3 activation [ 57 , 58 ] . pathogens can exploit the host ubiquitin system by expressing their own ubiquitin - specific enzymes , and the host cell can up- or downregulate expression and/or activity of host dubs . first , an example of a pathogen - encoded deubiquitinase disturbing the host innate immune pathways is salmonella 's avra , which is a dub that facilitates inhibition of the nf-b pathway . also , depletion of avra in salmonella leads to significantly increased secretion of cytokine il-6 in the host cell , which is dependent on nf-b pathway [ 6063 ] . since several known bacterial dubs directly target important functions in the host immune system , development of selective inhibitors for pathogenic dubs could be exploited as a therapeutic approach in the treatment of infections . bacterial infection can induce inflammasome activation in the host cell and deubiquitination has been implicated in this process . salmonella typhimurium infection leads to changes in the activity of several host dubs , such as usp4 , usp5 , uchl3 , and uchl5 , and increased activity of uchl5 was found to contribute to the inflammasome activation during this infection . although many of the studies mentioned in this review have been performed in vitro in cell culture models , the involvement of dubs in inflammatory responses has been also studied by using animal models , highlighting the relevance of these proteases in a relevant tissue and immune context ( table 1 ) . mutations in the cyld gene lead to a subtype of the benign cancer predisposition syndrome of skin appendages also known as brooke - spiegler syndrome , although inactivation or downregulation of cyld is also observed in a variety of other cancers , including melanoma , and breast , colon , lung , breast , cervical , and , recently , prostate cancer . usp18 has been thoroughly studied in the context of viral responses , since it regulates protein isgylation in response to viral infection . an example of this is usp7 , whose increased activity mediates the deubiquitination and destabilization of a number of critical tumour suppressors , including p53 or pten , and is by inference an oncogenic prosurvival protein . usp7 also interacts and stabilizes the icp0 ubiquitin e3 ligase of herpes simplex virus ( hsv ) , which is required for the effective initiation of the lytic cycle , facilitating lytic viral growth . usp7 can also interact with other viral proteins , such as the ebna1 protein of the epstein - barr virus ( ebv ) and the viral interferon regulatory factor 1 ( virf1 ) of a kaposi sarcoma herpesvirus protein . unfortunately usp7 mice are embryonically lethal explaining the lack of in vivo studies to further characterize the role of usp7 in immune responses and associated pathologies . given the importance of dubs in inflammatory and other pathological responses , it is certainly easy to think of dubs as potential therapeutic targets , whose modulation could be beneficial for inflammatory conditions . the identification and success of inhibitors that target other elements of the ubiquitin system suggest that altering inflammation by targeting the ubiquitin system , including dubs , could be a viable approach to develop novel anti - inflammatory treatments . an example of successful development of ups inhibitors has been achieved with the proteasomal inhibitors bortezomib or carfilzomib , which have been effected in multiple myeloma treatment . dubs present the advantage of being druggable targets since they have a catalytic domain , and unlike other ups members , such as the e3 ubiquitin ligase family with approx . given the clear evidence of the contribution of dubs to disease there is a considerable effort put into the development of compounds that modulate dub activity . despite these intensive efforts and great advances in the dub field although no dub - selective compound has yet reached clinical trials , the field is moving fast and in the right direction . this might not reflect the reality of dub behaviour in a tissue - specific context and more work has to be developed using in vivo mice models and primary human cells . to achieve this , new and more powerful tools are required , including in - cell based assays to discriminate selective dub function and cytotoxicity and the development of inducible mouse models , which would allow for the study of tissue - specific dub functions . based on our actual knowledge on dubs thinking that not all dubs will be good therapeutic targets is likely , since some of them might share more than one substrate , which play opposing roles in different tissues or be essential to maintain homeostasis and health . for instance , targeting usp7 in the oncology context would be a good therapeutic strategy ; however we need to very carefully consider the possible effects of inhibiting usp7 on the inflammatory response to the tumour . the presence of dubs in pathogens causative of disease , such as virus , bacteria , or parasites , has also highlighted the possibility of developing dub inhibitors , which specifically target the pathogen and not the host . immune responses are strongly regulated by the addition and removal of ubiquitin molecules , and although the roles of e3 ubiquitin ligases in these signalling pathways are well established , it is still unclear how dubs contribute to prr signalling . the advances in this field due to novel tools and approaches including advanced mass spectroscopic techniques , ubiquitin linkage - specific antibodies , and structural and biochemical studies will provide new insights into the regulatory mechanism of immune signalling molecules by dubs and vice versa . since the involvement of dubs in several inflammatory conditions is clear , development of potent and selective dub - specific inhibitors or agonists could provide new therapeutics to treat these conditions . for instance , given the high regulation of nod1/2 by ubiquitin and the contribution of nod mutations to inflammatory diseases such as inflammatory bowel disease ( ibd ) or crohn 's disease , it is possible that dubs could be used as a target in nod - associated inflammatory conditions . there are many challenges that remain to be solved to further advance our understanding of dub function , specificity , and activity and to develop compounds that inhibit this activity .

oral candidiasis is one of the most common opportunistic infections with varying clinical manifestations that affect the oral cavity . based on previous studies , candida albicans is the most common etiologic agent for this condition ( 1 , 2 ) . research has shown a significant correlation between candida colonies in children and early carious lesions during childhood ( 3 , 4 ) . polyenes ( nystatin ) and azoles ( fluconazole ) are the most commonly - used local agents for the treatment of candidiasis ; however , due to the presence of resistant species of candida , toxicity , and insufficient bioavailability , only a small number of these agents are effective therapeutically ( 5 ) . in recent decades one of these plants with excellent therapeutic properties is lippia citriodora from the verbena family . the plant is indigenous to north america ; however , it has been planted in some european countries and in iran in regions with moderate weather conditions ( northern areas ) under greenhouse conditions due to its beneficial properties ( 7 ) . the leaves of this plant have aromatic constituents , which are used to prepare traditional tea . l. citriodora has been listed as one of the safe plants and use of its alcoholic and traditional tea forms has been considered safe ( 8) . citral ( geranial , neral ) , geraniol , lemonene , and cineol are the main ingredients of the extract of l. citriodora ( 9 ) . this plant is used in traditional medicine for the treatment of asthma , spasms , colds , colic , diarrhea , malabsorption , and anxiety ( 10 ) . evaluation of the biological activity and ingredients isolated from the extract and leaf extract of l. citriodora showed its anti - oxidative , anti - microbial , anti - inflammatory , and anti - fungal activities and analgesic effects as a result of its phenolic acid , flavonoid , and terpenoid compounds . the essential oils of l. citriodora decrease cellular lysis due to oxidative stresses , and they are a good candidate for conditions leading to neuronal degradation ( 8 , 1013 ) . the aim of the present study was to determine the inhibitory effect of the aqueous and ethanolic extracts of l. citriodora leaves on candida albicans isolated from the dorsum of the tongue of patients and the standard strain in culture media . in this in vitro study , we used the standard strain of candida albicans ( ptcc 5027 ) procured from the industrial microorganism collection center of iran and 15 c. albicans samples isolated from the dorsum of the tongue of 412-year - old children who were referred to the department of pediatric dentistry of babol university of medical sciences . the inclusion criteria consisted of no use of anti - microbial mouthwashes during the past month and no use of anti - fungal agents , antihistamines and corticosteroids , and a caries index > 4 . the samples were collected after briefing the children s parents and obtaining informed written consent forms . the samples were immediately cultured on sabouraud dextrose agar plates ( scharlau - merck , germany ) in association with chloramphenicol . after fungal growth , a part of the colony was transferred to the chromagar culture medium ( chromagar candida , paris , france ) . c. albicans species was confirmed by the color produced on the chromagar medium and formation of a germ tube in serum and production of vesicles in the cornmeal agar median ( himedia laboratories , ltd , mumbai , india ) plus 1% tween 80 . then , a suspension of 48-hour yeast colonies was prepared in the sabouraud dextrose agar with chloramphenicol medium with the use of physiologic serum . then , a standard 0.5 mcfarland concentration was prepared using the well technique with the disk placemat method . microdilutions of yeasts were prepared at 510 2.510 in each ml with the use of rpmi medium ( sigma - aldrich , germany ) . l. citrodora leaves were collected from the research garden of babol university of medical sciences in may 2015 , rinsed , dried at room temperature for 27 hours , and milled . to prepare the aqueous extract , 30 g of the dry powder of the leaves were mixed with 100 ml of water at 7080 c , which had already been boiled , and stored at room temperature for 24 hours . 1 ) and placed in an oven at 4050 c for drying . the dry weight of the material was determined . this extract was considered the aqueous extract of l. citriodora . to prepare the ethanolic extract , 75 g of the dry material and 250 ml of 95% dehydrated ethanol were poured in a beaker , and , after 48 hours , the resultant solution was filtered through no . 1 whatman paper filters and dried in a vacuum machine . sterile paper disks were immersed in the final concentrations of 1562500 g / ml of the aqueous ( dissolved in water ) and ethanolic ( dissolved in dimethyl sulfoxide , dmso ) extracts , 464 g / ml of fluconazole ( dissolved in water ) and 8128 g / ml of nystatin ( dissolved in dimethyl sulfoxide , dmso ) and then dried . the suspension prepared from c. albicans in sterile physiologic serum was spread on a plate containing sc medium using a sterile swab . five disks containing different concentrations of the aqueous and ethanolic extracts of l. citriodora , nystatin and fluconazole were placed separately in each plate . the disks were placed at a distance of 15 mm from the plate margin and 24 mm from the center of the adjacent disk . a disk with no preparation and a disk with 0.2% dmso were considered as negative controls . all of the plates were prepared in duplicate and incubated at 37 c for 48 hours . the suspension prepared from c. albicans in sterile physiologic serum was inoculated on a plate containing sabouraud dextrose agar culture medium along with chloramphenicol with the use of a sterile swab . different concentrations of the aqueous extract ( dissolved in water ) , ethanolic extract ( dissolved in dmso ) , fluconazole ( dissolved in water ) and nystatin ( dissolved in dmso ) were added to the wells . all of the plates were prepared in duplicate and incubated at 37 c for 48 hours . mic was determined with the use of clsi - m27-a3 ( clinical and laboratory standard institute ) standard technique to determine the inhibitory and lethal effect of the medication ( 15 ) . nine series of different concentrations of the extract were prepared in duplicate at the final concentrations of 195000 g / ml , 0.2564 g / ml of fluconazole , 0.5128 g / ml nystatin in a total volume of 100 l in sterile 96-well elisa plates with flat bottoms , containing 100 l of rpmi . 2.510 yeast cells / ml was added to the plates at a volume of 100 l . all of the plates were prepared in duplicate and incubated at 35 c for 48 hours . the data were analyzed using tukey s post hoc and one - way anova tests using spss 18 software , and they were expressed as the mean the standard error of the mean ( sem ) . the data obtained from the ethanolic and aqueous extracts of lemon verbena were compared with the findings of the control group fluconazole and nystatin and p < 0.05 was considered statistically significant . in this in vitro study , we used the standard strain of candida albicans ( ptcc 5027 ) procured from the industrial microorganism collection center of iran and 15 c. albicans samples isolated from the dorsum of the tongue of 412-year - old children who were referred to the department of pediatric dentistry of babol university of medical sciences . the inclusion criteria consisted of no use of anti - microbial mouthwashes during the past month and no use of anti - fungal agents , antihistamines and corticosteroids , and a caries index > 4 . the samples were collected after briefing the children s parents and obtaining informed written consent forms . the samples were immediately cultured on sabouraud dextrose agar plates ( scharlau - merck , germany ) in association with chloramphenicol . after fungal growth , a part of the colony was transferred to the chromagar culture medium ( chromagar candida , paris , france ) . c. albicans species was confirmed by the color produced on the chromagar medium and formation of a germ tube in serum and production of vesicles in the cornmeal agar median ( himedia laboratories , ltd , mumbai , india ) plus 1% tween 80 . then , a suspension of 48-hour yeast colonies was prepared in the sabouraud dextrose agar with chloramphenicol medium with the use of physiologic serum . then , a standard 0.5 mcfarland concentration was prepared using the well technique with the disk placemat method . microdilutions of yeasts were prepared at 510 2.510 in each ml with the use of rpmi medium ( sigma - aldrich , germany ) . l. citrodora leaves were collected from the research garden of babol university of medical sciences in may 2015 , rinsed , dried at room temperature for 27 hours , and milled . to prepare the aqueous extract , 30 g of the dry powder of the leaves were mixed with 100 ml of water at 7080 c , which had already been boiled , and stored at room temperature for 24 hours . 1 ) and placed in an oven at 4050 c for drying . the dry weight of the material was determined . this extract was considered the aqueous extract of l. citriodora . to prepare the ethanolic extract , 75 g of the dry material and 250 ml of 95% dehydrated ethanol were poured in a beaker , and , after 48 hours , the resultant solution was filtered through no . 1 whatman paper filters and dried in a vacuum machine . sterile paper disks were immersed in the final concentrations of 1562500 g / ml of the aqueous ( dissolved in water ) and ethanolic ( dissolved in dimethyl sulfoxide , dmso ) extracts , 464 g / ml of fluconazole ( dissolved in water ) and 8128 g / ml of nystatin ( dissolved in dimethyl sulfoxide , dmso ) and then dried . the suspension prepared from c. albicans in sterile physiologic serum was spread on a plate containing sc medium using a sterile swab . five disks containing different concentrations of the aqueous and ethanolic extracts of l. citriodora , nystatin and fluconazole were placed separately in each plate . the disks were placed at a distance of 15 mm from the plate margin and 24 mm from the center of the adjacent disk . a disk with no preparation and a disk with 0.2% dmso were considered as negative controls . all of the plates were prepared in duplicate and incubated at 37 c for 48 hours . the suspension prepared from c. albicans in sterile physiologic serum was inoculated on a plate containing sabouraud dextrose agar culture medium along with chloramphenicol with the use of a sterile swab . different concentrations of the aqueous extract ( dissolved in water ) , ethanolic extract ( dissolved in dmso ) , fluconazole ( dissolved in water ) and nystatin ( dissolved in dmso ) were added to the wells . all of the plates were prepared in duplicate and incubated at 37 c for 48 hours . mic was determined with the use of clsi - m27-a3 ( clinical and laboratory standard institute ) standard technique to determine the inhibitory and lethal effect of the medication ( 15 ) . nine series of different concentrations of the extract were prepared in duplicate at the final concentrations of 195000 g / ml , 0.2564 g / ml of fluconazole , 0.5128 g / ml nystatin in a total volume of 100 l in sterile 96-well elisa plates with flat bottoms , containing 100 l of rpmi . then , the fungal suspension at concentrations of 510 2.510 yeast cells / ml was added to the plates at a volume of 100 l . all of the plates were prepared in duplicate and incubated at 35 c for 48 hours . the data were analyzed using tukey s post hoc and one - way anova tests using spss 18 software , and they were expressed as the mean the standard error of the mean ( sem ) . the data obtained from the ethanolic and aqueous extracts of lemon verbena were compared with the findings of the control group fluconazole and nystatin and p < 0.05 was considered statistically significant . in the present in vitro study , the samples were collected from 22 volunteers as follows : 10 subjects in the 46 age group and 12 in the 712 age group , with mean dmft score of 5.5 and 8.6 respectively . of the 22 samples cultured , two were not c. albicans samples , and no fungal growth was detected in five samples . the presence of green colonies in 15 chromagar culture media indicated the growth of c. albicans ( figure 1 ) . in addition , all of these microorganisms produced germ tubes and vesicles ( chlamydoconidium ) in the cornmeal agar along with 1% tween 80 in 13 plates ( figure 2 ) . in this study , the anti - fungal activities of different concentrations of aqueous and ethanolic extracts of l. citiodora leaves against c. albicans were evaluated with the use of disk diffusion , well , and microdilution techniques in comparison to nystatin and fluconazole . in the well and disk diffusion techniques , small growth inhibitory halos were observed around a number of clinically - isolated stains ; however , no inhibitory halos were observed around the standard strains . in addition , the aqueous extract exhibited no inhibitory halos in the disk diffusion and well techniques . the diameters of growth inhibitory halos with nystatin - containing disks ( with the minimum concentration of 8 g / ml around the isolated and standard stains ) were 0.53 0.53 and 0 ; the maximum diameters of growth inhibitory halos at a concentration of 128 g / ml around clinically - isolated and standard stains increased to 16.13 0.44 and 17 in a dose - dependent manner . in addition , the growth inhibitory halos in the same order in the well technique with the minimum nystatin concentration were 0.535 0.55 and 0 , and , with the maximum concentration , they were 17.8 0.59 and 17 . the growth inhibitory halo diameters for 4 g / ml of fluconazole with the clinically isolated and standard strains in the disk diffusion technique were 0.57 0.55 and 0 , and in the well technique they were 0 and 0 . with the maximum concentration ( 64 g / ml ) , the diameters were 15.53 1.28 and 17 , and 17.33 0.36 and 18 , respectively . the mic results showed a higher inhibitory effect of ethanolic extract of l. citriodora than its aqueous extract . in addition , the standard strain exhibited higher sensitivity than the clinically isolated c. albicans strains at the concentrations evaluated . although no growth inhibitory halos were detected in the disks and wells containing the aqueous extract , in the mic technique , the growth inhibitions for the clinical and standard strains were 833 78.5 and 2500 g / ml , respectively . the mic of the ethanolic extract was 833 78.5 and 625 g / ml for the isolated and standard strains , respectively , indicating higher inhibitory potential for the ethanolic extract than the aqueous extract ( p = 0 ) . in addition , the mic of nystatin for clinical and standard strains was 10.13 1.91 and 4 , respectively , with 1.97 0.25 and 1 g / ml , respectively for fluconazole . although the mic of the ethanolic extract of l. citriodora was higher than those of nystatin and fluconazole ( p = 0 ) , this extract exhibited anti - fungal activity . the present study evaluated the anti - fungal effects of aqueous and ethanolic extracts of l. citriodora leaves on c. albicans . the results showed that 15 colonies of these fungal species were green in color in the chromagar culture medium , indicating the presence of c. albicans . in addition , all of these colonies produced germ tubes , and 13 strains produced vesicles ( chlamydoconidium ) in the cornmeal agar medium . a previous study showed the anti - oxidative activity of leaf extract of l. citriodora . in addition , the anti - microbial and anti - fungal properties of l. citriodora leaves have been attributed to aromatic and polyphenolic compounds in the leaves ( 1618 ) . in the present study , the effects of aqueous and ethanolic extracts of l. citriodora leaves were evaluated using the disk diffusion , well diffusion , and mic techniques . in the well and disk diffusion techniques , limited growth inhibition halos were produced around some clinical isolates at different concentrations of the ethanolic extract ; however , no growth inhibitory halos were observed with any concentrations of the aqueous extract . absence of growth inhibitory halos around disks containing the extract might be attributed to the fact that , in the present study , a lower concentration of the extract was used compared to previous studies . in addition , since the effective material in the well and disk diffusion technique gradually diffuses into the environment and affects the margins of the growing colonies , the fungal species in question had the opportunity to make use of its defensive mechanisms to counteract the material s toxicity through a proper technique ; however , in the micro - dilution technique , mixing of the extract with the culture medium resulted in the presence of the effective material in all the areas of the environment . therefore , it can be expected that the threshold of response of each fungal species to the anti - fungal agent will be different ( 19 ) . the mics of ethanolic extract , in the present study , on clinically isolated and standard strains were 833 78.5 and 625 g / ml , respectively , which were much lower than those in a 2005 study by oskay , which showed that the mic of methanolic extract of l. citriodora leaves on c. albicans was 6 mg / ml ( 20 ) . in the present study , inhibition occurred at a lower concentration than that in the study by oskay , which might be attributed to higher levels of polyphenols and aromatic compounds in the extract ( 13 , 17 ) . in the present study , the mics of the aqueous extracts with clinically isolated and standard strains were 4156 67.4 and 2500 g / ml respectively , indicating a lower inhibitory potential of the aqueous extract than the ethanolic extract . in a 2013 study by koohsari , the antibacterial effects of aqueous and ethanolic extracts of l. citriodora were evaluated at concentrations of 1251000 mg / ml on nine pathogenic species with the use of disk diffusion and well techniques . the results showed that the ethanolic extract exhibited an antibacterial effect ; however , the aqueous extract had no inhibitory effect on the pathogenic species . the results showed that e. faecalis , s. epidermidis , and s. aureus were the most sensitive bacteria among gram - positive bacteria and that y. enterocolitica was the most resistant gram - negative bacterial species ( 14 ) . most plant extracts have anti - microbial properties and consist of saturated organic or aromatic compounds . in most cases , ethanolic or methanolic solvents are used for their initial extraction and , typically in the majority of studies , the use of water is avoided for extraction of plant derivatives ( 21 ) . since the presence of phenolic components in plant extracts is one of the main reasons for their anti - microbial effects , it is possible that the presence of very small amounts of phenolic compounds in the aqueous extract was the reason for the low anti - microbial effect of this extract ( 17 ) . lozada , in a 2012 study , evaluated the effect of the essential oils of l. citriodora and l. origanoid on growth inhibition of moniliophthora roreri , and the results showed complete inhibition of fungal growth at concentrations of 6001000 g / ml ( 22 ) . observations in relation to the essential oils of l. citriodora showed its high anti - bacterial activity against h. pylori ( 23 ) . a 2010 study by ramzi showed that l. citriodora extract has an inhibitory effect on gram - positive bacteria , but it had no effect on candida maltosa ( 24 ) . in 2012 , ansari conducted a study with 1/2 , 1/4 , 1/8 , and 1/16 concentrations of 100l of l. citriodora extract using disk diffusion technique to determine mic against s. aureus strain resistant to methicillin . the results showed that the inhibitory effect increased with an increase in the concentration of the extract up to 55 g / ml . in addition , the mic was determined at 15 g / ml , and the results showed a high anti - microbial activity against methicillin - resistant s. aureus despite very low toxicity against cells ( 25 ) . the anti - bacterial activity of the extract might be attributed to the presence of alkaloids , flavonoids , and tannin in its chemical structure ( 23 ) . tatsadjieu evaluated the antifungal activity of lippia rugosa leaves and showed that geraniol , neral , and geranial in the extract were the agents responsible for the anti - fungal activity against aspergillus flavus ( 26 ) . a study in 2009 by royaro showed that the extract of l. citriodora leaves exhibited the highest anti - fungal activity at inhibitory concentrations of 99.21 and 65.5 g / ml against c. krusei and aspergillus fumigatus , respectively ( 22 ) . a study in 2012 by lozada showed that the essential oils of l. citriodora and l. origanoid resulted in 90% inhibition of fungal growth at a concentration of 200 g / ml ; however , 100% inhibition was achieved at concentrations in the range of 8001000 g / ml ( 27 ) . a study in 2007 by oliviera on the antibacterial activity of l. origanoid with the use of drop diffusion technique showed its inhibitory effects on gram - positive microorganisms , including s. aureus and c. albicans ( 28 ) a study in 1994 by villon and chaumont showed the antifungal activity of lippia multiflora and lippia chevalieri extracts against aspergillus flavus . they reported that trepenoids , especially citral and geraniol that are present in extract of l. citriodora , were responsible for the anti - fungal activity ( 29 ) . other anti - fungal agents are beta - caryophyllene and beta - caryophyllene oxide that belong to cisquiterpens and are found in the plant extracts of lippia rehmanii and lippia citriodora ( 30 ) . despite the complications reported in previous studies in relation to other antifungal agents , no side effects have been reported after the use of leaf extract of l. citriodora ; on the contrary , its positive protective effects on the general health have been shown ( 7 , 10 , 11 , 16 , 30 , 31 ) . the results of the present study showed that the aqueous extract , and particularly the ethanolic extract , of the leaves of l. citriodora has activity against c. albicans , which might be attributed to the higher content of aromatic agents , including essences and polyphenols , in the ethanolic extract compared to the aqueous extract . use of this extract can be suggested to volunteers , after evaluating them , as an agent against c. albicans .

introductionbecause of resistance and side effects to common antifungal drugs activity , the research on herbal substances with antifungal activity is frequent . lemon verbena ( lippia citriodora ) is a member of verbenaceae family . the aim of this study was to determine the anti - candida activities of the ethanolic and aqueous extracts of the lemon verbena leaves and compare them with nystatin and fluconazole.methodsin this 2015 study , 15 clinical isolates and standard strain of candida albicans ptcc 5027 were used , and the inhibitory effects of the ethanolic and aqueous extracts , nystatin and fluconazole , were evaluated using disk and well diffusion methods . also , the minimal inhibitory concentration ( mic ) was determined . five concentrations of aqueous and ethanolic extracts ( 1562500 g / ml ) , nystatin ( 8128 g / ml ) and fluconazole ( 464 g / ml ) were used in disk and well diffusion methods , and nine concentrations of aqueous and ethanolic extracts ( 195000 g / ml ) , nystatin ( 0.5128 g / ml ) , and fluconazole ( 0.2564 g / ml ) were applied for mic . data were analyzed using tukey s post - hoc and one - way anova tests . the significant level was considered p < 0.05 in the current study.resultsin the well and disk diffusion techniques , limited growth inhibition halos were produced around some clinical isolates at different concentrations of ethanolic extract ; however , no growth inhibitory halo was observed with any concentrations of the aqueous extract . the mic values of ethanolic extract , aqueous extract , nystatin and fluconazole for clinical isolated and standard strain were 833 78.5and 625g / ml ; 4156 67.4 and 2500 g / ml ; 10.13 1.91 and 4 g / ml ; and 1.97 0.25 and 1 g / ml , respectively.conclusionthe results showed that the ethanolic extract was stronger than the aqueous extract of this plant , which can be used as an alternative for drugs . it is recommended that the ethanolic extract of this plant be investigated in vivo for better evaluation of its efficacy and properties .

1. Introduction 2. Material and Methods 2.1. Study design and setting 2.2. Preparation of aqueous and ethanolic leaf extract of L. citriodora 2.3. Evaluation of the antifungal activity with the use of disk diffusion technique 2.4. Evaluation of the antifungal activity using the well technique 2.5. Microdilution to determine the minimum inhibitory concentration (MIC) 2.6. Statistical analysis 3. Results 4. Discussion 5. Conclusions

polyenes ( nystatin ) and azoles ( fluconazole ) are the most commonly - used local agents for the treatment of candidiasis ; however , due to the presence of resistant species of candida , toxicity , and insufficient bioavailability , only a small number of these agents are effective therapeutically ( 5 ) . the plant is indigenous to north america ; however , it has been planted in some european countries and in iran in regions with moderate weather conditions ( northern areas ) under greenhouse conditions due to its beneficial properties ( 7 ) . the leaves of this plant have aromatic constituents , which are used to prepare traditional tea . l. citriodora has been listed as one of the safe plants and use of its alcoholic and traditional tea forms has been considered safe ( 8) . citral ( geranial , neral ) , geraniol , lemonene , and cineol are the main ingredients of the extract of l. citriodora ( 9 ) . this plant is used in traditional medicine for the treatment of asthma , spasms , colds , colic , diarrhea , malabsorption , and anxiety ( 10 ) . evaluation of the biological activity and ingredients isolated from the extract and leaf extract of l. citriodora showed its anti - oxidative , anti - microbial , anti - inflammatory , and anti - fungal activities and analgesic effects as a result of its phenolic acid , flavonoid , and terpenoid compounds . the essential oils of l. citriodora decrease cellular lysis due to oxidative stresses , and they are a good candidate for conditions leading to neuronal degradation ( 8 , 1013 ) . the aim of the present study was to determine the inhibitory effect of the aqueous and ethanolic extracts of l. citriodora leaves on candida albicans isolated from the dorsum of the tongue of patients and the standard strain in culture media . in this in vitro study , we used the standard strain of candida albicans ( ptcc 5027 ) procured from the industrial microorganism collection center of iran and 15 c. albicans samples isolated from the dorsum of the tongue of 412-year - old children who were referred to the department of pediatric dentistry of babol university of medical sciences . the inclusion criteria consisted of no use of anti - microbial mouthwashes during the past month and no use of anti - fungal agents , antihistamines and corticosteroids , and a caries index > 4 . l. citrodora leaves were collected from the research garden of babol university of medical sciences in may 2015 , rinsed , dried at room temperature for 27 hours , and milled . to prepare the aqueous extract , 30 g of the dry powder of the leaves were mixed with 100 ml of water at 7080 c , which had already been boiled , and stored at room temperature for 24 hours . 1 ) and placed in an oven at 4050 c for drying . the dry weight of the material was determined . this extract was considered the aqueous extract of l. citriodora . to prepare the ethanolic extract , 75 g of the dry material and 250 ml of 95% dehydrated ethanol were poured in a beaker , and , after 48 hours , the resultant solution was filtered through no . sterile paper disks were immersed in the final concentrations of 1562500 g / ml of the aqueous ( dissolved in water ) and ethanolic ( dissolved in dimethyl sulfoxide , dmso ) extracts , 464 g / ml of fluconazole ( dissolved in water ) and 8128 g / ml of nystatin ( dissolved in dimethyl sulfoxide , dmso ) and then dried . five disks containing different concentrations of the aqueous and ethanolic extracts of l. citriodora , nystatin and fluconazole were placed separately in each plate . the disks were placed at a distance of 15 mm from the plate margin and 24 mm from the center of the adjacent disk . all of the plates were prepared in duplicate and incubated at 37 c for 48 hours . different concentrations of the aqueous extract ( dissolved in water ) , ethanolic extract ( dissolved in dmso ) , fluconazole ( dissolved in water ) and nystatin ( dissolved in dmso ) were added to the wells . mic was determined with the use of clsi - m27-a3 ( clinical and laboratory standard institute ) standard technique to determine the inhibitory and lethal effect of the medication ( 15 ) . nine series of different concentrations of the extract were prepared in duplicate at the final concentrations of 195000 g / ml , 0.2564 g / ml of fluconazole , 0.5128 g / ml nystatin in a total volume of 100 l in sterile 96-well elisa plates with flat bottoms , containing 100 l of rpmi . the data were analyzed using tukey s post hoc and one - way anova tests using spss 18 software , and they were expressed as the mean the standard error of the mean ( sem ) . the data obtained from the ethanolic and aqueous extracts of lemon verbena were compared with the findings of the control group fluconazole and nystatin and p < 0.05 was considered statistically significant . in this in vitro study , we used the standard strain of candida albicans ( ptcc 5027 ) procured from the industrial microorganism collection center of iran and 15 c. albicans samples isolated from the dorsum of the tongue of 412-year - old children who were referred to the department of pediatric dentistry of babol university of medical sciences . the inclusion criteria consisted of no use of anti - microbial mouthwashes during the past month and no use of anti - fungal agents , antihistamines and corticosteroids , and a caries index > 4 . l. citrodora leaves were collected from the research garden of babol university of medical sciences in may 2015 , rinsed , dried at room temperature for 27 hours , and milled . to prepare the aqueous extract , 30 g of the dry powder of the leaves were mixed with 100 ml of water at 7080 c , which had already been boiled , and stored at room temperature for 24 hours . the dry weight of the material was determined . this extract was considered the aqueous extract of l. citriodora . to prepare the ethanolic extract , 75 g of the dry material and 250 ml of 95% dehydrated ethanol were poured in a beaker , and , after 48 hours , the resultant solution was filtered through no . sterile paper disks were immersed in the final concentrations of 1562500 g / ml of the aqueous ( dissolved in water ) and ethanolic ( dissolved in dimethyl sulfoxide , dmso ) extracts , 464 g / ml of fluconazole ( dissolved in water ) and 8128 g / ml of nystatin ( dissolved in dimethyl sulfoxide , dmso ) and then dried . five disks containing different concentrations of the aqueous and ethanolic extracts of l. citriodora , nystatin and fluconazole were placed separately in each plate . different concentrations of the aqueous extract ( dissolved in water ) , ethanolic extract ( dissolved in dmso ) , fluconazole ( dissolved in water ) and nystatin ( dissolved in dmso ) were added to the wells . mic was determined with the use of clsi - m27-a3 ( clinical and laboratory standard institute ) standard technique to determine the inhibitory and lethal effect of the medication ( 15 ) . nine series of different concentrations of the extract were prepared in duplicate at the final concentrations of 195000 g / ml , 0.2564 g / ml of fluconazole , 0.5128 g / ml nystatin in a total volume of 100 l in sterile 96-well elisa plates with flat bottoms , containing 100 l of rpmi . then , the fungal suspension at concentrations of 510 2.510 yeast cells / ml was added to the plates at a volume of 100 l . the data were analyzed using tukey s post hoc and one - way anova tests using spss 18 software , and they were expressed as the mean the standard error of the mean ( sem ) . the data obtained from the ethanolic and aqueous extracts of lemon verbena were compared with the findings of the control group fluconazole and nystatin and p < 0.05 was considered statistically significant . in the present in vitro study , the samples were collected from 22 volunteers as follows : 10 subjects in the 46 age group and 12 in the 712 age group , with mean dmft score of 5.5 and 8.6 respectively . of the 22 samples cultured , two were not c. albicans samples , and no fungal growth was detected in five samples . in this study , the anti - fungal activities of different concentrations of aqueous and ethanolic extracts of l. citiodora leaves against c. albicans were evaluated with the use of disk diffusion , well , and microdilution techniques in comparison to nystatin and fluconazole . in the well and disk diffusion techniques , small growth inhibitory halos were observed around a number of clinically - isolated stains ; however , no inhibitory halos were observed around the standard strains . in addition , the aqueous extract exhibited no inhibitory halos in the disk diffusion and well techniques . the diameters of growth inhibitory halos with nystatin - containing disks ( with the minimum concentration of 8 g / ml around the isolated and standard stains ) were 0.53 0.53 and 0 ; the maximum diameters of growth inhibitory halos at a concentration of 128 g / ml around clinically - isolated and standard stains increased to 16.13 0.44 and 17 in a dose - dependent manner . in addition , the growth inhibitory halos in the same order in the well technique with the minimum nystatin concentration were 0.535 0.55 and 0 , and , with the maximum concentration , they were 17.8 0.59 and 17 . the growth inhibitory halo diameters for 4 g / ml of fluconazole with the clinically isolated and standard strains in the disk diffusion technique were 0.57 0.55 and 0 , and in the well technique they were 0 and 0 . with the maximum concentration ( 64 g / ml ) , the diameters were 15.53 1.28 and 17 , and 17.33 0.36 and 18 , respectively . the mic results showed a higher inhibitory effect of ethanolic extract of l. citriodora than its aqueous extract . in addition , the standard strain exhibited higher sensitivity than the clinically isolated c. albicans strains at the concentrations evaluated . although no growth inhibitory halos were detected in the disks and wells containing the aqueous extract , in the mic technique , the growth inhibitions for the clinical and standard strains were 833 78.5 and 2500 g / ml , respectively . the mic of the ethanolic extract was 833 78.5 and 625 g / ml for the isolated and standard strains , respectively , indicating higher inhibitory potential for the ethanolic extract than the aqueous extract ( p = 0 ) . in addition , the mic of nystatin for clinical and standard strains was 10.13 1.91 and 4 , respectively , with 1.97 0.25 and 1 g / ml , respectively for fluconazole . although the mic of the ethanolic extract of l. citriodora was higher than those of nystatin and fluconazole ( p = 0 ) , this extract exhibited anti - fungal activity . the present study evaluated the anti - fungal effects of aqueous and ethanolic extracts of l. citriodora leaves on c. albicans . the results showed that 15 colonies of these fungal species were green in color in the chromagar culture medium , indicating the presence of c. albicans . in addition , all of these colonies produced germ tubes , and 13 strains produced vesicles ( chlamydoconidium ) in the cornmeal agar medium . a previous study showed the anti - oxidative activity of leaf extract of l. citriodora . in addition , the anti - microbial and anti - fungal properties of l. citriodora leaves have been attributed to aromatic and polyphenolic compounds in the leaves ( 1618 ) . in the present study , the effects of aqueous and ethanolic extracts of l. citriodora leaves were evaluated using the disk diffusion , well diffusion , and mic techniques . in the well and disk diffusion techniques , limited growth inhibition halos were produced around some clinical isolates at different concentrations of the ethanolic extract ; however , no growth inhibitory halos were observed with any concentrations of the aqueous extract . absence of growth inhibitory halos around disks containing the extract might be attributed to the fact that , in the present study , a lower concentration of the extract was used compared to previous studies . in addition , since the effective material in the well and disk diffusion technique gradually diffuses into the environment and affects the margins of the growing colonies , the fungal species in question had the opportunity to make use of its defensive mechanisms to counteract the material s toxicity through a proper technique ; however , in the micro - dilution technique , mixing of the extract with the culture medium resulted in the presence of the effective material in all the areas of the environment . therefore , it can be expected that the threshold of response of each fungal species to the anti - fungal agent will be different ( 19 ) . the mics of ethanolic extract , in the present study , on clinically isolated and standard strains were 833 78.5 and 625 g / ml , respectively , which were much lower than those in a 2005 study by oskay , which showed that the mic of methanolic extract of l. citriodora leaves on c. albicans was 6 mg / ml ( 20 ) . in the present study , inhibition occurred at a lower concentration than that in the study by oskay , which might be attributed to higher levels of polyphenols and aromatic compounds in the extract ( 13 , 17 ) . in the present study , the mics of the aqueous extracts with clinically isolated and standard strains were 4156 67.4 and 2500 g / ml respectively , indicating a lower inhibitory potential of the aqueous extract than the ethanolic extract . in a 2013 study by koohsari , the antibacterial effects of aqueous and ethanolic extracts of l. citriodora were evaluated at concentrations of 1251000 mg / ml on nine pathogenic species with the use of disk diffusion and well techniques . the results showed that the ethanolic extract exhibited an antibacterial effect ; however , the aqueous extract had no inhibitory effect on the pathogenic species . the results showed that e. faecalis , s. epidermidis , and s. aureus were the most sensitive bacteria among gram - positive bacteria and that y. enterocolitica was the most resistant gram - negative bacterial species ( 14 ) . most plant extracts have anti - microbial properties and consist of saturated organic or aromatic compounds . in most cases , ethanolic or methanolic solvents are used for their initial extraction and , typically in the majority of studies , the use of water is avoided for extraction of plant derivatives ( 21 ) . since the presence of phenolic components in plant extracts is one of the main reasons for their anti - microbial effects , it is possible that the presence of very small amounts of phenolic compounds in the aqueous extract was the reason for the low anti - microbial effect of this extract ( 17 ) . lozada , in a 2012 study , evaluated the effect of the essential oils of l. citriodora and l. origanoid on growth inhibition of moniliophthora roreri , and the results showed complete inhibition of fungal growth at concentrations of 6001000 g / ml ( 22 ) . in 2012 , ansari conducted a study with 1/2 , 1/4 , 1/8 , and 1/16 concentrations of 100l of l. citriodora extract using disk diffusion technique to determine mic against s. aureus strain resistant to methicillin . the results showed that the inhibitory effect increased with an increase in the concentration of the extract up to 55 g / ml . in addition , the mic was determined at 15 g / ml , and the results showed a high anti - microbial activity against methicillin - resistant s. aureus despite very low toxicity against cells ( 25 ) . the anti - bacterial activity of the extract might be attributed to the presence of alkaloids , flavonoids , and tannin in its chemical structure ( 23 ) . tatsadjieu evaluated the antifungal activity of lippia rugosa leaves and showed that geraniol , neral , and geranial in the extract were the agents responsible for the anti - fungal activity against aspergillus flavus ( 26 ) . a study in 2009 by royaro showed that the extract of l. citriodora leaves exhibited the highest anti - fungal activity at inhibitory concentrations of 99.21 and 65.5 g / ml against c. krusei and aspergillus fumigatus , respectively ( 22 ) . a study in 2012 by lozada showed that the essential oils of l. citriodora and l. origanoid resulted in 90% inhibition of fungal growth at a concentration of 200 g / ml ; however , 100% inhibition was achieved at concentrations in the range of 8001000 g / ml ( 27 ) . a study in 2007 by oliviera on the antibacterial activity of l. origanoid with the use of drop diffusion technique showed its inhibitory effects on gram - positive microorganisms , including s. aureus and c. albicans ( 28 ) a study in 1994 by villon and chaumont showed the antifungal activity of lippia multiflora and lippia chevalieri extracts against aspergillus flavus . they reported that trepenoids , especially citral and geraniol that are present in extract of l. citriodora , were responsible for the anti - fungal activity ( 29 ) . other anti - fungal agents are beta - caryophyllene and beta - caryophyllene oxide that belong to cisquiterpens and are found in the plant extracts of lippia rehmanii and lippia citriodora ( 30 ) . despite the complications reported in previous studies in relation to other antifungal agents , no side effects have been reported after the use of leaf extract of l. citriodora ; on the contrary , its positive protective effects on the general health have been shown ( 7 , 10 , 11 , 16 , 30 , 31 ) . the results of the present study showed that the aqueous extract , and particularly the ethanolic extract , of the leaves of l. citriodora has activity against c. albicans , which might be attributed to the higher content of aromatic agents , including essences and polyphenols , in the ethanolic extract compared to the aqueous extract . use of this extract can be suggested to volunteers , after evaluating them , as an agent against c. albicans .

mental health nurses frequently care for service users with a diagnosis of borderline personality disorder ( bpd ) in both hospital and community settings . the literature suggests that bpd is the most prevalent of all personality disorders with an estimated 2 - 3% of the population meeting the diagnostic criteria . international research indicates that bpd has a higher incidence of occurrence than schizophrenia or bipolar disorder . it is estimated that between 10 percent of service users in outpatient clinical settings and 15 to 20 percent of those in inpatient psychiatric settings meet the diagnostic criteria for bpd . suicidal or self - harming behaviour is one of the core diagnostic criteria in dsm iv - tr for bpd , and management of and recovery from this personality disorder can be complex and challenging . suicide rates among those diagnosed with bpd are approximately 8 to 10 percent [ 5 , 6 ] . bpd is also characterized by service users having a pattern of unstable and intense interpersonal relationships , affective instability , poor impulse control , and self - mutilating behaviour . many professionals find these service users difficult to interact with , treat and show empathy towards , perhaps because bpd behaviours may aversely effect interpersonal relationships , including relationships with nursing staff . a number of studies have examined attitudes of nurses towards service users with bpd [ 815 ] . these studies report nurses ' perceptions of service users with bpd being powerful and destructive in their behaviours . the literature also describes the ability of persons with a bpd to split staff and display manipulative behaviour [ 7 , 11 , 15 ] . in addition , it is reported that service users with a bpd tend to evoke fewer relaxed feelings and quite a high level of aggressive feelings in staff . in contrast , nurses are more likely to respond with sad and self - critical feelings towards psychotic service users and with warm and helpful feelings towards those with neurosis . other findings suggest that nurses may perceive service users with a bpd to have a greater degree of control over the negative behaviours they display , when compared to those with other disorders [ 12 , 14 ] . over recent years there has been some focus on the lived experience of those diagnosed with bpd [ 1723 ] . women diagnosed with bpd have reported feeling that they were living with a pejorative label , with self - destructive behaviour perceived as manipulative , and having limited access to care because of this . according to the participants in one study , health care providers held preconceived and unfavourable opinions of people with bpd , and they referred to their experience as been labelled , not diagnosed . some service users have spoken about being terrified of disapproval or rejection , particularly from key professionals such as their therapist , and frequently withheld information to defend against this . this sense of being judged negatively by professionals is also reported elsewhere . in terms of living with the diagnosis , service users have described the hopelessness and misery they felt as well as the role of self - harm as a sort - term intervention employed to release pent up emotions and tensions . those with bpd also report the service being reluctant to give them the diagnosis with two participants in one study only being told their diagnosis when they were recruited for the study . the perception of service users that there is a reluctance to diagnose them with bpd is also reported elsewhere . a number of studies [ 1821 , 24 ] recommend that health professionals examine their own attitudes and beliefs surrounding self - harm and bpd and that they engage in meaningful , therapeutic dialogue with these service users . to improve the management and care outcomes for these service users , there is general consensus on the need for more training for mental health professionals [ 10 , 1214 ] . there is little published exploring the empathic interactions of psychiatric nurses towards service users with bpd . however , a recent study reports that nurses scored lower than psychiatrists and psychologists on empathy towards those with a bpd . in addition , nurses are reported to have the lowest self - ratings on empathy towards those with bpd when compared to other mental health clinicians . however , considerable work on this topic has been undertaken on the responses of nursing staff to service users diagnosed with a bpd and also the characteristics and stereotypes of service users with bpd [ 24 , 2731 ] . it is suggested that one way to measure empathy is to consider the verbally expressed empathy of the nurse , and this approach was undertaken with a group of nurses ( n = 113 ) using the staff - patient interaction response scale ( spirs ) . the spirs was developed from theoretical views of therapeutic empathy as a multiphase time - sequenced process . this view of empathy as a process was first proposed by the german philosopher edith stein ( 19171970 ) and is one that combines the philosophical , psychological , aesthetic , and the interpersonal . it is described as a three - level model of empathy where a field of tension between views on closeness and distancing in relationships is evident , and sympathy is considered part of empathy . this empathy three - phased process is dependent upon the nurse being attentive to expressed meanings and interpretations that service users attribute to their experience . therefore , mediators such as nurses ' knowledge - beliefs , service users ' age , and contextual variables such as hospital site may influence outcomes of each phase of the empathic process . the spirs focuses on phase 2 ( expressed empathy ) of the three - phase process of empathy . the scale uses the written responses to hypothetical patient stimuli to assess the expressed empathy of staff towards service users diagnosed with bpd and schizophrenia . the ten scoring categorises represent three levels of empathic care , which are ( 1 ) no care , ( 2 ) solution , and ( 3 ) affective involvement . using the spirs and semistructured interviewing , the study described here aimed to explore registered psychiatric nurses ' interactions with and empathy towards service users with a diagnosis of bpd in their care . the specific objectives of this study were to ( i ) identify common themes from an analysis of the nurses ' reported interactions with service users diagnosed with bpd and ( ii ) describe the level of empathy of rpns towards service users with bpd using the spirs . inclusion criteria for the study were that participants had to be registered psychiatric nurses with a minimum of three - year postregistration experience , working in a mental health setting for a minimum of two years , and experience of working with service users diagnosed with bpd . following ethical approval , a letter was sent to each registered psychiatric nurse meeting the inclusion criteria ( n = 31 ) working in the community mental health service inviting their participation in the study . seventeen nurses contacted the first author ( twelve females and five males ) and agreed to take part in the study . six participants worked in a psychiatric community day setting , one worked as a community psychiatric nurse ( cpn ) , and the other eleven participants worked in a community psychiatric residential setting . eleven participants had obtained a higher - qualification after registration in mental health nursing , and one participant had a masters degree . one participant had undertaken specific training on bpd after registration ( a one day workshop ) . ten participants reported that they had daily contact with a service user with a diagnosis of bpd . four reported that they had contact with service users diagnosed with bpd two or three times a week , and a further three reported that they had contact with these service users less than 5 times within a month ( these were staff working in community day settings ) . the first author has ten years postregistration experience in the discipline of psychiatric nursing and works in a community mental health setting within the service where the study was undertaken . a semistructured interview guide was used which explored participants ' interactions and experiences of caring for service users with a diagnosis of bpd . permission was given by the original authors to use the instrument . although developed as a questionnaire , the scale was used in the study here to describe the level of empathy for service users with bpd expressed by the participants . the participants were asked to provide their typical response to the scenarios posed in the spirs ( boxes 1 and 2 ) . data analysis incorporated two methods of thematic analysis [ 36 , 37 ] for the responses to the open questioning . a deductive approach was employed to explore the level of empathy expressed by participants to the questions asked using the spirs . following analysis , all the participants were asked to read their transcribed interview for accuracy and to ascertain if the identified themes accurately reflected their views . when returning to the participants after analysis of the data , some participants expressed surprise and concern regarding their level of empathy . two participants requested for a line to be removed because they were uncomfortable with their responses . following thematic analysis of the open questioning , four themes were identified : ( 1 ) challenging and difficult , ( 2 ) manipulative , destructive and threatening behaviour , ( 3 ) preying on the vulnerable resulting in splitting staff and other service users , and ( 4 ) boundaries and structure . the majority of nurses ' responses to the scenarios presented in the spirs were at level one ( no care ) and level two ( solution ) empathy . the theme challenging and difficult reflected the participants ' description of their experiences and attitudes in care delivery to service users with a bpd . good level of care to those with bpd due to previous experiences with other service users similarly diagnosed . this is demonstrated in the following viewpoints from helen : it is difficult because you try to be open and non judgmental and give them the opportunity to take responsibility for their actions and improve their circumstances but they usually sabotage things and take no responsibility blaming others or life events for things not working out . it is difficult because you try to be open and non judgmental and give them the opportunity to take responsibility for their actions and improve their circumstances but they usually sabotage things and take no responsibility blaming others or life events for things not working out . jane expressed similar views as helen , saying that persons with a bpd are : totally difficult patient to manage .. how would i say that .they are totally self obsessed . totally difficult patient to manage .. how would i say that .they are totally self obsessed . .manipulating you and also they always seem to exaggerate their feeling . many of participants ' comments were related to the inappropriate behaviour or symptoms that those with bpd display . yvonne described service users with a bpd as.difficult to deal with a lot of behavioural problems . symptom focused approach to care and this was distracting attention from the true emotional or psychological difficulties of service users with a bpd . while the majority of participants expressed the view that it was not the service user 's personally but their behaviour that they found challenging ; most also acknowledged that service users with a bpd do not take responsibility for their behaviour . people with attention seeking behaviour and a lot of the time they have unresolved issues and they largely take this out on everyone else ( john ) . they usually refuse to accept the diagnosis alone without stating there are other diagnoses such as an underlying mood disorder to which again they can contribute blame for their actions . ( helen ) . participants also commented on the attention seeking behaviour of service users with bpd : sometimes they are trying to be on your right side all of the time saying the right things and looking for attention . sometimes they are trying to be on your right side all of the time saying the right things and looking for attention . ( mary ) . they can give a staff member .. what i call hedgehog syndrome you know they all come out with the same kind of stuff but you know maybe different ways .. ( emma ) . they can give a staff member .. what i call hedgehog syndrome oh here we go here comes another bpd you know they all come out with the same kind of stuff but you know maybe different ways .. ( emma ) . two participants stated that they would avoid providing a service user with bpd any level of care or just a minimal level . furthermore , they stated that they would avoid any interaction with the service users with bpd until it was completely necessary and they would do this at the end of the day where they knew that there would be no time to explore the issues in depth . emma 's views , however , were more questioning than other participants : they do not present with bpd ; they present with anxiety or an emotional disorder than any thing else .. or maybe after any episodes of self harm or maybe referred in from a&e after an over dose .. so .. em .. to describe them em they can be a challenging group of clients .. however they are all individual and i suppose you do get a glance of the real person underneath all that once the crisis has passed a bit . i suppose that .. they arrive at the service and that they have a diagnosis of bpd it is like they have a red light flashing .. on them and i suppose .. whatever that brings up for the health professional in question and i suppose , it always brings up your association with previous clients that you have had with bpd . they do not present with bpd ; they present with anxiety or an emotional disorder than any thing else .. or maybe after any episodes of self harm or maybe referred in from a&e after an over dose .. so .. em .. to describe them em they can be a challenging group of clients .. however they are all individual and i suppose you do get a glance of the real person underneath all that once the crisis has passed a bit . i suppose that .. they arrive at the service and that they have a diagnosis of bpd it is like they have a red light flashing .. on them and i suppose .. whatever that brings up for the health professional in question and i suppose , it always brings up your association with previous clients that you have had with bpd . similarly lorraine stated that she gained a better insight into herself after her encounters with service users with bpd : that i am not saying that they are easy to deal with but you have to have a good level of understanding and insight about yourself .. you know that you are not reacting to people all the time . .. you have to realise that it is not about you it is about them , . that i am not saying that they are easy to deal with but you have to have a good level of understanding and insight about yourself .. you know that you are not reacting to people all the time . you know if someone says they are going to kill themselves .. you have to realise that it is not about you it is about them , . so you will have to realise i can i help them . the theme manipulative , destructive and threatening behaviour reflected the views of all participants who used the terms manipulative , destructive , and threatening . all the participants associated manipulation with the idea that all service users with a bpd have a hidden agenda and they try to find out the real agenda behind their actions . helen describes her interactions with service users who have a bpd as superficial and calculated in order to get their needs met by staff . a few participants referred to the term manipulation because they found some service users with bpd to be dishonest and not genuine in nature . most participants admitted that being manipulated brought about feelings of frustration and being used . some can be manipulating in behaviour , like playing one nurse off another by making a request to one nurse which is turned down due to policies / care plan in place and then telling another nurse that this request was granted in the first place well i suppose you just feel so used when they do this to you as they have used you to get what they want and when that happens you sometimes .. well .. em .. i suppose you might question your profession ? which leaves you feeling so frustrated as they rope you in the term threatening was used as an umbrella term to describe self - harm or threats of causing harm to other people or property if their needs are not meet . fifteen participants reported that they found the threat of suicide as the most distressing of all behaviours . well in any treat of self harm is stressful on the nurse and patient but i mean has to be taken seriously regardless if they have a diagnosis of bpd ( well in any treat of self harm is stressful on the nurse and patient but i mean has to be taken seriously regardless if they have a diagnosis of bpd ( emma ) . you give them time , support and encouragement and in turn they usually continue with behaviours such as deliberate self harm , threatening suicide or absconding . it 's difficult to build a therapeutic rapport as their behaviours and actions are manipulative and attention seeking ( helen ) . you give them time , support and encouragement and in turn they usually continue with behaviours such as deliberate self harm , threatening suicide or absconding . it 's difficult to build a therapeutic rapport as their behaviours and actions are manipulative and attention seeking ( helen ) . mary described an incident where a service user with a bpd engaged in this type of behaviour and how it caused her personal stress : someone with bpd was having an argument with another patient . they totally am ignored me first of all then they decided to turn all their anger and aggression on to me and i ended up am pinned up against the wall i then .. set off the alarm .. the personal alarm the more staff that came to help the bpd person enjoyed it more seem to enjoy it more got more aggressive and angry and .. acted and play out . the whole situation was a huge learning curve for me and took me a long time to de - stress ! they totally am ignored me first of all then they decided to turn all their anger and aggression on to me and i ended up am pinned up against the wall i then .. set off the alarm .. the personal alarm the more staff that came to help the bpd person enjoyed it more seem to enjoy it more got more aggressive and angry and .. acted and play out . the whole situation was a huge learning curve for me and took me a long time to de - stress ! mary also reported that she had received professional debriefing after this incident . the theme preying on the vulnerable resulting in splitting staff and other service users reflected participants ' experiences of interactions between service users with a bpd and staff and other service users . em .. they will pick out one weaker one .. get closer to them initiate relationships which can be inappropriate at times . staff splitting all the time ( jane ) . em .. they will pick out one weaker one .. get closer to them initiate relationships which can be inappropriate at times . they usually lean on people that they believe that are vulnerable and weak be it staff or patients and they play one off the other again .. you know giving conflicting reports and thoughts ( john ) . they usually lean on people that they believe that are vulnerable and weak be it staff or patients and they play one off the other again .. you know giving conflicting reports and thoughts ( john ) . mary described service users with a bpd interactions with other service users : well at the beginning the other patients believe that the bpd patient is very nice , pleasant , very helpful , getting involved in their care giving advice .. telling them what they should and should not do . and then suddenly there will be a big bust up .. fighting and arguing and not getting on splitting of patients and different groups . well at the beginning the other patients believe that the bpd patient is very nice , pleasant , very helpful , getting involved in their care giving advice .. telling them what they should and should not do . and then suddenly there will be a big bust up .. fighting and arguing and not getting on splitting of patients and different groups . this brought about tension and stress within staff members : you will always know who is on duty because the two bpd will not leave the bloody office because they know they can get what they want from x .. when they are on .. this is so frustrating because what is the point of a plan when they just give into the demands ( bernie ) . you will always know who is on duty because the two bpd will not leave the bloody office because they know they can get what they want from x .. when they are on .. this is so frustrating because what is the point of a plan when they just give into the demands lorraine reported a feeling of anger , frustration , hurt and disappointment when she described an incident where a staff member had given out to her for doing a task for a particular service user with bpd : i am not weak , how dare they think i am i am not weak , how dare they think i am i was just doing my job and sticking to the plan . two participants reported a feeling of paranoia and feeling of mistrust due to service users with a bpd telling them that the opposite shift was giving out about them . they spoke about how it was difficult to confront a colleague about the accusation made . also they spoke about how they became distrustful of the service user with a bpd and their colleagues . the final theme of boundaries and structure referred to participants ' need for strict boundaries and firm limit setting when interacting with these service users . at the beginning you can be drawn in or sucked in by someone with bpd and it is only from experience and from dealing with people that you find out that you have to be very strong and that all the staff will have to have a set programme and everyone has to follow that programme ( mary ) . at the beginning you can be drawn in or sucked in by someone with bpd and it is only from experience and from dealing with people that you find out that you have to be very strong and that all the staff will have to have a set programme and everyone has to follow that programme ( mary ) . emma , however , explored reasons why service users with a bpd have no boundaries . well certainly with the bpd they have poor em uh ego control they have no sense of boundaries .. bpd they are a high percentage of them would be subjected to some sort of abuse .. so they do n't know their own boundaries so therefore they kinda very much infringe in another people 's boundaries .. and they do n't know they have no concept of themselves like you know ! they do n't know where their problems end and someone else 's problems start . so when they are upset they expect you to be equally upset they do n't empathise very well and they do n't when you give them empathy they can see that this is very dismissive they are looking for a lot of sympathy ! ( emma ) . well certainly with the bpd they have poor em uh ego control they have no sense of boundaries .. bpd they are a high percentage of them would be subjected to some sort of abuse .. so they do n't know their own boundaries so therefore they kinda very much infringe in another people 's boundaries .. and they do n't know they have no concept of themselves like you know ! they do n't know where their problems end and someone else 's problems start . they do n't empathise very well and they do n't when you give them empathy they can see that this is very dismissive they are looking for a lot of sympathy ! ( emma ) . some participants spoke about the safety of other service users if service users with a bpd were in the unit for a long admission . lorraine commented on one particular experience and how distressing it was for staff and other service users : when they are in hospital for ages , they teach other service users harmful behaviours i saw one bpd teaching a young girl of 18 how to cut herself so she could get attention .. how was that benefiting either of them ? we separated them , put a strict rule in place but that just seemed to make them want to get together more ! all staff at some point gave up with them .. it was like banging your head off a wall ! ! when they are in hospital for ages , they teach other service users harmful behaviours i saw one bpd teaching a young girl of 18 how to cut herself so she could get attention .. how was that benefiting either of them ? we separated them , put a strict rule in place but that just seemed to make them want to get together more ! all staff at some point gave up with them .. it was like banging your head off a wall ! ! analysis of the participants ' responses to the scenarios presented to them in the spirs ( boxes 1 and 2 ) involved scoring their responses on a ten - category response scale . the scale represents an ascending hierarchy of expressed empathy : ( 1 ) no care , ( 2 ) solution , and ( 3 ) affective involvement ( box 3 ) . the majority of participants ' responses to the questions asked in the spirs scenario regarding a first admission ( box 1 ) offered responses categorised as level one ( no care ) and level two ( offer solution ) empathy . a typical category 3 response which explains why rules or processes take place each day is represented by rebecca 's statement : i would explain to her that we work as a team and am .. that there is primary nursing and that you will be assigned a nurse each day so that they will give you time so you can talk about your worries and concerns i would explain to her that we work as a team and am .. that there is primary nursing and that you will be assigned a nurse each day so that they will give you time so you can talk about your worries and concerns emma was the only participant whose responses reflected level three empathy to the questions posed in the first scenario . emma responded by addressing the self - esteem of the girl : well what it means to her for me to be listening to her .. so i would look at it what does it mean for her to be listened to .. and what is her association with that you know .. when before did she ever feel listened to well what it means to her for me to be listening to her .. so i would look at it what does it mean for her to be listened to .. and what is her association with that you know .. when before did she ever feel listened to i would take that as a precursor to someone being angry and i would certainly ask them their feelings .. and go through it with them ( emma ) . i would take that as a precursor to someone being angry and i would certainly ask them their feelings .. and go through it with them ( emma ) . participants ' responses to the second scenario ( multiple admissions ) presented to them were mostly in the level one empathy . for instance , jane responded by telling the service user about the rules : i am the nurse on duty and these are the rules again limits are set.if you are her key worker then you will deal with her appropriately again limits are set.if you are her key worker then you will deal with her appropriately participants explained that their response to the service user was prompted by their view that he was displaying attention seeking behaviour . emma was the only participant whose responses reflected empathy at level three ( affective involvement ) . invited care and concern : maybe they did n't want to get dressed . i would ask them to sit out anywhere but the bedroom and i would make a cup of tea for them and talk to them ( emma ) . maybe they did n't want to get dressed . i would ask them to sit out anywhere but the bedroom and i would make a cup of tea for them and talk to them ( emma ) . the majority of participants offered responses in category 2 ( platitudes , clichs , or rules ) . a typical category 2 response is where the nurse tells the service user about the rules as jane did when she answered they need strict guidelines this is your plan for the day . the participants scored the highest level of empathy for the last statement asked in both scenarios ( i wish staff would just let me kill myself the majority of responses invited exploration with the service users in response to these statements . the participants stated that they would respond in this way because any threat of self - harm has to be taken seriously regardless if they have a diagnosis of bpd or not . nine participants did not change their overall level of empathy from the first scenario ( first admission : box 1 ) to the second scenario ( multiple admissions : box 2 ) ( table 1 ) . in harmony with the existing literature , the participants of this study perceived service users with bpd in a negative manner [ 11 , 15 ] . as discussed earlier , these service users are challenging and difficult to deliver care to [ 8 , 11 , 15 , 38 , 39 ] , and this theme was also echoed by the participants of this study . nurses ' feelings of being used and devalued in their experiences are also reported elsewhere . the term honeymoon to chaos stage is used to describe nurses ' experience of caring for those with bpd in which at the initial stage of treatment it is like a honeymoon where the relationships between service users and nurses were peaceful as they were getting to know each other . however , this is for a brief time frame only and is followed by the chaos stage where service users begin to demonstrate various disruptive behaviours , annoying nurses . nurses admit feeling tempted to abandon positive expectations for care outcomes at this chaos stage . they also report both positive and negative care expectations as well as both positive and negative care outcomes . similar to the findings in this study , where some nurses admitted providing minimal care and ignoring or avoiding service users with bpd , it is also reported elsewhere that some nurses withdraw and distance themselves from service users with bpd . the level of care provided by nurses to service users with bpd is also questioned . the level of empathy expressed by the study participants for the first admission of a service user with bpd was categorised overall at level 2 ( offer solution ) . with the scenario of multiple admissions , the overall score was lower , categorised at a level one score ( no care ) . these findings are similar to those reported elsewhere where the spirs was used , and nurses displayed a low level of empathy for service users with bpd in comparison for those with schizophrenia . participants who expressed level 1 empathy responses may believe this response is needed because they believe the behaviour of the service user bpd is manipulative or dangerous . it is reported that nurses believe it is socially acceptable to respond to the service user with bpd this way because of their diagnosis . these include feelings of helplessness , being used , therapeutic failure , devalued and unappreciated , anger , and frustration [ 11 , 15 ] . therefore , this level of response may be a defence mechanism against the intense affect generated in nurses by service users with bpd . the interview data in this study reveals that participants have a clear understanding of the characteristics of service users with bpd . almost collectively they agreed that those with bpd were difficult and challenging to provide care for . the majority of participants also believed that service users with bpd should not be cared for in a hospital environment because the services are inadequate and this has a negative impact on care delivery . the development of a specialist community service and improved education and skills training workshops were viewed by the participants as being the most probable way to improve the service and care received by service users with bpd . these service users ' behaviours are complex and arise from many causes , biological , psychodynamic , and sociocultural , which all work together to create the behaviours . therefore , nurses need to understand both the origins and the functions of these problems . the more the nurses understand the complexity of bpd , the more likely a higher level of empathy may be displayed . therefore , it may become less demanding for the nurses to respond therapeutically and consistently without anger , frustration , and fear of service users with bpd . a number of suggestions have been proposed about the content of such training ; for example , one study recommended cognitive behavioural therapy because of its perceived evidence base . the development and delivery of a brief training workshop on bpd for public mental health clinicians is reported in . the aim of the workshop was for participants to develop practical skills in carrying out treatment plans and to become more positive about working with service users with a diagnosis of bpd . it was thought that this would also improve relationships between service users with bpd and nurses by helping nurses to overcome negative perceptions that they associate with these service users . after the workshop , the participants ' confidence and willingness to work with people with bpd had statistically improved . it was also reported that the workshop provided a forum to improve understanding of the challenges and complexities faced in different work settings . practice guidelines set out also recommend supervision for staff caring for individuals with bpd . with appropriate training and support , nurses can be educated about realistic expectations of treatment outcomes to counter later pessimism that may arise . addressing these issues can modify negative nursing responses and help alleviate negative working experiences with service users with bpd . nurses need to receive regular supervision ; this will provide them the opportunity and space to process any perceived unpleasant experiences generated from caring for service users with bpd and help prevent burnout . in conclusion , a better understanding of the complexity of bpd may help nurses to respond therapeutically and consistently without anger , frustration , and fear . service users with bpd symbolise a challenge to nurses ; however , with improved education , training , and clinical supervision , a new era in bpd treatment can begin . nurses who view these service users in a holistic manner can frame research and practice in a way that can positively affect these service users ' lives [ 21 , 44 ] . nurses who hold positive attitudes about service users , have a sense of moral commitment , are skilled interpersonally , and are able to stay in a rational state of mind in the midst of conflict and can apply knowledge about the personality disorder and work skillfully with these service users . although the study findings add further evidence to suggest the need for training and education for nurses caring for service users with bpd , the study findings are limited for a number of reasons . the participants all work in one mental health service area and including nurses from other mental health service areas may have yielded different findings . the sample was self - selecting , and those who came forward for interview may have had a greater interest in bpd than those who did not . moreover , it is possible that the views of those who did not choose to take part in the study may differ from those who were interviewed . finally , social desirable answering

this study explored registered psychiatric nurses ' ( rpns ' ) interactions and level of empathy towards service users with a diagnosis of borderline personality disorder ( bpd ) . a qualitative approach was used , and 17 rpns were interviewed using a semistructured interview schedule incorporating the staff - patient interaction response scale ( spirs ) . four themes emerged following data analysis : challenging and difficult , manipulative , destructive and threatening behaviour , preying on the vulnerable resulting in splitting staff and other service users , and boundaries and structure . additionally , low levels of empathy were evident in the majority of participants ' responses to the spirs . the findings provide further insight on nurses ' empathy responses and views on caring for service users with bpd and further evidence for the need for training and education for nurses in the care of service users diagnosed with bpd .

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

mental health nurses frequently care for service users with a diagnosis of borderline personality disorder ( bpd ) in both hospital and community settings . it is estimated that between 10 percent of service users in outpatient clinical settings and 15 to 20 percent of those in inpatient psychiatric settings meet the diagnostic criteria for bpd . suicidal or self - harming behaviour is one of the core diagnostic criteria in dsm iv - tr for bpd , and management of and recovery from this personality disorder can be complex and challenging . suicide rates among those diagnosed with bpd are approximately 8 to 10 percent [ 5 , 6 ] . bpd is also characterized by service users having a pattern of unstable and intense interpersonal relationships , affective instability , poor impulse control , and self - mutilating behaviour . many professionals find these service users difficult to interact with , treat and show empathy towards , perhaps because bpd behaviours may aversely effect interpersonal relationships , including relationships with nursing staff . a number of studies have examined attitudes of nurses towards service users with bpd [ 815 ] . these studies report nurses ' perceptions of service users with bpd being powerful and destructive in their behaviours . the literature also describes the ability of persons with a bpd to split staff and display manipulative behaviour [ 7 , 11 , 15 ] . in addition , it is reported that service users with a bpd tend to evoke fewer relaxed feelings and quite a high level of aggressive feelings in staff . other findings suggest that nurses may perceive service users with a bpd to have a greater degree of control over the negative behaviours they display , when compared to those with other disorders [ 12 , 14 ] . over recent years there has been some focus on the lived experience of those diagnosed with bpd [ 1723 ] . women diagnosed with bpd have reported feeling that they were living with a pejorative label , with self - destructive behaviour perceived as manipulative , and having limited access to care because of this . according to the participants in one study , health care providers held preconceived and unfavourable opinions of people with bpd , and they referred to their experience as been labelled , not diagnosed . some service users have spoken about being terrified of disapproval or rejection , particularly from key professionals such as their therapist , and frequently withheld information to defend against this . those with bpd also report the service being reluctant to give them the diagnosis with two participants in one study only being told their diagnosis when they were recruited for the study . the perception of service users that there is a reluctance to diagnose them with bpd is also reported elsewhere . a number of studies [ 1821 , 24 ] recommend that health professionals examine their own attitudes and beliefs surrounding self - harm and bpd and that they engage in meaningful , therapeutic dialogue with these service users . to improve the management and care outcomes for these service users , there is general consensus on the need for more training for mental health professionals [ 10 , 1214 ] . there is little published exploring the empathic interactions of psychiatric nurses towards service users with bpd . however , a recent study reports that nurses scored lower than psychiatrists and psychologists on empathy towards those with a bpd . in addition , nurses are reported to have the lowest self - ratings on empathy towards those with bpd when compared to other mental health clinicians . however , considerable work on this topic has been undertaken on the responses of nursing staff to service users diagnosed with a bpd and also the characteristics and stereotypes of service users with bpd [ 24 , 2731 ] . it is suggested that one way to measure empathy is to consider the verbally expressed empathy of the nurse , and this approach was undertaken with a group of nurses ( n = 113 ) using the staff - patient interaction response scale ( spirs ) . this view of empathy as a process was first proposed by the german philosopher edith stein ( 19171970 ) and is one that combines the philosophical , psychological , aesthetic , and the interpersonal . it is described as a three - level model of empathy where a field of tension between views on closeness and distancing in relationships is evident , and sympathy is considered part of empathy . therefore , mediators such as nurses ' knowledge - beliefs , service users ' age , and contextual variables such as hospital site may influence outcomes of each phase of the empathic process . the spirs focuses on phase 2 ( expressed empathy ) of the three - phase process of empathy . the scale uses the written responses to hypothetical patient stimuli to assess the expressed empathy of staff towards service users diagnosed with bpd and schizophrenia . the ten scoring categorises represent three levels of empathic care , which are ( 1 ) no care , ( 2 ) solution , and ( 3 ) affective involvement . using the spirs and semistructured interviewing , the study described here aimed to explore registered psychiatric nurses ' interactions with and empathy towards service users with a diagnosis of bpd in their care . the specific objectives of this study were to ( i ) identify common themes from an analysis of the nurses ' reported interactions with service users diagnosed with bpd and ( ii ) describe the level of empathy of rpns towards service users with bpd using the spirs . inclusion criteria for the study were that participants had to be registered psychiatric nurses with a minimum of three - year postregistration experience , working in a mental health setting for a minimum of two years , and experience of working with service users diagnosed with bpd . following ethical approval , a letter was sent to each registered psychiatric nurse meeting the inclusion criteria ( n = 31 ) working in the community mental health service inviting their participation in the study . ten participants reported that they had daily contact with a service user with a diagnosis of bpd . four reported that they had contact with service users diagnosed with bpd two or three times a week , and a further three reported that they had contact with these service users less than 5 times within a month ( these were staff working in community day settings ) . a semistructured interview guide was used which explored participants ' interactions and experiences of caring for service users with a diagnosis of bpd . although developed as a questionnaire , the scale was used in the study here to describe the level of empathy for service users with bpd expressed by the participants . the participants were asked to provide their typical response to the scenarios posed in the spirs ( boxes 1 and 2 ) . data analysis incorporated two methods of thematic analysis [ 36 , 37 ] for the responses to the open questioning . a deductive approach was employed to explore the level of empathy expressed by participants to the questions asked using the spirs . when returning to the participants after analysis of the data , some participants expressed surprise and concern regarding their level of empathy . following thematic analysis of the open questioning , four themes were identified : ( 1 ) challenging and difficult , ( 2 ) manipulative , destructive and threatening behaviour , ( 3 ) preying on the vulnerable resulting in splitting staff and other service users , and ( 4 ) boundaries and structure . the majority of nurses ' responses to the scenarios presented in the spirs were at level one ( no care ) and level two ( solution ) empathy . the theme challenging and difficult reflected the participants ' description of their experiences and attitudes in care delivery to service users with a bpd . good level of care to those with bpd due to previous experiences with other service users similarly diagnosed . many of participants ' comments were related to the inappropriate behaviour or symptoms that those with bpd display . yvonne described service users with a bpd as.difficult to deal with a lot of behavioural problems . symptom focused approach to care and this was distracting attention from the true emotional or psychological difficulties of service users with a bpd . while the majority of participants expressed the view that it was not the service user 's personally but their behaviour that they found challenging ; most also acknowledged that service users with a bpd do not take responsibility for their behaviour . participants also commented on the attention seeking behaviour of service users with bpd : sometimes they are trying to be on your right side all of the time saying the right things and looking for attention . two participants stated that they would avoid providing a service user with bpd any level of care or just a minimal level . furthermore , they stated that they would avoid any interaction with the service users with bpd until it was completely necessary and they would do this at the end of the day where they knew that there would be no time to explore the issues in depth . i suppose that .. they arrive at the service and that they have a diagnosis of bpd it is like they have a red light flashing .. on them and i suppose .. whatever that brings up for the health professional in question and i suppose , it always brings up your association with previous clients that you have had with bpd . i suppose that .. they arrive at the service and that they have a diagnosis of bpd it is like they have a red light flashing .. on them and i suppose .. whatever that brings up for the health professional in question and i suppose , it always brings up your association with previous clients that you have had with bpd . similarly lorraine stated that she gained a better insight into herself after her encounters with service users with bpd : that i am not saying that they are easy to deal with but you have to have a good level of understanding and insight about yourself .. you know that you are not reacting to people all the time . the theme manipulative , destructive and threatening behaviour reflected the views of all participants who used the terms manipulative , destructive , and threatening . all the participants associated manipulation with the idea that all service users with a bpd have a hidden agenda and they try to find out the real agenda behind their actions . a few participants referred to the term manipulation because they found some service users with bpd to be dishonest and not genuine in nature . some can be manipulating in behaviour , like playing one nurse off another by making a request to one nurse which is turned down due to policies / care plan in place and then telling another nurse that this request was granted in the first place well i suppose you just feel so used when they do this to you as they have used you to get what they want and when that happens you sometimes .. well .. em .. i suppose you might question your profession ? which leaves you feeling so frustrated as they rope you in the term threatening was used as an umbrella term to describe self - harm or threats of causing harm to other people or property if their needs are not meet . well in any treat of self harm is stressful on the nurse and patient but i mean has to be taken seriously regardless if they have a diagnosis of bpd ( well in any treat of self harm is stressful on the nurse and patient but i mean has to be taken seriously regardless if they have a diagnosis of bpd ( emma ) . mary described an incident where a service user with a bpd engaged in this type of behaviour and how it caused her personal stress : someone with bpd was having an argument with another patient . the theme preying on the vulnerable resulting in splitting staff and other service users reflected participants ' experiences of interactions between service users with a bpd and staff and other service users . mary described service users with a bpd interactions with other service users : well at the beginning the other patients believe that the bpd patient is very nice , pleasant , very helpful , getting involved in their care giving advice .. telling them what they should and should not do . you will always know who is on duty because the two bpd will not leave the bloody office because they know they can get what they want from x .. when they are on .. this is so frustrating because what is the point of a plan when they just give into the demands lorraine reported a feeling of anger , frustration , hurt and disappointment when she described an incident where a staff member had given out to her for doing a task for a particular service user with bpd : i am not weak , how dare they think i am i am not weak , how dare they think i am i was just doing my job and sticking to the plan . two participants reported a feeling of paranoia and feeling of mistrust due to service users with a bpd telling them that the opposite shift was giving out about them . also they spoke about how they became distrustful of the service user with a bpd and their colleagues . the final theme of boundaries and structure referred to participants ' need for strict boundaries and firm limit setting when interacting with these service users . at the beginning you can be drawn in or sucked in by someone with bpd and it is only from experience and from dealing with people that you find out that you have to be very strong and that all the staff will have to have a set programme and everyone has to follow that programme ( mary ) . at the beginning you can be drawn in or sucked in by someone with bpd and it is only from experience and from dealing with people that you find out that you have to be very strong and that all the staff will have to have a set programme and everyone has to follow that programme ( mary ) . emma , however , explored reasons why service users with a bpd have no boundaries . some participants spoke about the safety of other service users if service users with a bpd were in the unit for a long admission . lorraine commented on one particular experience and how distressing it was for staff and other service users : when they are in hospital for ages , they teach other service users harmful behaviours i saw one bpd teaching a young girl of 18 how to cut herself so she could get attention .. how was that benefiting either of them ? when they are in hospital for ages , they teach other service users harmful behaviours i saw one bpd teaching a young girl of 18 how to cut herself so she could get attention .. how was that benefiting either of them ? analysis of the participants ' responses to the scenarios presented to them in the spirs ( boxes 1 and 2 ) involved scoring their responses on a ten - category response scale . the majority of participants ' responses to the questions asked in the spirs scenario regarding a first admission ( box 1 ) offered responses categorised as level one ( no care ) and level two ( offer solution ) empathy . a typical category 3 response which explains why rules or processes take place each day is represented by rebecca 's statement : i would explain to her that we work as a team and am .. that there is primary nursing and that you will be assigned a nurse each day so that they will give you time so you can talk about your worries and concerns i would explain to her that we work as a team and am .. that there is primary nursing and that you will be assigned a nurse each day so that they will give you time so you can talk about your worries and concerns emma was the only participant whose responses reflected level three empathy to the questions posed in the first scenario . participants ' responses to the second scenario ( multiple admissions ) presented to them were mostly in the level one empathy . the majority of participants offered responses in category 2 ( platitudes , clichs , or rules ) . the participants scored the highest level of empathy for the last statement asked in both scenarios ( i wish staff would just let me kill myself the majority of responses invited exploration with the service users in response to these statements . the participants stated that they would respond in this way because any threat of self - harm has to be taken seriously regardless if they have a diagnosis of bpd or not . nine participants did not change their overall level of empathy from the first scenario ( first admission : box 1 ) to the second scenario ( multiple admissions : box 2 ) ( table 1 ) . in harmony with the existing literature , the participants of this study perceived service users with bpd in a negative manner [ 11 , 15 ] . as discussed earlier , these service users are challenging and difficult to deliver care to [ 8 , 11 , 15 , 38 , 39 ] , and this theme was also echoed by the participants of this study . the term honeymoon to chaos stage is used to describe nurses ' experience of caring for those with bpd in which at the initial stage of treatment it is like a honeymoon where the relationships between service users and nurses were peaceful as they were getting to know each other . similar to the findings in this study , where some nurses admitted providing minimal care and ignoring or avoiding service users with bpd , it is also reported elsewhere that some nurses withdraw and distance themselves from service users with bpd . the level of care provided by nurses to service users with bpd is also questioned . the level of empathy expressed by the study participants for the first admission of a service user with bpd was categorised overall at level 2 ( offer solution ) . these findings are similar to those reported elsewhere where the spirs was used , and nurses displayed a low level of empathy for service users with bpd in comparison for those with schizophrenia . it is reported that nurses believe it is socially acceptable to respond to the service user with bpd this way because of their diagnosis . these include feelings of helplessness , being used , therapeutic failure , devalued and unappreciated , anger , and frustration [ 11 , 15 ] . therefore , this level of response may be a defence mechanism against the intense affect generated in nurses by service users with bpd . the interview data in this study reveals that participants have a clear understanding of the characteristics of service users with bpd . the majority of participants also believed that service users with bpd should not be cared for in a hospital environment because the services are inadequate and this has a negative impact on care delivery . the development of a specialist community service and improved education and skills training workshops were viewed by the participants as being the most probable way to improve the service and care received by service users with bpd . these service users ' behaviours are complex and arise from many causes , biological , psychodynamic , and sociocultural , which all work together to create the behaviours . the more the nurses understand the complexity of bpd , the more likely a higher level of empathy may be displayed . therefore , it may become less demanding for the nurses to respond therapeutically and consistently without anger , frustration , and fear of service users with bpd . the aim of the workshop was for participants to develop practical skills in carrying out treatment plans and to become more positive about working with service users with a diagnosis of bpd . it was thought that this would also improve relationships between service users with bpd and nurses by helping nurses to overcome negative perceptions that they associate with these service users . after the workshop , the participants ' confidence and willingness to work with people with bpd had statistically improved . practice guidelines set out also recommend supervision for staff caring for individuals with bpd . addressing these issues can modify negative nursing responses and help alleviate negative working experiences with service users with bpd . nurses need to receive regular supervision ; this will provide them the opportunity and space to process any perceived unpleasant experiences generated from caring for service users with bpd and help prevent burnout . service users with bpd symbolise a challenge to nurses ; however , with improved education , training , and clinical supervision , a new era in bpd treatment can begin . nurses who hold positive attitudes about service users , have a sense of moral commitment , are skilled interpersonally , and are able to stay in a rational state of mind in the midst of conflict and can apply knowledge about the personality disorder and work skillfully with these service users . although the study findings add further evidence to suggest the need for training and education for nurses caring for service users with bpd , the study findings are limited for a number of reasons . moreover , it is possible that the views of those who did not choose to take part in the study may differ from those who were interviewed .

trauma remains a major cause of death and disability , especially in persons younger than 45 years [ 13 ] . in the past 30 years , several scoring systems have been proposed for assessing trauma patients initial status , describing injuries , and eventually predicting outcome [ 49 ] . these scoring systems , like the trauma injury severity score ( triss ) , can be used to develop models for predicting the probability of survival in a population base . despite advances in trauma care and the identification of numerous limitations of triss , this method continues to be the most commonly used tool for monitoring trauma outcomes and assessing trauma unit performance [ 11 , 12 ] . the triss method has led to the major trauma outcome score ( mtos ) , which allows comparative evaluation of the hospital care of the injured patient [ 10 , 13 ] . the triss method was developed in 1987 and revised a couple of years later [ 10 , 14 ] . however , computed tomography ( ct ) was not used extensively in emergency departments at that time , whereas it has found increasing use ever since . moreover , due to technological advances , the sensitivity of ct has increased considerably , which has led to improved injury detection . as a consequence of these and other factors , ct has been increasingly used in the initial evaluation of blunt trauma patients during the last few decades . while many institutions still prefer to use thoracoabdominal ct only in selected situations , others prefer to use ct routinely in every patient after high - energy blunt trauma . we hypothesized that the use of routine thoracoabdominal ct will interfere with injury - based survival analyses such as the triss method , since routine ct will result in an improved injury detection and therefore in higher injury severity scores , which in turn will lead to higher predicted mortality rates . although this seems logical , to the best of our knowledge , no study has demonstrated and quantified this before . however , when interpreting survival results of individual institutions and comparing several clinics , it is crucial to comprehend the influence of routine ct on the survival analyses . moreover , knowledge of this mathematical influence is essential when interpreting studies about the effect of routine ct on outcome parameters such as survival . therefore , the purpose of this study was to evaluate the influence of routine thoracoabdominal ct scanning on predicted survival calculations according to the triss method . a prospective observational cohort study was performed in a 953-bed teaching hospital with a full 24-h surgical capability that serves as a level 1 trauma center for an area with a population of 1.6 million . patients with a high index of suspicion for serious injuries after trauma are directly transported to our hospital . in the period of may 2005 until june 2008 , all patients who sustained high - energy blunt trauma were prospectively registered and included in a trauma ct database that was originally designed to evaluate the additional value of routine versus selective ct of the cervical spine , chest , abdomen and pelvis in blunt trauma patients . high - energy trauma was defined as a fall from a height of 3 m , a car collision at 50 km / h ( or at 30 km / h when a seatbelt was not worn ) , collisions between bicyclists or moped drivers and motor vehicles at a speed of 30 km / h , or being jammed , stuck , buried or crushed between heavy objects . the results from studies of the additional value of ct have been described in previous publications [ 17 , 18 ] . in this trauma ct database , radiological and clinical data were collected from all blunt trauma patients of 16 years and older . patients who had been transferred from another hospital and patients who had sustained penetrating trauma were excluded . we also excluded patients with class iii or iv shock requiring immediate surgical intervention , cases with neurological conditions or deterioration requiring immediate neurosurgical intervention without any further diagnostic delay , and patients with a suspected or known pregnancy . at admission , a multidisciplinary trauma team examined each patient according to the hospital s protocol based on the guidelines for advanced trauma life support ( atls ) . data were prospectively collected in a standardized database using microsoft access version 2000 ( microsoft inc . , all patients underwent primary and secondary surveys according to the atls guidelines and conventional radiography , consisting of radiography of the chest , pelvis and spine , and a focused abdominal sonography . after this , the trauma team prospectively established whether there was an indication to perform an additional ( selective ) ct of the chest , abdomen , pelvis or thoracolumbar spine ( table 1 ) [ 17 , 18 ] . subsequently , instead of a selective mdct , all patients underwent a routine thoracoabdominal ct . after this , it was determined whether the final diagnoses should be made based on selective ct that was performed on indication or by routine ct . ct on indication and routine ct ( fig . 1 ) , and calculated the rts , iss , and predicted survival for both algorithms using published methods [ 10 , 14 ] . for the calculations of the iss and the predicted survival in the ct on indication algorithm , we only used the injuries found by physical examination , conventional radiography , and selectively performed ct . in the routine ct algorithm , we used all injuries found on physical examination and total radiological work - up , including routine thoracoabdominal ct.table 1indications for selective ct of specific body regionsregionindicationthorax>3 rib fractures on conventional radiographysuspicion of hemothorax on conventional radiographysuspicion of lung contusion on conventional radiographysuspicion of pneumothorax on conventional radiographyabnormal mediastinum / suspicion of aortic lesion on conventional radiographyabdomenabdominal tendernessfree fluid on sonographyparenchymal injuries on sonographymacroscopic hematuriapelvispelvic fracture on conventional radiographyinadequate quality of conventional radiographythoracolumbar spinespinal cord injuryosseous painvertebral fracture on conventional radiographyinadequate quality of conventional radiographyfig . 1two algorithms were compared in this study : ct scan on indication and routine ct scan . however , patients who met criteria for the ct on indication algorithm were also prospectively defined . this resulted in a number of diagnoses , ais , iss , and a predicted survival based on physical examination , conventional radiologic examination , ultrasound , and ct for patients who met these criteria for ct on indication . in the right arm , the total number of diagnoses was based solely on routine ct in all patients indications for selective ct of specific body regions two algorithms were compared in this study : ct scan on indication and routine ct scan . all 1,047 blunt trauma patients underwent thoracoabdominal ct . however , patients who met criteria for the ct on indication algorithm were also prospectively defined . this resulted in a number of diagnoses , ais , iss , and a predicted survival based on physical examination , conventional radiologic examination , ultrasound , and ct for patients who met these criteria for ct on indication . in the right arm , the total number of diagnoses was based solely on routine ct in all patients the rts was derived from the respiratory rate , the systolic blood pressure , and the ( on - scene ) glasgow coma score . the iss was calculated using the square of the three highest scores on the ais , following the six body regions of : face ; head and neck ; chest and thoracic spine ; abdomen , lumbar spine and pelvic contents ; bony pelvis and limb and body surface . the triss was calculated from the rts , iss , the age of the patient and the nature of the injury ( blunt or penetrating ) , and this subsequently provided a probability of survival ( ps ) . the triss was calculated using the formula presented in the original publication from boyd et al . . the m statistic is a measure of how closely the injury severities of the study subset and the mtos match . it is defined by adding the lowest of all fractions of patients falling into each of six predicted survival ranges ( ps ) of the study subset and the baseline subset ( mtos ) together . a value of 1 represents an excellent match between the study group and the baseline patient group ( mtos ) . the z score is a statistic that compares the outcomes of two subsets of a population . it quantifies the difference between the actual number of deaths in the test ( e.g. , our hospital ) and the predicted number of deaths based on the mtos norm . the statistic w describes the clinical and practical significance of the difference between the actual and expected survivors . the z and w scores were calculated as presented in the publication from flora et al . . a prospective observational cohort study was performed in a 953-bed teaching hospital with a full 24-h surgical capability that serves as a level 1 trauma center for an area with a population of 1.6 million . patients with a high index of suspicion for serious injuries after trauma are directly transported to our hospital . in the period of may 2005 until june 2008 , all patients who sustained high - energy blunt trauma were prospectively registered and included in a trauma ct database that was originally designed to evaluate the additional value of routine versus selective ct of the cervical spine , chest , abdomen and pelvis in blunt trauma patients . high - energy trauma was defined as a fall from a height of 3 m , a car collision at 50 km / h ( or at 30 km / h when a seatbelt was not worn ) , collisions between bicyclists or moped drivers and motor vehicles at a speed of 30 km / h , or being jammed , stuck , buried or crushed between heavy objects . the results from studies of the additional value of ct have been described in previous publications [ 17 , 18 ] . in this trauma ct database , radiological and clinical data were collected from all blunt trauma patients of 16 years and older . patients who had been transferred from another hospital and patients who had sustained penetrating trauma were excluded . we also excluded patients with class iii or iv shock requiring immediate surgical intervention , cases with neurological conditions or deterioration requiring immediate neurosurgical intervention without any further diagnostic delay , and patients with a suspected or known pregnancy . at admission , a multidisciplinary trauma team examined each patient according to the hospital s protocol based on the guidelines for advanced trauma life support ( atls ) . data were prospectively collected in a standardized database using microsoft access version 2000 ( microsoft inc . , all patients underwent primary and secondary surveys according to the atls guidelines and conventional radiography , consisting of radiography of the chest , pelvis and spine , and a focused abdominal sonography . after this , the trauma team prospectively established whether there was an indication to perform an additional ( selective ) ct of the chest , abdomen , pelvis or thoracolumbar spine ( table 1 ) [ 17 , 18 ] . subsequently , instead of a selective mdct , all patients underwent a routine thoracoabdominal ct . after this , it was determined whether the final diagnoses should be made based on selective ct that was performed on indication or by routine ct . for all patients , we processed the data using two different algorithms : ct on indication and routine ct ( fig . 1 ) , and calculated the rts , iss , and predicted survival for both algorithms using published methods [ 10 , 14 ] . for the calculations of the iss and the predicted survival in the ct on indication algorithm , we only used the injuries found by physical examination , conventional radiography , and selectively performed ct . in the routine ct algorithm , we used all injuries found on physical examination and total radiological work - up , including routine thoracoabdominal ct.table 1indications for selective ct of specific body regionsregionindicationthorax>3 rib fractures on conventional radiographysuspicion of hemothorax on conventional radiographysuspicion of lung contusion on conventional radiographysuspicion of pneumothorax on conventional radiographyabnormal mediastinum / suspicion of aortic lesion on conventional radiographyabdomenabdominal tendernessfree fluid on sonographyparenchymal injuries on sonographymacroscopic hematuriapelvispelvic fracture on conventional radiographyinadequate quality of conventional radiographythoracolumbar spinespinal cord injuryosseous painvertebral fracture on conventional radiographyinadequate quality of conventional radiographyfig . 1two algorithms were compared in this study : ct scan on indication and routine ct scan . all 1,047 blunt trauma patients underwent thoracoabdominal ct . however , patients who met criteria for the ct on indication algorithm were also prospectively defined . this resulted in a number of diagnoses , ais , iss , and a predicted survival based on physical examination , conventional radiologic examination , ultrasound , and ct for patients who met these criteria for ct on indication . in the right arm , the total number of diagnoses was based solely on routine ct in all patients indications for selective ct of specific body regions two algorithms were compared in this study : ct scan on indication and routine ct scan . however , patients who met criteria for the ct on indication algorithm were also prospectively defined . this resulted in a number of diagnoses , ais , iss , and a predicted survival based on physical examination , conventional radiologic examination , ultrasound , and ct for patients who met these criteria for ct on indication . in the right arm , the total number of diagnoses was based solely on routine ct in all patients the rts was derived from the respiratory rate , the systolic blood pressure , and the ( on - scene ) glasgow coma score . the iss was calculated using the square of the three highest scores on the ais , following the six body regions of : face ; head and neck ; chest and thoracic spine ; abdomen , lumbar spine and pelvic contents ; bony pelvis and limb and body surface . the triss was calculated from the rts , iss , the age of the patient and the nature of the injury ( blunt or penetrating ) , and this subsequently provided a probability of survival ( ps ) . the triss was calculated using the formula presented in the original publication from boyd et al . . the m statistic is a measure of how closely the injury severities of the study subset and the mtos match . it is defined by adding the lowest of all fractions of patients falling into each of six predicted survival ranges ( ps ) of the study subset and the baseline subset ( mtos ) together . a value of 1 represents an excellent match between the study group and the baseline patient group ( mtos ) . the z score is a statistic that compares the outcomes of two subsets of a population . it quantifies the difference between the actual number of deaths in the test ( e.g. , our hospital ) and the predicted number of deaths based on the mtos norm . the statistic w describes the clinical and practical significance of the difference between the actual and expected survivors . the z and w scores were calculated as presented in the publication from flora et al . . from may 2005 to june 2008 , a total of 1,199 patients who had sustained a high - energy blunt trauma were admitted to our emergency department . the predominant mechanism of trauma was traffic incidents . in this study , 569 patients were admitted after being involved in a motor vehicle accident , 217 were bicyclist or moped drivers , and 37 were pedestrians . sixty - six patients fell from a height of 3 m. other high - energy mechanisms ( n = 158 ) were being jammed , stuck , buried or crushed between heavy objects . mean age was 39.5 ( range 1695 , sd 17.8 ) , mean rts was 11.3 , and mean gcs was 12.7 . of the 265 patients who presented with an rts of 11 , 231 had a decreased glasgow coma score of 12 or less . fifty - seven patients died during their hospital stay , most of them ( n = 46 ) due to associated neurologic injury ( 81.0% ) . of the 1,047 patients included , 115 ( 11.0% ) were indicated for complete thoracoabdominal ct based on abnormalities during physical examination , conventional radiography , and abdominal sonography . for 205 patients there was an indication to perform solely ct of the thorax , and 211 patients had an indication to perform ct of the abdomen and pelvis . for 516 patients ( 49.3% ) these patients received thoracoabdominal ct based solely on their high - energy trauma mechanism . in the algorithm based on the injuries detected solely by ct scan on indication , the mean injury severity score was 14.6 ( sd 13.9 ) . calculations according to the triss methodology gave a mean predicted survival of 87.5% , representing a predicted mortality of 12.5% . in the routine ct algorithm , based on all injuries detected by routine thoracoabdominal ct , the mean injury severity score was 17.0 ( sd 13.1 ) . using the triss methodology , the predicted survival in the routine ct algorithm was 86.3% . this implies that the predicted mortality rates according to the triss methodology were 13.7% using ct routinely and 12.5% using ct on indication ( table 2 ) . this is a significant difference ( p = 0.016 ) . in our study population , the w score was 13 , implying a difference in the predicted number of deaths between the two algorithms of 13 patients . the most important reasons for an increase in the iss due to routine thoracoabdominal ct as compared ct on indication were diagnoses of ( bilateral ) lung contusion , multiple rib fractures with or without pneumothorax , or laceration of the abdominal organs which were not detected by physical examination , conventional radiography , sonography , and indicated radiological work-up.table 2differences in iss and probable survival between two different algorithmsct on indicationroutine ctinjury severity score*14.616.9 range075075 sd13.913.1predicted survival ( mtos)87.5%86.3% * significant difference between routine ct and ct on indication ( p < 0.05 ) differences in iss and probable survival between two different algorithms * significant difference between routine ct and ct on indication this was significantly lower than predicted for both the routine ct group and the ct on indication group according to the triss methodology . in our study , the m score was 0.866 ( table 3 ) . after applying the values derived from our study data ( d = 57 ; qi = 143.7 ; qips = 54.9 ) to the formula used to establish the z score , the overall z statistic for our hospital was 11.7 . applying our values ( a = 990 , e = 903 , n = 1,047 ) to the previously mentioned formula for the w statistic of flora et al . , a w value of 8.3 was calculated . this means that , in our hospital , per hundred patients treated , 8.3 more adults with blunt trauma injuries survive than would be expected from the mtos norm . table 4 outlines the number of patients with additional injuries found through routine ct as compared to the injuries found on ct by indication.table 3m - score definitionrange of predicted survivalno . of patientsfraction of patients within rangestudy subsetbaseline subset ( mtos)0.961.007280.6950.8280.910.95600.0570.0450.760.90580.0550.0440.510.75770.0740.0290.260.50550.0530.0170.000.25690.0660.036the m score was defined by summing the smaller of the two fractions ( in italics ) , resulting in an m score of 0.866table 4the number of patients ( study group n = 1,047 ) and percentages with additional injuries found through routine ct as compared to the injuries found on ct by indicationct on indicationroutine ctabsolute differencethorax198 ( 18.9%)409 ( 39.1%)211 ( 20.2%)abdomen116 ( 11.1%)362 ( 34.6%)245 ( 23.4% ) the m score was defined by summing the smaller of the two fractions ( in italics ) , resulting in an m score of 0.866 the number of patients ( study group n = 1,047 ) and percentages with additional injuries found through routine ct as compared to the injuries found on ct by indication from may 2005 to june 2008 , a total of 1,199 patients who had sustained a high - energy blunt trauma were admitted to our emergency department . the predominant mechanism of trauma was traffic incidents . in this study , 569 patients were admitted after being involved in a motor vehicle accident , 217 were bicyclist or moped drivers , and 37 were pedestrians . sixty - six patients fell from a height of 3 m. other high - energy mechanisms ( n = 158 ) were being jammed , stuck , buried or crushed between heavy objects . mean age was 39.5 ( range 1695 , sd 17.8 ) , mean rts was 11.3 , and mean gcs was 12.7 . of the 265 patients who presented with an rts of 11 , 231 had a decreased glasgow coma score of 12 or less . fifty - seven patients died during their hospital stay , most of them ( n = 46 ) due to associated neurologic injury ( 81.0% ) . of the 1,047 patients included , 115 ( 11.0% ) were indicated for complete thoracoabdominal ct based on abnormalities during physical examination , conventional radiography , and abdominal sonography . for 205 patients there was an indication to perform solely ct of the thorax , and 211 patients had an indication to perform ct of the abdomen and pelvis . for 516 patients ( 49.3% ) in the algorithm based on the injuries detected solely by ct scan on indication , the mean injury severity score was 14.6 ( sd 13.9 ) . calculations according to the triss methodology gave a mean predicted survival of 87.5% , representing a predicted mortality of 12.5% . in the routine ct algorithm , based on all injuries detected by routine thoracoabdominal ct , the mean injury severity score was 17.0 ( sd 13.1 ) . using the triss methodology , the predicted survival in the routine ct algorithm was 86.3% . this implies that the predicted mortality rates according to the triss methodology were 13.7% using ct routinely and 12.5% using ct on indication ( table 2 ) . this is a significant difference ( p = 0.016 ) . in our study population , the w score was 13 , implying a difference in the predicted number of deaths between the two algorithms of 13 patients . the most important reasons for an increase in the iss due to routine thoracoabdominal ct as compared ct on indication were diagnoses of ( bilateral ) lung contusion , multiple rib fractures with or without pneumothorax , or laceration of the abdominal organs which were not detected by physical examination , conventional radiography , sonography , and indicated radiological work-up.table 2differences in iss and probable survival between two different algorithmsct on indicationroutine ctinjury severity score*14.616.9 range075075 sd13.913.1predicted survival ( mtos)87.5%86.3% * significant difference between routine ct and ct on indication ( p < 0.05 ) differences in iss and probable survival between two different algorithms * significant difference between routine ct and ct on indication this was significantly lower than predicted for both the routine ct group and the ct on indication group according to the triss methodology . in our study , the m score was 0.866 ( table 3 ) . after applying the values derived from our study data ( d = 57 ; qi = 143.7 ; qips = 54.9 ) to the formula used to establish the z score , the overall z statistic for our hospital was 11.7 . applying our values ( a = 990 , e = 903 , n = 1,047 ) to the previously mentioned formula for the w statistic of flora et al . , a w value of 8.3 was calculated . this means that , in our hospital , per hundred patients treated , 8.3 more adults with blunt trauma injuries survive than would be expected from the mtos norm . table 4 outlines the number of patients with additional injuries found through routine ct as compared to the injuries found on ct by indication.table 3m - score definitionrange of predicted survivalno . of patientsfraction of patients within rangestudy subsetbaseline subset ( mtos)0.961.007280.6950.8280.910.95600.0570.0450.760.90580.0550.0440.510.75770.0740.0290.260.50550.0530.0170.000.25690.0660.036the m score was defined by summing the smaller of the two fractions ( in italics ) , resulting in an m score of 0.866table 4the number of patients ( study group n = 1,047 ) and percentages with additional injuries found through routine ct as compared to the injuries found on ct by indicationct on indicationroutine ctabsolute differencethorax198 ( 18.9%)409 ( 39.1%)211 ( 20.2%)abdomen116 ( 11.1%)362 ( 34.6%)245 ( 23.4% ) the m score was defined by summing the smaller of the two fractions ( in italics ) , resulting in an m score of 0.866 the number of patients ( study group n = 1,047 ) and percentages with additional injuries found through routine ct as compared to the injuries found on ct by indication despite its widespread use , the triss methodology for calculating the predicted survival has many limitations and is criticized widely in the literature [ 4 , 79 , 2123 ] . it has been found to have high misclassification rates in severely traumatized patients . in many studies , attempts have been made to improve either physiologically or anatomically based outcome estimates in trauma [ 7 , 9 ] . nevertheless , it is still the most commonly used method for predicting outcomes in trauma populations . in this study we evaluated the influence of improved injury detection by routine thoracoabdominal ct on the triss survival analysis . we found a significant difference for both the injury severity scores and the predicted survival rates within the same group of patients when comparing the two different diagnostic algorithms ct on indication and routine ct . predicted mortality in the two algorithms was 12.5 and 13.7% ( p = 0.016 ) , respectively . performing a routine thoracoabdominal ct resulted in the detection of an increased number of diagnoses , resulting in higher injury severity scores and consequently in a significant decrease in predicted survival . based on the results of this study , it is demonstrated that the interpretation of outcome data in blunt trauma patients is highly dependent on the diagnostic modalities used . this is not only important when comparing the outcomes of different institutions , but also when comparing historical cohorts and drawing conclusions from them . another finding of our study is the disparity between the observed and predicted outcomes using the triss methodology . the predicted mortality based on the triss and mtos data was 13.7% , while the actually observed mortality was only 5.4% . although mathematical calculations using m statistics showed that the case mix of our study was slightly different from that of the mtos database , the distribution of the m statistics showed that our study population was more severely injured than the triss population . nevertheless , we still found a significantly better observed survival than predicted according to the triss methodology . this significant difference between the predicted and observed survival may be explained by several arguments . first , this might be explained by the fact that the predicted ( reference ) survival rates according to the mtos seem to be too pessimistic when ct is performed routinely . although it is unclear which diagnostic tools were used in the mtos population , it is unlikely that each patient received routine ct at that time . without the use of routine ct , it is likely that some injuries in the original mtos population remained undetected , resulting in an underestimation of the actual injury severity in some cases , thus leading to an deceivingly low iss for the mtos population , while the population was actually more injured than assumed . on the other hand , routine ct scanning leads to the detection of many additional injuries ( for example small and clinically irrelevant pulmonary contusions ) that can consequently lead to a higher iss , thus leading to a higher predicted mortality . moreover , the missing / underestimated injuries due to incomprehensive diagnostics in the mtos population may have had a negative result on the actual outcome of the mtos population , thereby leading to worse reference mortality rates . taken together , this implies that the mtos data are outdated and need adjustments , or that new ( ct - based ) scoring systems should be created in the future . the mortality rate in the excluded patients with class iii or iv shock requiring immediate surgical intervention , cases of neurological condition or deterioration needing immediate neurosurgical intervention without any further diagnostic delay , and patients with suspected or known pregnancy should also be noted . if we add these numbers to the dataset , the overall mortality rate may become closer to the mtos . besides the overly pessimistic predicted survival rates , the significant difference in the predicted and observed survival rates can also be explained by improved care in our population as compared to the care of patients described in the mtos . this in turn may be explained by our use of better diagnostic tools , like routine thoracoabdominal ct , resulting in more specific diagnoses and thereby enabling better - tailored care . recently , huber - wagner et al . tried to demonstrate improved outcome due to routine ct in a large retrospective study . they concluded that the use of whole - body ct increases the probability of survival in polytrauma patients . however , it could be argued that the discrepancy between the predicted and the actual survival rates in their study was not caused by an improved diagnostic algorithm ( i.e. , routine ct ) , but rather by the fact that the use of routine ct resulted in an increase in iss due to the diagnosis of more injuries , which will consequently result in an underestimation of the predicted survival rates . this is backed up by the results of our present study . to demonstrate the influence of routine ct on the outcome of blunt trauma patients definitively , finally , the improved outcome of our populations as compared to the mtos might be explained by the ongoing evaluation of damage control surgery and the improved care of current intensive care units , as well as the logistical procedure and improved prehospital care . for instance , in the netherlands , distances to well - equipped level ii hospitals or level i trauma centers are relatively short , and 24/7 helicopter emergency medical services are provided in cases where patients require special assistance , for instance full anesthesia and intubation . although the results of our study seem to be important for interpreting outcome data concerning blunt trauma patients , some limitations of our study should be addressed . first of all , estimations of the performances of conventional radiography and ct were not done independently of clinical information . surgeons and radiologists work in close cooperation ; the radiologist is present in the trauma bay during resuscitation . however , because of the purpose and design of our study , this was not considered a major problem . secondly , we eliminated hindsight bias as much as possible by insisting that clinicians and radiologists thoroughly assess conventional radiography before ct was performed . however , in the middle of the night , no investigator was present to protect and ensure the prospective nature of selective ct classification , and clinicians and radiologists were trusted with respect to their reports . this may have induced hindsight bias in the interpretation of radiography and clinical evaluations . in a minority of the cases , this may have resulted in misjudgments of physical examination and conventional radiography performance and in the misclassification of an indicated or routine ct . thirdly , although ct was indicated prospectively , trauma scores of the indication algorithm were calculated retrospectively . routine thoracoabdominal ct in patients after high - energy blunt trauma leads to the detection of more injuries , thus resulting in higher injury severity scores and a lower predicted probability of survival as compared to a diagnostic work - up with the selective use of ct on indication . calculated predicted survival by the triss methodology does not seem to be representative of the observed survival if thoracoabdominal ct is used routinely . re - evaluations of current trauma scores and survival prediction methods appear mandatory if these are to be applied to blunt trauma populations who undergo routine ct in a standard fashion .

introductionmany scoring systems have been proposed to predict the survival of trauma patients . this study was performed to evaluate the influence of routine thoracoabdominal computed tomography ( ct ) on the predicted survival according to the trauma injury severity score ( triss).patients and methods1,047 patients who had sustained a high - energy blunt trauma over a 3-year period were prospectively included in the study . all patients underwent physical examination , conventional radiography of the chest , thoracolumbar spine and pelvis , abdominal sonography , and routine thoracoabdominal ct . from this group with routine ct , we prospectively defined a selective ct ( sub)group for cases with abnormal physical examination and/or conventional radiography and/or sonography . type and extent of injuries were recorded for both the selective and the routine ct groups . based on the injuries found by the two different ct algorithms , we calculated the injury severity scores ( iss ) and predicted survivals according to the triss methodology for the routine and the selective ct algorithms.resultsbased on injuries detected by the selective ct algorithm , the mean iss was 14.6 , resulting in a predicted mortality of 12.5% . because additional injuries were found by the routine ct algorithm , the mean iss increased to 16.9 , resulting in a predicted mortality of 13.7% . the actual observed mortality was 5.4%.conclusionroutine thoracoabdominal ct in high - energy blunt trauma patients reveals more injuries than a selective ct algorithm , resulting in a higher iss . according to the triss , this results in higher predicted mortalities . observed mortality , however , was significantly lower than predicted . the predicted survival according to mtos seems to underestimate the actual survival when routine ct is used .

Introduction Patients and methods Subjects Data collection Statistical analysis Results Characteristics of subjects Analysis predicted survival Evaluation of survival with routine CT versus MTOS Discussion Conclusions

in the past 30 years , several scoring systems have been proposed for assessing trauma patients initial status , describing injuries , and eventually predicting outcome [ 49 ] . these scoring systems , like the trauma injury severity score ( triss ) , can be used to develop models for predicting the probability of survival in a population base . however , computed tomography ( ct ) was not used extensively in emergency departments at that time , whereas it has found increasing use ever since . while many institutions still prefer to use thoracoabdominal ct only in selected situations , others prefer to use ct routinely in every patient after high - energy blunt trauma . we hypothesized that the use of routine thoracoabdominal ct will interfere with injury - based survival analyses such as the triss method , since routine ct will result in an improved injury detection and therefore in higher injury severity scores , which in turn will lead to higher predicted mortality rates . however , when interpreting survival results of individual institutions and comparing several clinics , it is crucial to comprehend the influence of routine ct on the survival analyses . therefore , the purpose of this study was to evaluate the influence of routine thoracoabdominal ct scanning on predicted survival calculations according to the triss method . in the period of may 2005 until june 2008 , all patients who sustained high - energy blunt trauma were prospectively registered and included in a trauma ct database that was originally designed to evaluate the additional value of routine versus selective ct of the cervical spine , chest , abdomen and pelvis in blunt trauma patients . at admission , a multidisciplinary trauma team examined each patient according to the hospital s protocol based on the guidelines for advanced trauma life support ( atls ) . , all patients underwent primary and secondary surveys according to the atls guidelines and conventional radiography , consisting of radiography of the chest , pelvis and spine , and a focused abdominal sonography . after this , the trauma team prospectively established whether there was an indication to perform an additional ( selective ) ct of the chest , abdomen , pelvis or thoracolumbar spine ( table 1 ) [ 17 , 18 ] . subsequently , instead of a selective mdct , all patients underwent a routine thoracoabdominal ct . 1 ) , and calculated the rts , iss , and predicted survival for both algorithms using published methods [ 10 , 14 ] . for the calculations of the iss and the predicted survival in the ct on indication algorithm , we only used the injuries found by physical examination , conventional radiography , and selectively performed ct . in the routine ct algorithm , we used all injuries found on physical examination and total radiological work - up , including routine thoracoabdominal ct.table 1indications for selective ct of specific body regionsregionindicationthorax>3 rib fractures on conventional radiographysuspicion of hemothorax on conventional radiographysuspicion of lung contusion on conventional radiographysuspicion of pneumothorax on conventional radiographyabnormal mediastinum / suspicion of aortic lesion on conventional radiographyabdomenabdominal tendernessfree fluid on sonographyparenchymal injuries on sonographymacroscopic hematuriapelvispelvic fracture on conventional radiographyinadequate quality of conventional radiographythoracolumbar spinespinal cord injuryosseous painvertebral fracture on conventional radiographyinadequate quality of conventional radiographyfig . this resulted in a number of diagnoses , ais , iss , and a predicted survival based on physical examination , conventional radiologic examination , ultrasound , and ct for patients who met these criteria for ct on indication . in the right arm , the total number of diagnoses was based solely on routine ct in all patients indications for selective ct of specific body regions two algorithms were compared in this study : ct scan on indication and routine ct scan . this resulted in a number of diagnoses , ais , iss , and a predicted survival based on physical examination , conventional radiologic examination , ultrasound , and ct for patients who met these criteria for ct on indication . in the right arm , the total number of diagnoses was based solely on routine ct in all patients the rts was derived from the respiratory rate , the systolic blood pressure , and the ( on - scene ) glasgow coma score . the triss was calculated from the rts , iss , the age of the patient and the nature of the injury ( blunt or penetrating ) , and this subsequently provided a probability of survival ( ps ) . it is defined by adding the lowest of all fractions of patients falling into each of six predicted survival ranges ( ps ) of the study subset and the baseline subset ( mtos ) together . , our hospital ) and the predicted number of deaths based on the mtos norm . in the period of may 2005 until june 2008 , all patients who sustained high - energy blunt trauma were prospectively registered and included in a trauma ct database that was originally designed to evaluate the additional value of routine versus selective ct of the cervical spine , chest , abdomen and pelvis in blunt trauma patients . , all patients underwent primary and secondary surveys according to the atls guidelines and conventional radiography , consisting of radiography of the chest , pelvis and spine , and a focused abdominal sonography . after this , the trauma team prospectively established whether there was an indication to perform an additional ( selective ) ct of the chest , abdomen , pelvis or thoracolumbar spine ( table 1 ) [ 17 , 18 ] . subsequently , instead of a selective mdct , all patients underwent a routine thoracoabdominal ct . for all patients , we processed the data using two different algorithms : ct on indication and routine ct ( fig . 1 ) , and calculated the rts , iss , and predicted survival for both algorithms using published methods [ 10 , 14 ] . for the calculations of the iss and the predicted survival in the ct on indication algorithm , we only used the injuries found by physical examination , conventional radiography , and selectively performed ct . in the routine ct algorithm , we used all injuries found on physical examination and total radiological work - up , including routine thoracoabdominal ct.table 1indications for selective ct of specific body regionsregionindicationthorax>3 rib fractures on conventional radiographysuspicion of hemothorax on conventional radiographysuspicion of lung contusion on conventional radiographysuspicion of pneumothorax on conventional radiographyabnormal mediastinum / suspicion of aortic lesion on conventional radiographyabdomenabdominal tendernessfree fluid on sonographyparenchymal injuries on sonographymacroscopic hematuriapelvispelvic fracture on conventional radiographyinadequate quality of conventional radiographythoracolumbar spinespinal cord injuryosseous painvertebral fracture on conventional radiographyinadequate quality of conventional radiographyfig . this resulted in a number of diagnoses , ais , iss , and a predicted survival based on physical examination , conventional radiologic examination , ultrasound , and ct for patients who met these criteria for ct on indication . in the right arm , the total number of diagnoses was based solely on routine ct in all patients indications for selective ct of specific body regions two algorithms were compared in this study : ct scan on indication and routine ct scan . this resulted in a number of diagnoses , ais , iss , and a predicted survival based on physical examination , conventional radiologic examination , ultrasound , and ct for patients who met these criteria for ct on indication . in the right arm , the total number of diagnoses was based solely on routine ct in all patients the rts was derived from the respiratory rate , the systolic blood pressure , and the ( on - scene ) glasgow coma score . the triss was calculated from the rts , iss , the age of the patient and the nature of the injury ( blunt or penetrating ) , and this subsequently provided a probability of survival ( ps ) . , our hospital ) and the predicted number of deaths based on the mtos norm . from may 2005 to june 2008 , a total of 1,199 patients who had sustained a high - energy blunt trauma were admitted to our emergency department . of the 1,047 patients included , 115 ( 11.0% ) were indicated for complete thoracoabdominal ct based on abnormalities during physical examination , conventional radiography , and abdominal sonography . in the algorithm based on the injuries detected solely by ct scan on indication , the mean injury severity score was 14.6 ( sd 13.9 ) . calculations according to the triss methodology gave a mean predicted survival of 87.5% , representing a predicted mortality of 12.5% . in the routine ct algorithm , based on all injuries detected by routine thoracoabdominal ct , the mean injury severity score was 17.0 ( sd 13.1 ) . using the triss methodology , the predicted survival in the routine ct algorithm was 86.3% . this implies that the predicted mortality rates according to the triss methodology were 13.7% using ct routinely and 12.5% using ct on indication ( table 2 ) . in our study population , the w score was 13 , implying a difference in the predicted number of deaths between the two algorithms of 13 patients . the most important reasons for an increase in the iss due to routine thoracoabdominal ct as compared ct on indication were diagnoses of ( bilateral ) lung contusion , multiple rib fractures with or without pneumothorax , or laceration of the abdominal organs which were not detected by physical examination , conventional radiography , sonography , and indicated radiological work-up.table 2differences in iss and probable survival between two different algorithmsct on indicationroutine ctinjury severity score*14.616.9 range075075 sd13.913.1predicted survival ( mtos)87.5%86.3% * significant difference between routine ct and ct on indication ( p < 0.05 ) differences in iss and probable survival between two different algorithms * significant difference between routine ct and ct on indication this was significantly lower than predicted for both the routine ct group and the ct on indication group according to the triss methodology . table 4 outlines the number of patients with additional injuries found through routine ct as compared to the injuries found on ct by indication.table 3m - score definitionrange of predicted survivalno . of patientsfraction of patients within rangestudy subsetbaseline subset ( mtos)0.961.007280.6950.8280.910.95600.0570.0450.760.90580.0550.0440.510.75770.0740.0290.260.50550.0530.0170.000.25690.0660.036the m score was defined by summing the smaller of the two fractions ( in italics ) , resulting in an m score of 0.866table 4the number of patients ( study group n = 1,047 ) and percentages with additional injuries found through routine ct as compared to the injuries found on ct by indicationct on indicationroutine ctabsolute differencethorax198 ( 18.9%)409 ( 39.1%)211 ( 20.2%)abdomen116 ( 11.1%)362 ( 34.6%)245 ( 23.4% ) the m score was defined by summing the smaller of the two fractions ( in italics ) , resulting in an m score of 0.866 the number of patients ( study group n = 1,047 ) and percentages with additional injuries found through routine ct as compared to the injuries found on ct by indication from may 2005 to june 2008 , a total of 1,199 patients who had sustained a high - energy blunt trauma were admitted to our emergency department . of the 1,047 patients included , 115 ( 11.0% ) were indicated for complete thoracoabdominal ct based on abnormalities during physical examination , conventional radiography , and abdominal sonography . for 516 patients ( 49.3% ) in the algorithm based on the injuries detected solely by ct scan on indication , the mean injury severity score was 14.6 ( sd 13.9 ) . calculations according to the triss methodology gave a mean predicted survival of 87.5% , representing a predicted mortality of 12.5% . in the routine ct algorithm , based on all injuries detected by routine thoracoabdominal ct , the mean injury severity score was 17.0 ( sd 13.1 ) . using the triss methodology , the predicted survival in the routine ct algorithm was 86.3% . this implies that the predicted mortality rates according to the triss methodology were 13.7% using ct routinely and 12.5% using ct on indication ( table 2 ) . in our study population , the w score was 13 , implying a difference in the predicted number of deaths between the two algorithms of 13 patients . the most important reasons for an increase in the iss due to routine thoracoabdominal ct as compared ct on indication were diagnoses of ( bilateral ) lung contusion , multiple rib fractures with or without pneumothorax , or laceration of the abdominal organs which were not detected by physical examination , conventional radiography , sonography , and indicated radiological work-up.table 2differences in iss and probable survival between two different algorithmsct on indicationroutine ctinjury severity score*14.616.9 range075075 sd13.913.1predicted survival ( mtos)87.5%86.3% * significant difference between routine ct and ct on indication ( p < 0.05 ) differences in iss and probable survival between two different algorithms * significant difference between routine ct and ct on indication this was significantly lower than predicted for both the routine ct group and the ct on indication group according to the triss methodology . table 4 outlines the number of patients with additional injuries found through routine ct as compared to the injuries found on ct by indication.table 3m - score definitionrange of predicted survivalno . of patientsfraction of patients within rangestudy subsetbaseline subset ( mtos)0.961.007280.6950.8280.910.95600.0570.0450.760.90580.0550.0440.510.75770.0740.0290.260.50550.0530.0170.000.25690.0660.036the m score was defined by summing the smaller of the two fractions ( in italics ) , resulting in an m score of 0.866table 4the number of patients ( study group n = 1,047 ) and percentages with additional injuries found through routine ct as compared to the injuries found on ct by indicationct on indicationroutine ctabsolute differencethorax198 ( 18.9%)409 ( 39.1%)211 ( 20.2%)abdomen116 ( 11.1%)362 ( 34.6%)245 ( 23.4% ) the m score was defined by summing the smaller of the two fractions ( in italics ) , resulting in an m score of 0.866 the number of patients ( study group n = 1,047 ) and percentages with additional injuries found through routine ct as compared to the injuries found on ct by indication despite its widespread use , the triss methodology for calculating the predicted survival has many limitations and is criticized widely in the literature [ 4 , 79 , 2123 ] . in this study we evaluated the influence of improved injury detection by routine thoracoabdominal ct on the triss survival analysis . we found a significant difference for both the injury severity scores and the predicted survival rates within the same group of patients when comparing the two different diagnostic algorithms ct on indication and routine ct . performing a routine thoracoabdominal ct resulted in the detection of an increased number of diagnoses , resulting in higher injury severity scores and consequently in a significant decrease in predicted survival . based on the results of this study , it is demonstrated that the interpretation of outcome data in blunt trauma patients is highly dependent on the diagnostic modalities used . the predicted mortality based on the triss and mtos data was 13.7% , while the actually observed mortality was only 5.4% . nevertheless , we still found a significantly better observed survival than predicted according to the triss methodology . first , this might be explained by the fact that the predicted ( reference ) survival rates according to the mtos seem to be too pessimistic when ct is performed routinely . without the use of routine ct , it is likely that some injuries in the original mtos population remained undetected , resulting in an underestimation of the actual injury severity in some cases , thus leading to an deceivingly low iss for the mtos population , while the population was actually more injured than assumed . on the other hand , routine ct scanning leads to the detection of many additional injuries ( for example small and clinically irrelevant pulmonary contusions ) that can consequently lead to a higher iss , thus leading to a higher predicted mortality . moreover , the missing / underestimated injuries due to incomprehensive diagnostics in the mtos population may have had a negative result on the actual outcome of the mtos population , thereby leading to worse reference mortality rates . besides the overly pessimistic predicted survival rates , the significant difference in the predicted and observed survival rates can also be explained by improved care in our population as compared to the care of patients described in the mtos . this in turn may be explained by our use of better diagnostic tools , like routine thoracoabdominal ct , resulting in more specific diagnoses and thereby enabling better - tailored care . however , it could be argued that the discrepancy between the predicted and the actual survival rates in their study was not caused by an improved diagnostic algorithm ( i.e. , routine ct ) , but rather by the fact that the use of routine ct resulted in an increase in iss due to the diagnosis of more injuries , which will consequently result in an underestimation of the predicted survival rates . to demonstrate the influence of routine ct on the outcome of blunt trauma patients definitively , finally , the improved outcome of our populations as compared to the mtos might be explained by the ongoing evaluation of damage control surgery and the improved care of current intensive care units , as well as the logistical procedure and improved prehospital care . however , in the middle of the night , no investigator was present to protect and ensure the prospective nature of selective ct classification , and clinicians and radiologists were trusted with respect to their reports . in a minority of the cases , this may have resulted in misjudgments of physical examination and conventional radiography performance and in the misclassification of an indicated or routine ct . routine thoracoabdominal ct in patients after high - energy blunt trauma leads to the detection of more injuries , thus resulting in higher injury severity scores and a lower predicted probability of survival as compared to a diagnostic work - up with the selective use of ct on indication . calculated predicted survival by the triss methodology does not seem to be representative of the observed survival if thoracoabdominal ct is used routinely .

governments across the developed world are concerned with enabling older people to maintain their active contribution to society , and thereby their quality of life ( qol).1 qol has become a commonly used end point in the evaluation of multisector public policy , including health , social , community and environmental policy actions . for policy outcomes to be measured with any validity , measures of qol need to have social , as well as policy , relevance , to be meaningful to people 's lives , and to be carefully conceptualised and constructed . lawton26 developed a popularly cited quadripartite concept of qol , proposing that the good life ( qol ) may be represented by behavioural and social competence ( health , cognition , time use , social behaviour ) , perceptions of qol ( subjective evaluation of each domain of life ) , psychological well - being ( mental health , cognitive judgements of life satisfaction , positive - negative emotions ) and the external , objective , physical environment ( housing , economic indicators ) . however , there is no consensus about its conceptual definition or measurement,7 and most investigators have based their concepts on expert opinions rather than the perspectives of lay people . this has the consequence that there are few empirical data on the extent to which the items included in measurement scales have any relevance to people . thus , it is increasingly important to develop a multidimensional model and measure of quality of life , for use in descriptive and evaluative multisector policy research , which reflects the views of the population concerned , with cross - sectional and longitudinal applicability . elicitation of people 's own views of qol in this process is particularly important because qol is a subjective concept . survey and qualitative research with people aged 65 + , living at home in britain , reported that the central planks of qol emphasised by respondents were psychological well - being and positive outlook , having health and functioning , social relationships , leisure activities , neighbourhood resources , adequate financial circumstances and independence.710 this research led to the development of the older people 's quality of life questionnaire ( opqol ) , which is unique in being derived from the views of a representative sample of older people , cross - checked against theoretical models for assessment comprehensiveness . the aim here is to compare the psychometric properties of the opqol , with the casp-19 and whoqol - old among people 65 + participating in three national surveys of older people living at home in britain . two of these three surveys were cross - sectional , and the third was longitudinal ( see supplementary appendix 1 ) : ethnibus survey of people aged 65 + responding to two waves of the national ethni surveys ( http://www.ethnicfocus.com ) in 2008 . this is a rolling face - to - face interview survey with adults aged 16 + , living at home , based on focused enumeration , stratified random sampling of postcodes in britain , and statistically robust sampling of people in common ethnic minority groups in britain ; the response rate was 70% ( n=400 ) . ons survey of people aged 65 + responding to two waves of the office for national statistics ( ons ) national omnibus survey ( http://www.statistics.gov.uk ) in 2008 . this is a rolling face - to - face interview survey with adults aged 16 + , living at home , based on a stratified random sample of postcodes across britain ; the response rate was 61% ( n=589 ) . qol follow - up survey in 20072008 , of people living at home in britain , aged 65 + at baseline , who had responded to four ons national omnibus interview surveys . these were based on stratified random samples of postcodes across britain during 1999/2000 ; response was 77% ( n=999 ) at baseline and 58% among survivors ( n=287 ) at 20072008 follow - up . the qol follow - up survey is included here as the longitudinal design provided the opportunity to test the causal model of the opqol , as well as a willing sample for test - retest reliability assessment . the opqol was administered in all three surveys . prior to administration in the surveys reported here , the items in the opqol were pretested with 179 older people and three focus groups , reduced to 32-item and 35-item versions , and statistical tests of reliability and validity were applied . the casp-1911 and the whoqol - old12 13 were administered in the two face - to - face interview surveys only ; it would have been too cognitively burdensome to have included all three scales in the postal , self - administration mode . supplementary appendix 2 displays the opqol , summarises its development and briefly summarises the casp-19 and whoqol - old . independent self - ratings of global qol , and of its domains , were included in the questionnaire in order to distinguish between the constituents of , and influences on , qol.14 also included were standard sociodemographic items , self - rated active ageing , items measuring health and psychosocial circumstances.7 ethnic status was measured using a standard item about ethnic identity in the uk . this would not necessarily be applicable to populations in other countries , because it reflects close connections between new commonwealth countries and ethnic minority groups in the uk.15 descriptive analyses included frequencies , tests , and spearman 's r correlations . tests of scale reliability were applied in order to assess the extent to which scale items measure the same construct , with freedom from random error ( internal consistency ) . this is the strength of the association between each scale item and the full scale , item - item and item - total correlations . test - retest reliability of the stability of the newly developed opqol was assessed by mailing a second copy of the questionnaire to a random subsample of 50 follow - up qol survey respondents , 4 weeks after return of the first questionnaire ( response rate : 76%/38 ) . criterion ( concurrent ) validity is the independent corroboration that the scale is measuring what it intends to measure . this can only be measured by proxy with subjective measures , as there is no gold standard . proxy variables used here included independent self - ratings of qol overall and of qol domains ( health , social relationships , independence / control over life / freedom , home and neighbourhood , psychological / emotional well - being , financial circumstances , social and leisure activities ) . construct ( convergent and discriminant ) validity requires corroboration that scales measure the underlying construct they purport to measure . this was tested by assessing spearman 's r correlations between the qol scales and similar variables ( for convergent validity that the scale should correlate with similar or hypothesised variables ) and dissimilar variables ( for discriminant validity that there should be low correlations between scales and variables not expected to be associated ) . multiple regression was used to assess validity further by examining the ability of theoretically relevant variables to predict total qol scores . a hierarchical approach was used , with independent variables entered in their theoretical order of importance . statistical significance was set at p<0.05 . the variables entered did not correlate by more than 0.732 ; tests for multicollinearity were satisfied . the opqol was administered in all three surveys . prior to administration in the surveys reported here , the items in the opqol were pretested with 179 older people and three focus groups , reduced to 32-item and 35-item versions , and statistical tests of reliability and validity were applied . the casp-1911 and the whoqol - old12 13 were administered in the two face - to - face interview surveys only ; it would have been too cognitively burdensome to have included all three scales in the postal , self - administration mode . supplementary appendix 2 displays the opqol , summarises its development and briefly summarises the casp-19 and whoqol - old . independent self - ratings of global qol , and of its domains , were included in the questionnaire in order to distinguish between the constituents of , and influences on , qol.14 also included were standard sociodemographic items , self - rated active ageing , items measuring health and psychosocial circumstances.7 ethnic status was measured using a standard item about ethnic identity in the uk . this would not necessarily be applicable to populations in other countries , because it reflects close connections between new commonwealth countries and ethnic minority groups in the uk.15 tests of scale reliability were applied in order to assess the extent to which scale items measure the same construct , with freedom from random error ( internal consistency ) . this is the strength of the association between each scale item and the full scale , item - item and item - total correlations . test - retest reliability of the stability of the newly developed opqol was assessed by mailing a second copy of the questionnaire to a random subsample of 50 follow - up qol survey respondents , 4 weeks after return of the first questionnaire ( response rate : 76%/38 ) . criterion ( concurrent ) validity is the independent corroboration that the scale is measuring what it intends to measure . this can only be measured by proxy with subjective measures , as there is no gold standard . proxy variables used here included independent self - ratings of qol overall and of qol domains ( health , social relationships , independence / control over life / freedom , home and neighbourhood , psychological / emotional well - being , financial circumstances , social and leisure activities ) . construct ( convergent and discriminant ) validity requires corroboration that scales measure the underlying construct they purport to measure . this was tested by assessing spearman 's r correlations between the qol scales and similar variables ( for convergent validity that the scale should correlate with similar or hypothesised variables ) and dissimilar variables ( for discriminant validity that there should be low correlations between scales and variables not expected to be associated ) . multiple regression was used to assess validity further by examining the ability of theoretically relevant variables to predict total qol scores . a hierarchical approach was used , with independent variables entered in their theoretical order of importance . the variables entered did not correlate by more than 0.732 ; tests for multicollinearity were satisfied . just over half of each sample comprised women ( 52%/207 ethnibus , 55%/324 ons , 54%/154 qol follow - up ) . whereas most ethnibus respondents were aged 65<75 ( 91%/363 ) , just over half of ons omnibus ( 55%/326 ) , and less than a fifth of qol follow - up respondents ( 17%/47 ) , were aged 65<75 . thirty - eight per cent ( 152 ) of the ethnibus sample were indian , 29% ( 117 ) were pakistani , 22% ( 86 ) were black caribbean and 11% ( 45 ) were chinese . most , 94% ( 555 ) of the ons omnibus sample were white british ; all qol follow - up respondents were white british . in reflection of their younger age , more of the ethnibus than other respondents were married or cohabiting ( 58%/230 , 49%/285 , 49%/138 respectively ) . fewer ethnibus than other respondents were home - owners ( 532%/208 , 73%/429 , 85%/239 respectively ) and fewer lived alone ( 5%/19 , 48%/286 , 49%/137 respectively ) ( all differences were statistically significant at least at p<0.01 . ) for detailed characteristics of the samples , see supplementary table 1 . few , 12%/70 , of the ons omnibus sample , compared with more , 45%/113 of the older qol follow - up sample , and 73%/290 of the ethnibus sample were in the lowest two opqol categories ( < 119 ) , indicating worse qol ( see supplementary table 2 ) . the ethnibus and ons cross - sectional samples only were administered the casp-19 and whoqol - old . consistent with the opqol findings , 23%/94 of ethnibus respondents were in the worst two casp-19 categories ( < 29 ) , compared with 8%/43 of ons respondents ; 25%/100 of the ethnibus sample fell in the worst two whoqol - old categories , compared with 15%/80 of the ons respondents ( see supplementary tables 3 and 4 ) . further analyses by total qol scores and ethnicity in the ethnibus sample showed that 58% ( 26 ) of chinese people scored a good qol with the opqol , compared with 28% ( 33 ) of pakistani , 20% ( 31 ) of indian and 23% ( 31 ) of black caribbean people ( test 28.064 , 2 degrees of freedom , differences by ethnicity were not analysed in the other samples due to their low numbers in ethnic minority groups . the reliability criterion for item - total correlations ( the correlation of the item with the scale total with that item omitted ) is that the item should correlate with the total scale by at least 0.20 . with three exceptions , the 35 full opqol items met this criterion for all three samples ( the exceptions were in the ethnibus sample with items 10 , 12 and 32 ; but as cronbach 's was not improved by their removal , and they performed well in validity tests , they were retained ) . six of the 19 casp items failed to meet this criterion ( ethnibus : items 1 , 2 , 5 , 17 , 18 ; ons : item 6 ) . fourteen of the 24 whoqol - old items failed this criterion in the ethnibus sample only . as expected , all items correlated more highly with similar , than dissimilar , items in the scales . cronbach 's for the opqol in all three samples satisfied the 0.70<0.90 threshold for internal consistency : 0.748 ( ethnibus survey ) , 0.876 ( ons omnibus survey ) , 0.901 ( qol follow - up survey ) . the casp-19 and the whoqol - old satisfied the threshold for cronbach 's in the ons sample ( 0.866 and 0.849 respectively ) , but neither met this in ethnibus ( 0.553 and 0.415 respectively ) ( see earlier , neither were administered in the qol follow - up sample ) . the 4 week test - retest correlations , assessed among qol follow - up survey respondents , ranged from moderate to high ( r 0.4030.782 ) . lower correlations were explained by reported life changes in the intervening month , demonstrating the difficulties of test - retest exercises in older populations . respondents ' comments at follow - up about life changes in the last 4 weeks illustrate this:about 4 days ago the plaster was taken off my left hand so now i can go on buses again - my only means of regular transport apart from volunteer drivers , a few friends and taxis . anyway it means i am free;my husband of nearly 60 years was told he has lung cancer so it has changed very much how i feel . we are trying to be as normal as possible but it 's very hard;my daughter and her young son have now left our home and acquired her own house . we miss them a lot;my husband has just come home after spending another 2 weeks in hospital ( suspected heart attack). about 4 days ago the plaster was taken off my left hand so now i can go on buses again - my only means of regular transport apart from volunteer drivers , a few friends and taxis . anyway it means i am free ; my husband of nearly 60 years was told he has lung cancer so it has changed very much how i feel . we are trying to be as normal as possible but it 's very hard ; my daughter and her young son have now left our home and acquired her own house . we miss them a lot ; my husband has just come home after spending another 2 weeks in hospital ( suspected heart attack). in order to test the criterion ( also known as concurrent ) validity of the qol scales , all respondents were asked to rate the qol of their lives overall and by area of life ( qol domain ) , using five - point scales from very good to very bad. the criterion validity of all three qol scales was indicated by their moderate to strong , significant correlations with global self - rated qol : the spearman 's r correlations for the opqol by self - rated qol overall in each sample were ethnibus 0.347 , ons 0.602 and qol follow - up 0.659 . for the casp , in the two cross - sectional samples , they were ethnibus 0.273 , ons 0.577 , and for the whoqol - old , in the two cross - sectional samples , they were ethnibus 0.128 and ons 0.466 . all correlations were significant at least at p<0.01 , with the exception of whoqol - old in the ethnibus sample which was p<0.05 . the validity of the opqol was further supported by significant correlations between its subscales and the independent qol domain ratings , in theoretically expected , similar directions7 ( eg , opqol health and functioning subscale correlated with self - rated health : spearman 's r ethnibus 0.122 ( p<0.05 ) , ons omnibus 0.679 ( p<0.01 ) and qol follow - up 0.713 ( p<0.01 ) . there were no significant correlations with dissimilar pairs ( eg , health and religion ) , again as expected . the casp-19 control and autonomy subscales and the whoqol - old autonomy subscale also correlated significantly , as expected in similar directions , with self - rated independence , control over life and freedom in the ons sample ( r 0.472 , p<0.01 ; r 0.466 , p<0.01 respectively ) , but not in the ethnibus sample . the whoqol - old sensory abilities subscale correlated significantly , again as expected , with self - rated health in the ons ( r 0.322 , p<0.01 ) , but not the ethnibus sample . the whoqol - old intimacy subscale correlated significantly , also as expected , with the social relationships domain in the ons sample ( r 0.330 , p<0.01 ) , but not in the ethnibus sample . in support of construct ( convergent ) validity , the opqol correlated moderately strongly in the same direction , as hypothesised,7 with self - rated health status ( compared with others of same age ) in each sample : opqol ethnibus 0.364 , ons 0.543 and qol follow - up 0.628 . the casp-19 and whoqol - old correlations in the two cross - sectional samples were also in the same direction and significant , although slightly weaker ( casp-19 ethnibus 0.238 , ons 0.530 ; whoqol - old ethnibus 0.138 , ons 0.465 ; all p<0.01 ) . multivariable analyses were conducted with each sample in order to examine independent predictors of the opqol , casp-19 and whoqol - old . for comparability , the same independent variables were entered into each model . on the basis of the literature,7 optimum scores on each measure were hypothesised to be associated with optimum qol : self - rated active ageing , independent self - ratings of qol domains , social activities and help from social network members , self - rated health status and physical functioning ( adl ) , age , sex , marital status and housing tenure . the qol follow - up sample also provided an opportunity to test the causal model underpinning the opqol . the cross - sectional model for the qol follow - up sample was highly significant ( see table 1 ) . perceptions of ageing more actively , having optimal self - ratings of health , independence , home and neighbourhood , psychological well - being and finances , more social activities and female sex significantly , and independently , predicted optimal opqol scores . the amount of explained variance of opqol scores in the model was high at 77% ( adjusted r 0.774 ) . multiple regression of predictors of opqol : qol follow - up sample ( final model ) adl , activities of daily living ; ns , not significant ; opqol , older people 's quality of life ; qol , quality of life . the opqol models in the ons and ethnibus samples were also highly significant . again , optimal ratings of active ageing , most self - rated qol domains and also self - rated health status were significant in both samples . the model explained 65% of the variance in opqol scores ( adjusted r 0.653 ) in the ons sample and 43% ( adjusted r 0.430 ) in the ethnibus sample ( table 2 ) . multiple regression of predictors of opqol : ons omnibus and ethnibus samples ( final models ) adl , activities of daily living ; ns , not significant ; qol , quality of life . the variables included in the test of the causal model underpinning the opqol , in the qol follow - up sample , were the baseline indicators that reflected the components chosen for the opqol domains ( health and functional status , practical help received , social support and activities , perceived quality of neighbourhood , psychological outlook , gap score for social comparisons and expectations and self - efficacy ) , plus standard sociodemographic indications to control for their effects . this model explained 56% of the variance in opqol scores ( adjusted r : 0.563 ) . as number of different social activities was not significant in the model , a reduced model was conducted excluding this variable . health status and number of diagnosed medical conditions , help and social support , perceptions of neighbourhood and feeling safe , social comparisons ( comparing one 's financial and living circumstances with others who are worse off ) , feelings of self - efficacy and control , then explained 48% of the variance in opqol scores in expected directions ( adjusted r 0.481 ) . the overall model was highly significant in general support of the opqol ( see table 3 ) . causal model underpinning opqol adl , activities of daily living ; ns , not statistically significant at least the 0.05 level ; opqol , older people 's quality of life . multiple regression of baseline ( 1999/2000 ) predictors of opqol at follow - up ( 2007/2008 ) : qol follow - up sample ( final model ) . the amount of explained variance in casp-19 scores in the ons sample explained by the model was 57% ( adjusted r 0.568 ) ; the model was highly significant , and in expected directions . the variables that retained significance in the model were five of the domain ratings , health and functioning . in contrast , the casp-19 model for the ethnibus sample was weak : the amount of explained variance in casp-19 scores was just 14% ( adjusted r 0.141 ) , although the model was still significant . the variables that were significant were self - rated active ageing , and three of the seven qol domain self - ratings , health status , but not physical functioning ( see table 4 ) . multiple regression of predictors of casp-19:ons omnibus and ethnibus ( final models ) adl , activities of daily living ; ns , not significant ; qol , quality of life . the whoqol - old was assessed in the ons and ethnibus samples the amount of explained variance in whoqol - old scores in the ons omnibus survey was 45% ( adjusted r 0.448 ) ; the model was highly significant , again in expected directions . the significant variables were self - rated active ageing , three of the seven qol domain ratings and the number of social activities and helpers , health status and housing tenure . however , the whoqol - old model for the ethnibus sample was weak , although significant : the amount of explained variance in whoqol - old scores was just 5% ( adjusted r 0.048 ) . the significant variables were three of the seven domain ratings , and number of social activities ( see table 5 ) . multiple regression of predictors of whoqol - old : ons omnibus and ethnibus ( final model ) adl , activities of daily living ; ns , not significant ; qol , quality of life . just over half of each sample comprised women ( 52%/207 ethnibus , 55%/324 ons , 54%/154 qol follow - up ) . whereas most ethnibus respondents were aged 65<75 ( 91%/363 ) , just over half of ons omnibus ( 55%/326 ) , and less than a fifth of qol follow - up respondents ( 17%/47 ) , were aged 65<75 . thirty - eight per cent ( 152 ) of the ethnibus sample were indian , 29% ( 117 ) were pakistani , 22% ( 86 ) were black caribbean and 11% ( 45 ) were chinese . most , 94% ( 555 ) of the ons omnibus sample were white british ; all qol follow - up respondents were white british . in reflection of their younger age , more of the ethnibus than other respondents were married or cohabiting ( 58%/230 , 49%/285 , 49%/138 respectively ) . fewer ethnibus than other respondents were home - owners ( 532%/208 , 73%/429 , 85%/239 respectively ) and fewer lived alone ( 5%/19 , 48%/286 , 49%/137 respectively ) ( all differences were statistically significant at least at p<0.01 . ) for detailed characteristics of the samples , see supplementary table 1 . few , 12%/70 , of the ons omnibus sample , compared with more , 45%/113 of the older qol follow - up sample , and 73%/290 of the ethnibus sample were in the lowest two opqol categories ( < 119 ) , indicating worse qol ( see supplementary table 2 ) . the ethnibus and ons cross - sectional samples only were administered the casp-19 and whoqol - old . consistent with the opqol findings , 23%/94 of ethnibus respondents were in the worst two casp-19 categories ( < 29 ) , compared with 8%/43 of ons respondents ; 25%/100 of the ethnibus sample fell in the worst two whoqol - old categories , compared with 15%/80 of the ons respondents ( see supplementary tables 3 and 4 ) . further analyses by total qol scores and ethnicity in the ethnibus sample showed that 58% ( 26 ) of chinese people scored a good qol with the opqol , compared with 28% ( 33 ) of pakistani , 20% ( 31 ) of indian and 23% ( 31 ) of black caribbean people ( test 28.064 , 2 degrees of freedom , differences by ethnicity were not analysed in the other samples due to their low numbers in ethnic minority groups . the reliability criterion for item - total correlations ( the correlation of the item with the scale total with that item omitted ) is that the item should correlate with the total scale by at least 0.20 . with three exceptions , the 35 full opqol items met this criterion for all three samples ( the exceptions were in the ethnibus sample with items 10 , 12 and 32 ; but as cronbach 's was not improved by their removal , and they performed well in validity tests , they were retained ) . six of the 19 casp items failed to meet this criterion ( ethnibus : items 1 , 2 , 5 , 17 , 18 ; ons : item 6 ) . fourteen of the 24 whoqol - old items failed this criterion in the ethnibus sample only . as expected , all items correlated more highly with similar , than dissimilar , items in the scales . cronbach 's for the opqol in all three samples satisfied the 0.70<0.90 threshold for internal consistency : 0.748 ( ethnibus survey ) , 0.876 ( ons omnibus survey ) , 0.901 ( qol follow - up survey ) . the casp-19 and the whoqol - old satisfied the threshold for cronbach 's in the ons sample ( 0.866 and 0.849 respectively ) , but neither met this in ethnibus ( 0.553 and 0.415 respectively ) ( see earlier , neither were administered in the qol follow - up sample ) . the 4 week test - retest correlations , assessed among qol follow - up survey respondents , ranged from moderate to high ( r 0.4030.782 ) . lower correlations were explained by reported life changes in the intervening month , demonstrating the difficulties of test - retest exercises in older populations . respondents ' comments at follow - up about life changes in the last 4 weeks illustrate this:about 4 days ago the plaster was taken off my left hand so now i can go on buses again - my only means of regular transport apart from volunteer drivers , a few friends and taxis . anyway it means i am free;my husband of nearly 60 years was told he has lung cancer so it has changed very much how i feel . we are trying to be as normal as possible but it 's very hard;my daughter and her young son have now left our home and acquired her own house . we miss them a lot;my husband has just come home after spending another 2 weeks in hospital ( suspected heart attack). about 4 days ago the plaster was taken off my left hand so now i can go on buses again - my only means of regular transport apart from volunteer drivers , a few friends and taxis . anyway it means i am free ; my husband of nearly 60 years was told he has lung cancer so it has changed very much how i feel . we are trying to be as normal as possible but it 's very hard ; my daughter and her young son have now left our home and acquired her own house . we miss them a lot ; my husband has just come home after spending another 2 weeks in hospital ( suspected heart attack). in order to test the criterion ( also known as concurrent ) validity of the qol scales , all respondents were asked to rate the qol of their lives overall and by area of life ( qol domain ) , using five - point scales from very good to very bad. the criterion validity of all three qol scales was indicated by their moderate to strong , significant correlations with global self - rated qol : the spearman 's r correlations for the opqol by self - rated qol overall in each sample were ethnibus 0.347 , ons 0.602 and qol follow - up 0.659 . for the casp , in the two cross - sectional samples , they were ethnibus 0.273 , ons 0.577 , and for the whoqol - old , in the two cross - sectional samples , they were ethnibus 0.128 and ons 0.466 . all correlations were significant at least at p<0.01 , with the exception of whoqol - old in the ethnibus sample which was p<0.05 . the validity of the opqol was further supported by significant correlations between its subscales and the independent qol domain ratings , in theoretically expected , similar directions7 ( eg , opqol health and functioning subscale correlated with self - rated health : spearman 's r ethnibus 0.122 ( p<0.05 ) , ons omnibus 0.679 ( p<0.01 ) and qol follow - up 0.713 ( p<0.01 ) . there were no significant correlations with dissimilar pairs ( eg , health and religion ) , again as expected . the casp-19 control and autonomy subscales and the whoqol - old autonomy subscale also correlated significantly , as expected in similar directions , with self - rated independence , control over life and freedom in the ons sample ( r 0.472 , p<0.01 ; r 0.466 , p<0.01 respectively ) , but not in the ethnibus sample . the whoqol - old sensory abilities subscale correlated significantly , again as expected , with self - rated health in the ons ( r 0.322 , p<0.01 ) , but not the ethnibus sample . the whoqol - old intimacy subscale correlated significantly , also as expected , with the social relationships domain in the ons sample ( r 0.330 , p<0.01 ) , but not in the ethnibus sample . in support of construct ( convergent ) validity , the opqol correlated moderately strongly in the same direction , as hypothesised,7 with self - rated health status ( compared with others of same age ) in each sample : opqol ethnibus 0.364 , ons 0.543 and qol follow - up 0.628 . the casp-19 and whoqol - old correlations in the two cross - sectional samples were also in the same direction and significant , although slightly weaker ( casp-19 ethnibus 0.238 , ons 0.530 ; whoqol - old ethnibus 0.138 , ons 0.465 ; all p<0.01 ) . multivariable analyses were conducted with each sample in order to examine independent predictors of the opqol , casp-19 and whoqol - old . for comparability , the same independent variables were entered into each model . on the basis of the literature,7 optimum scores on each measure were hypothesised to be associated with optimum qol : self - rated active ageing , independent self - ratings of qol domains , social activities and help from social network members , self - rated health status and physical functioning ( adl ) , age , sex , marital status and housing tenure . the qol follow - up sample also provided an opportunity to test the causal model underpinning the opqol . the cross - sectional model for the qol follow - up sample was highly significant ( see table 1 ) . perceptions of ageing more actively , having optimal self - ratings of health , independence , home and neighbourhood , psychological well - being and finances , more social activities and female sex significantly , and independently , predicted optimal opqol scores . the amount of explained variance of opqol scores in the model was high at 77% ( adjusted r 0.774 ) . multiple regression of predictors of opqol : qol follow - up sample ( final model ) adl , activities of daily living ; ns , not significant ; opqol , older people 's quality of life ; qol , quality of life . the opqol models in the ons and ethnibus samples were also highly significant . again , optimal ratings of active ageing , most self - rated qol domains and also self - rated health status were significant in both samples . the model explained 65% of the variance in opqol scores ( adjusted r 0.653 ) in the ons sample and 43% ( adjusted r 0.430 ) in the ethnibus sample ( table 2 ) . multiple regression of predictors of opqol : ons omnibus and ethnibus samples ( final models ) adl , activities of daily living ; ns , not significant ; qol , quality of life . the variables included in the test of the causal model underpinning the opqol , in the qol follow - up sample , were the baseline indicators that reflected the components chosen for the opqol domains ( health and functional status , practical help received , social support and activities , perceived quality of neighbourhood , psychological outlook , gap score for social comparisons and expectations and self - efficacy ) , plus standard sociodemographic indications to control for their effects . this model explained 56% of the variance in opqol scores ( adjusted r : 0.563 ) . as number of different social activities was not significant in the model , a reduced model was conducted excluding this variable . health status and number of diagnosed medical conditions , help and social support , perceptions of neighbourhood and feeling safe , social comparisons ( comparing one 's financial and living circumstances with others who are worse off ) , feelings of self - efficacy and control , then explained 48% of the variance in opqol scores in expected directions ( adjusted r 0.481 ) . the overall model was highly significant in general support of the opqol ( see table 3 ) . causal model underpinning opqol adl , activities of daily living ; ns , not statistically significant at least the 0.05 level ; opqol , older people 's quality of life . multiple regression of baseline ( 1999/2000 ) predictors of opqol at follow - up ( 2007/2008 ) : qol follow - up sample ( final model ) . the amount of explained variance in casp-19 scores in the ons sample explained by the model was 57% ( adjusted r 0.568 ) ; the model was highly significant , and in expected directions . the variables that retained significance in the model were five of the domain ratings , health and functioning . in contrast , the casp-19 model for the ethnibus sample was weak : the amount of explained variance in casp-19 scores was just 14% ( adjusted r 0.141 ) , although the model was still significant . the variables that were significant were self - rated active ageing , and three of the seven qol domain self - ratings , health status , but not physical functioning ( see table 4 ) . multiple regression of predictors of casp-19:ons omnibus and ethnibus ( final models ) adl , activities of daily living ; ns , not significant ; qol , quality of life . the whoqol - old was assessed in the ons and ethnibus samples the amount of explained variance in whoqol - old scores in the ons omnibus survey was 45% ( adjusted r 0.448 ) ; the model was highly significant , again in expected directions . the significant variables were self - rated active ageing , three of the seven qol domain ratings and the number of social activities and helpers , health status and housing tenure . however , the whoqol - old model for the ethnibus sample was weak , although significant : the amount of explained variance in whoqol - old scores was just 5% ( adjusted r 0.048 ) . the significant variables were three of the seven domain ratings , and number of social activities ( see table 5 ) . multiple regression of predictors of whoqol - old : ons omnibus and ethnibus ( final model ) adl , activities of daily living ; ns , not significant ; qol , quality of life . the cross - sectional model for the qol follow - up sample was highly significant ( see table 1 ) . perceptions of ageing more actively , having optimal self - ratings of health , independence , home and neighbourhood , psychological well - being and finances , more social activities and female sex significantly , and independently , predicted optimal opqol scores . the amount of explained variance of opqol scores in the model was high at 77% ( adjusted r 0.774 ) . multiple regression of predictors of opqol : qol follow - up sample ( final model ) adl , activities of daily living ; ns , not significant ; opqol , older people 's quality of life ; qol , quality of life . the opqol models in the ons and ethnibus samples were also highly significant . again , optimal ratings of active ageing , most self - rated qol domains and also self - rated health status were significant in both samples . the model explained 65% of the variance in opqol scores ( adjusted r 0.653 ) in the ons sample and 43% ( adjusted r 0.430 ) in the ethnibus sample ( table 2 ) . multiple regression of predictors of opqol : ons omnibus and ethnibus samples ( final models ) adl , activities of daily living ; ns , not significant ; qol , quality of life . the variables included in the test of the causal model underpinning the opqol , in the qol follow - up sample , were the baseline indicators that reflected the components chosen for the opqol domains ( health and functional status , practical help received , social support and activities , perceived quality of neighbourhood , psychological outlook , gap score for social comparisons and expectations and self - efficacy ) , plus standard sociodemographic indications to control for their effects . this model explained 56% of the variance in opqol scores ( adjusted r : 0.563 ) . as number of different social activities was not significant in the model , a reduced model was conducted excluding this variable . health status and number of diagnosed medical conditions , help and social support , perceptions of neighbourhood and feeling safe , social comparisons ( comparing one 's financial and living circumstances with others who are worse off ) , feelings of self - efficacy and control , then explained 48% of the variance in opqol scores in expected directions ( adjusted r 0.481 ) . the overall model was highly significant in general support of the opqol ( see table 3 ) . causal model underpinning opqol adl , activities of daily living ; ns , not statistically significant at least the 0.05 level ; opqol , older people 's quality of life . multiple regression of baseline ( 1999/2000 ) predictors of opqol at follow - up ( 2007/2008 ) : qol follow - up sample ( final model ) . the amount of explained variance in casp-19 scores in the ons sample explained by the model was 57% ( adjusted r 0.568 ) ; the model was highly significant , and in expected directions . the variables that retained significance in the model were five of the domain ratings , health and functioning . in contrast , the casp-19 model for the ethnibus sample was weak : the amount of explained variance in casp-19 scores was just 14% ( adjusted r 0.141 ) , although the model was still significant . the variables that were significant were self - rated active ageing , and three of the seven qol domain self - ratings , health status , but not physical functioning ( see table 4 ) . multiple regression of predictors of casp-19:ons omnibus and ethnibus ( final models ) adl , activities of daily living ; ns , not significant ; qol , quality of life . the whoqol - old was assessed in the ons and ethnibus samples the amount of explained variance in whoqol - old scores in the ons omnibus survey was 45% ( adjusted r 0.448 ) ; the model was highly significant , again in expected directions . the significant variables were self - rated active ageing , three of the seven qol domain ratings and the number of social activities and helpers , health status and housing tenure . however , the whoqol - old model for the ethnibus sample was weak , although significant : the amount of explained variance in whoqol - old scores was just 5% ( adjusted r 0.048 ) . the significant variables were three of the seven domain ratings , and number of social activities ( see table 5 ) . multiple regression of predictors of whoqol - old : ons omnibus and ethnibus ( final model ) adl , activities of daily living ; ns , not significant ; qol , quality of life . this study describes the psychometric performance of a qol questionnaire , developed from the perspectives of older people themselves : the opqol . it was tested in two cross - sectional , and one longitudinal , surveys of older people across britain . the longitudinal survey enabled the opqol to be tested in a dynamic , ageing population and an assessment of its underlying model , although its self - administration mode necessitated the assessment of the opqol only ( and not the casp-19 or whoqol ) in this older sample . the surveys used statistically robust sampling methods , and the response rates were fairly to very good . the characteristics of respondents to the ons omnibus and ethnibus surveys ( and the qol survey at baseline ) were comparable with population estimates from the last census . the qol follow - up sample , by its longitudinal design , reflected the healthy survivors . also , although the sampling approach of the ethnibus survey was statistically robust , it used focused enumeration . there is no other practical methodology for attempting to obtain representative samples of people in ethnic minority groups in national samples . this study reported that ethnibus respondents obtained poorer ( worse ) qol scores than the other sample respondents , with the opqol , casp-19 and whoqol - old . this is not unexpected given that people in ethnic minority groups are often more economically disadvantaged than the wider population.15 further research is needed to examine whether differences in qol reflect real variations , methodology , and cultural variations in expectations or in reporting . ethnic minority groups in britain live in a wide range of different communities , and their diversity may also have affected responses in some way . it should also be noted that the standard question for ethnic status used , largely reflected britain 's new commonwealth groups , and may not be appropriate for use in other countries . multiple regression models supported its validity and underlying constructs . despite the ethnibus sample 's consistently worse qol scores , compared with the other samples , the casp-19 and whoqol - old did not meet all criteria for internal consistency ( reliability ) in the ethnically diverse ethnibus sample . the casp-19 and whoqol - old also had relatively large numbers of items that failed to meet the reliability criterion for item - total scale correlations ; they frequently failed correlation tests for validity in the ethnibus sample . this may have been due to this sample 's ethnic diversity , or because the casp-19 and whoqol - old were not sufficiently sensitive . the opqol is currently being tested with older people living in italy ; initial results for cultural equivalence and understanding are positive ( personal communication , dr claudio bilotta , university of milan).what is already known on this subjectincreasing numbers of older people , higher expectations for a good life , and demands for health and social care , have led to international interest in the enhancement , and measurement , of quality of life ( qol ) in older age.qol is a subjective concept , yet most measures of qol are based primarily or partly on expert opinions.what this study addsthis study focuses on the testing of a new measure of qol , the older people 's qol questionnaire ( opqol ) , which was derived entirely from the views of older people in britain , cross - checked against theoretical models for comprehensiveness.the opqol performed well in three samples of older people in britain , one of which comprised people from ethnic minority groups . it is of potential value in the outcome assessment of health and social interventions , which can have a multidimensional impact on people 's lives . increasing numbers of older people , higher expectations for a good life , and demands for health and social care , have led to international interest in the enhancement , and measurement , of quality of life ( qol ) in older age . qol is a subjective concept , yet most measures of qol are based primarily or partly on expert opinions . this study focuses on the testing of a new measure of qol , the older people 's qol questionnaire ( opqol ) , which was derived entirely from the views of older people in britain , cross - checked against theoretical models for comprehensiveness . the opqol performed well in three samples of older people in britain , one of which comprised people from ethnic minority groups . it is of potential value in the outcome assessment of health and social interventions , which can have a multidimensional impact on people 's lives .

backgroundmost measures of quality of life ( qol ) are based on expert opinions . this study describes a new measure of qol in older age , the older people 's qol questionnaire ( opqol ) , which is unique in being derived from the views of lay people , cross - checked against theoretical models for assessment of comprehensiveness . its performance was assessed cross - sectionally and longitudinally . it was compared with two existing qol measures in the cross - sectional studies in order to identify the optimal measure for use with older populations.methodsdata were taken from three surveys of older people living at home in britain in 20072008 : one population survey of people aged 65 + , one focused enumeration survey of ethnically diverse older people aged 65 + , one follow - up of a population survey of people aged 65 + at baseline in 1999/2000 . measures were qol ( using opqol , control , autonomy , satisfaction , pleasure - 19 items ( casp-19 ) , world health organization quality of life questionnaire - version for older people ( whoqol - old ) ) , health , social and socioeconomic circumstances . the casp-19 and whoqol - old were not administered to the longitudinal sample in order to reduce respondent burden.resultspsychometric tests were applied to each qol measure . the opqol , casp-19 and whoqol - old performed well with the cross - sectional samples ; however , only the opqol met criteria for internal consistency in the ethnibus samples.conclusionthe opqol is of potential value in the outcome assessment of health and social interventions , which can have a multidimensional impact on people 's lives . further research is needed to examine whether differences by ethnicity reflect real differences in qol , methodological issues , variations in expectations or cultural differences in reporting .

Background Methods Measures Statistical analysis Results Characteristics of samples Distributions of samples on QoL scales Reliability Validity Multivariable analyses OPQOL CASP-19 WHOQOL-OLD Conclusion

governments across the developed world are concerned with enabling older people to maintain their active contribution to society , and thereby their quality of life ( qol).1 qol has become a commonly used end point in the evaluation of multisector public policy , including health , social , community and environmental policy actions . lawton26 developed a popularly cited quadripartite concept of qol , proposing that the good life ( qol ) may be represented by behavioural and social competence ( health , cognition , time use , social behaviour ) , perceptions of qol ( subjective evaluation of each domain of life ) , psychological well - being ( mental health , cognitive judgements of life satisfaction , positive - negative emotions ) and the external , objective , physical environment ( housing , economic indicators ) . however , there is no consensus about its conceptual definition or measurement,7 and most investigators have based their concepts on expert opinions rather than the perspectives of lay people . thus , it is increasingly important to develop a multidimensional model and measure of quality of life , for use in descriptive and evaluative multisector policy research , which reflects the views of the population concerned , with cross - sectional and longitudinal applicability . elicitation of people 's own views of qol in this process is particularly important because qol is a subjective concept . survey and qualitative research with people aged 65 + , living at home in britain , reported that the central planks of qol emphasised by respondents were psychological well - being and positive outlook , having health and functioning , social relationships , leisure activities , neighbourhood resources , adequate financial circumstances and independence.710 this research led to the development of the older people 's quality of life questionnaire ( opqol ) , which is unique in being derived from the views of a representative sample of older people , cross - checked against theoretical models for assessment comprehensiveness . the aim here is to compare the psychometric properties of the opqol , with the casp-19 and whoqol - old among people 65 + participating in three national surveys of older people living at home in britain . two of these three surveys were cross - sectional , and the third was longitudinal ( see supplementary appendix 1 ) : ethnibus survey of people aged 65 + responding to two waves of the national ethni surveys ( http://www.ethnicfocus.com ) in 2008 . this is a rolling face - to - face interview survey with adults aged 16 + , living at home , based on focused enumeration , stratified random sampling of postcodes in britain , and statistically robust sampling of people in common ethnic minority groups in britain ; the response rate was 70% ( n=400 ) . qol follow - up survey in 20072008 , of people living at home in britain , aged 65 + at baseline , who had responded to four ons national omnibus interview surveys . the qol follow - up survey is included here as the longitudinal design provided the opportunity to test the causal model of the opqol , as well as a willing sample for test - retest reliability assessment . prior to administration in the surveys reported here , the items in the opqol were pretested with 179 older people and three focus groups , reduced to 32-item and 35-item versions , and statistical tests of reliability and validity were applied . supplementary appendix 2 displays the opqol , summarises its development and briefly summarises the casp-19 and whoqol - old . independent self - ratings of global qol , and of its domains , were included in the questionnaire in order to distinguish between the constituents of , and influences on , qol.14 also included were standard sociodemographic items , self - rated active ageing , items measuring health and psychosocial circumstances.7 ethnic status was measured using a standard item about ethnic identity in the uk . prior to administration in the surveys reported here , the items in the opqol were pretested with 179 older people and three focus groups , reduced to 32-item and 35-item versions , and statistical tests of reliability and validity were applied . supplementary appendix 2 displays the opqol , summarises its development and briefly summarises the casp-19 and whoqol - old . independent self - ratings of global qol , and of its domains , were included in the questionnaire in order to distinguish between the constituents of , and influences on , qol.14 also included were standard sociodemographic items , self - rated active ageing , items measuring health and psychosocial circumstances.7 ethnic status was measured using a standard item about ethnic identity in the uk . this would not necessarily be applicable to populations in other countries , because it reflects close connections between new commonwealth countries and ethnic minority groups in the uk.15 tests of scale reliability were applied in order to assess the extent to which scale items measure the same construct , with freedom from random error ( internal consistency ) . few , 12%/70 , of the ons omnibus sample , compared with more , 45%/113 of the older qol follow - up sample , and 73%/290 of the ethnibus sample were in the lowest two opqol categories ( < 119 ) , indicating worse qol ( see supplementary table 2 ) . the ethnibus and ons cross - sectional samples only were administered the casp-19 and whoqol - old . consistent with the opqol findings , 23%/94 of ethnibus respondents were in the worst two casp-19 categories ( < 29 ) , compared with 8%/43 of ons respondents ; 25%/100 of the ethnibus sample fell in the worst two whoqol - old categories , compared with 15%/80 of the ons respondents ( see supplementary tables 3 and 4 ) . further analyses by total qol scores and ethnicity in the ethnibus sample showed that 58% ( 26 ) of chinese people scored a good qol with the opqol , compared with 28% ( 33 ) of pakistani , 20% ( 31 ) of indian and 23% ( 31 ) of black caribbean people ( test 28.064 , 2 degrees of freedom , differences by ethnicity were not analysed in the other samples due to their low numbers in ethnic minority groups . cronbach 's for the opqol in all three samples satisfied the 0.70<0.90 threshold for internal consistency : 0.748 ( ethnibus survey ) , 0.876 ( ons omnibus survey ) , 0.901 ( qol follow - up survey ) . the casp-19 and the whoqol - old satisfied the threshold for cronbach 's in the ons sample ( 0.866 and 0.849 respectively ) , but neither met this in ethnibus ( 0.553 and 0.415 respectively ) ( see earlier , neither were administered in the qol follow - up sample ) . in order to test the criterion ( also known as concurrent ) validity of the qol scales , all respondents were asked to rate the qol of their lives overall and by area of life ( qol domain ) , using five - point scales from very good to very bad. for the casp , in the two cross - sectional samples , they were ethnibus 0.273 , ons 0.577 , and for the whoqol - old , in the two cross - sectional samples , they were ethnibus 0.128 and ons 0.466 . the casp-19 control and autonomy subscales and the whoqol - old autonomy subscale also correlated significantly , as expected in similar directions , with self - rated independence , control over life and freedom in the ons sample ( r 0.472 , p<0.01 ; r 0.466 , p<0.01 respectively ) , but not in the ethnibus sample . the whoqol - old sensory abilities subscale correlated significantly , again as expected , with self - rated health in the ons ( r 0.322 , p<0.01 ) , but not the ethnibus sample . the whoqol - old intimacy subscale correlated significantly , also as expected , with the social relationships domain in the ons sample ( r 0.330 , p<0.01 ) , but not in the ethnibus sample . in support of construct ( convergent ) validity , the opqol correlated moderately strongly in the same direction , as hypothesised,7 with self - rated health status ( compared with others of same age ) in each sample : opqol ethnibus 0.364 , ons 0.543 and qol follow - up 0.628 . the casp-19 and whoqol - old correlations in the two cross - sectional samples were also in the same direction and significant , although slightly weaker ( casp-19 ethnibus 0.238 , ons 0.530 ; whoqol - old ethnibus 0.138 , ons 0.465 ; all p<0.01 ) . multivariable analyses were conducted with each sample in order to examine independent predictors of the opqol , casp-19 and whoqol - old . multiple regression of predictors of opqol : qol follow - up sample ( final model ) adl , activities of daily living ; ns , not significant ; opqol , older people 's quality of life ; qol , quality of life . the variables included in the test of the causal model underpinning the opqol , in the qol follow - up sample , were the baseline indicators that reflected the components chosen for the opqol domains ( health and functional status , practical help received , social support and activities , perceived quality of neighbourhood , psychological outlook , gap score for social comparisons and expectations and self - efficacy ) , plus standard sociodemographic indications to control for their effects . causal model underpinning opqol adl , activities of daily living ; ns , not statistically significant at least the 0.05 level ; opqol , older people 's quality of life . multiple regression of predictors of whoqol - old : ons omnibus and ethnibus ( final model ) adl , activities of daily living ; ns , not significant ; qol , quality of life . few , 12%/70 , of the ons omnibus sample , compared with more , 45%/113 of the older qol follow - up sample , and 73%/290 of the ethnibus sample were in the lowest two opqol categories ( < 119 ) , indicating worse qol ( see supplementary table 2 ) . the ethnibus and ons cross - sectional samples only were administered the casp-19 and whoqol - old . consistent with the opqol findings , 23%/94 of ethnibus respondents were in the worst two casp-19 categories ( < 29 ) , compared with 8%/43 of ons respondents ; 25%/100 of the ethnibus sample fell in the worst two whoqol - old categories , compared with 15%/80 of the ons respondents ( see supplementary tables 3 and 4 ) . further analyses by total qol scores and ethnicity in the ethnibus sample showed that 58% ( 26 ) of chinese people scored a good qol with the opqol , compared with 28% ( 33 ) of pakistani , 20% ( 31 ) of indian and 23% ( 31 ) of black caribbean people ( test 28.064 , 2 degrees of freedom , differences by ethnicity were not analysed in the other samples due to their low numbers in ethnic minority groups . cronbach 's for the opqol in all three samples satisfied the 0.70<0.90 threshold for internal consistency : 0.748 ( ethnibus survey ) , 0.876 ( ons omnibus survey ) , 0.901 ( qol follow - up survey ) . the casp-19 and the whoqol - old satisfied the threshold for cronbach 's in the ons sample ( 0.866 and 0.849 respectively ) , but neither met this in ethnibus ( 0.553 and 0.415 respectively ) ( see earlier , neither were administered in the qol follow - up sample ) . in order to test the criterion ( also known as concurrent ) validity of the qol scales , all respondents were asked to rate the qol of their lives overall and by area of life ( qol domain ) , using five - point scales from very good to very bad. for the casp , in the two cross - sectional samples , they were ethnibus 0.273 , ons 0.577 , and for the whoqol - old , in the two cross - sectional samples , they were ethnibus 0.128 and ons 0.466 . all correlations were significant at least at p<0.01 , with the exception of whoqol - old in the ethnibus sample which was p<0.05 . the casp-19 control and autonomy subscales and the whoqol - old autonomy subscale also correlated significantly , as expected in similar directions , with self - rated independence , control over life and freedom in the ons sample ( r 0.472 , p<0.01 ; r 0.466 , p<0.01 respectively ) , but not in the ethnibus sample . the whoqol - old intimacy subscale correlated significantly , also as expected , with the social relationships domain in the ons sample ( r 0.330 , p<0.01 ) , but not in the ethnibus sample . in support of construct ( convergent ) validity , the opqol correlated moderately strongly in the same direction , as hypothesised,7 with self - rated health status ( compared with others of same age ) in each sample : opqol ethnibus 0.364 , ons 0.543 and qol follow - up 0.628 . the casp-19 and whoqol - old correlations in the two cross - sectional samples were also in the same direction and significant , although slightly weaker ( casp-19 ethnibus 0.238 , ons 0.530 ; whoqol - old ethnibus 0.138 , ons 0.465 ; all p<0.01 ) . multivariable analyses were conducted with each sample in order to examine independent predictors of the opqol , casp-19 and whoqol - old . multiple regression of predictors of opqol : qol follow - up sample ( final model ) adl , activities of daily living ; ns , not significant ; opqol , older people 's quality of life ; qol , quality of life . the variables included in the test of the causal model underpinning the opqol , in the qol follow - up sample , were the baseline indicators that reflected the components chosen for the opqol domains ( health and functional status , practical help received , social support and activities , perceived quality of neighbourhood , psychological outlook , gap score for social comparisons and expectations and self - efficacy ) , plus standard sociodemographic indications to control for their effects . the cross - sectional model for the qol follow - up sample was highly significant ( see table 1 ) . multiple regression of predictors of opqol : qol follow - up sample ( final model ) adl , activities of daily living ; ns , not significant ; opqol , older people 's quality of life ; qol , quality of life . the variables included in the test of the causal model underpinning the opqol , in the qol follow - up sample , were the baseline indicators that reflected the components chosen for the opqol domains ( health and functional status , practical help received , social support and activities , perceived quality of neighbourhood , psychological outlook , gap score for social comparisons and expectations and self - efficacy ) , plus standard sociodemographic indications to control for their effects . causal model underpinning opqol adl , activities of daily living ; ns , not statistically significant at least the 0.05 level ; opqol , older people 's quality of life . the whoqol - old was assessed in the ons and ethnibus samples the amount of explained variance in whoqol - old scores in the ons omnibus survey was 45% ( adjusted r 0.448 ) ; the model was highly significant , again in expected directions . this study describes the psychometric performance of a qol questionnaire , developed from the perspectives of older people themselves : the opqol . it was tested in two cross - sectional , and one longitudinal , surveys of older people across britain . this study reported that ethnibus respondents obtained poorer ( worse ) qol scores than the other sample respondents , with the opqol , casp-19 and whoqol - old . this is not unexpected given that people in ethnic minority groups are often more economically disadvantaged than the wider population.15 further research is needed to examine whether differences in qol reflect real variations , methodology , and cultural variations in expectations or in reporting . despite the ethnibus sample 's consistently worse qol scores , compared with the other samples , the casp-19 and whoqol - old did not meet all criteria for internal consistency ( reliability ) in the ethnically diverse ethnibus sample . the casp-19 and whoqol - old also had relatively large numbers of items that failed to meet the reliability criterion for item - total scale correlations ; they frequently failed correlation tests for validity in the ethnibus sample . this may have been due to this sample 's ethnic diversity , or because the casp-19 and whoqol - old were not sufficiently sensitive . the opqol is currently being tested with older people living in italy ; initial results for cultural equivalence and understanding are positive ( personal communication , dr claudio bilotta , university of milan).what is already known on this subjectincreasing numbers of older people , higher expectations for a good life , and demands for health and social care , have led to international interest in the enhancement , and measurement , of quality of life ( qol ) in older age.qol is a subjective concept , yet most measures of qol are based primarily or partly on expert opinions.what this study addsthis study focuses on the testing of a new measure of qol , the older people 's qol questionnaire ( opqol ) , which was derived entirely from the views of older people in britain , cross - checked against theoretical models for comprehensiveness.the opqol performed well in three samples of older people in britain , one of which comprised people from ethnic minority groups . it is of potential value in the outcome assessment of health and social interventions , which can have a multidimensional impact on people 's lives . increasing numbers of older people , higher expectations for a good life , and demands for health and social care , have led to international interest in the enhancement , and measurement , of quality of life ( qol ) in older age . qol is a subjective concept , yet most measures of qol are based primarily or partly on expert opinions . this study focuses on the testing of a new measure of qol , the older people 's qol questionnaire ( opqol ) , which was derived entirely from the views of older people in britain , cross - checked against theoretical models for comprehensiveness . the opqol performed well in three samples of older people in britain , one of which comprised people from ethnic minority groups . it is of potential value in the outcome assessment of health and social interventions , which can have a multidimensional impact on people 's lives .

we reviewed the image database of all consecutive pediatric patients referred to the pediatric retina department of our institute , and who underwent imaging with a handheld sd - oct device . the envisu 2300 has a maximum camera line rate of 36 khz with a typical imaging line rate of 32 khz and an axial resolution of < 4 m in tissue . the maximum imaging depth in tissue is 2.5 mm with a digital resolution of 2.4 m / pixel . during each imaging session , the scans were obtained in rectangular volume scans and/or radial volume scans . for each imaging session , the reference arm length was adjusted according to the axial length of the patient 's eye and the focus was adjusted ( 10 to + 11 diopter ( d ) ; retinal lens ; 70 field ) on the handheld probe according to the spherical equivalent of the patient 's refractive error . during the study period ( january 2011 to december 2014 ) , 975 unique patients with good quality oct images , adequate for analysis and with complete corroborative clinical diagnoses documented were reviewed for this manuscript . of these , most of the children ( 37.7% ; 368/975 ) were examined for evaluation of premature infants with and without retinopathy of prematurity ( rop ) . the clinical diagnoses that required other children to undergo an oct are summarized in table 1 for the remaining 607 children and are the focus of the current manuscript . clinical diagnosis of pediatric patients who underwent handheld spectral domain oct in our institute ( n=975 children ) based on the utility of the handheld oct in our series , we have categorized the indications that warrant the investigation and include : evaluation of premature childrensuboptimal vision and unexplained vision lossnystagmus and night blindnessintraocular tumors and mass lesionsmiscellaneous indications . evaluation of premature children suboptimal vision and unexplained vision loss nystagmus and night blindness intraocular tumors and mass lesions miscellaneous indications . when the 975 cases were classified according to the clinical utility of the sd - oct in our practice , we found that those that 75% ( 731 ) were clinically indicated , 20.3% ( 198 ) were performed for understanding the pathology further and 4.7% ( 46 ) did not add more information to case management than what was known through clinical examination [ table 1 ] . the foveal architecture and other retinal features in children with and without rop were studied in 368 children and have previously reported the utility of sd - oct imaging in premature infants . , , broadly these cases can be described as those with normal looking foveae ( clinically ) and those with abnormal foveae . fovea , but with subnormal visual acuity who are referred from the amblyopia clinic , those not improving visually despite compliant patching therapy , refractive errors which can not explain the full extent of visual deficit , premature infants who are undergoing longitudinal and serial imaging for correlation of visual milestones , infants who are undergoing vision rehabilitation including those with a diagnosis of delayed visual maturation and cortical vision impairment and other causes of unexplained visual deficit or suboptimal vision that can not be explained despite a there have been reports of oct changes resembling macular edema of adults in stage 2 rop in asian indian infants and subsequently in other ethnicities and in other stages of rop as well . although these changes spontaneously resolved as early as 52 weeks postmenstrual age , we found that the group with macular edema demonstrated early visual and refractive changes compared to age - matched positive and negative controls at the first year follow - up ( unpublished data ) . the retinal microstructure on oct has been correlated with visual acuity both retrospectively ( unpublished data ) and prospectively . our current understanding of foveal development in these infants suggests that the delay or absence of the inner retinal layer maturation , the growth of the photoreceptor complex and the foveal tent can influence visual acuity in these infants . the handheld oct has been used to evaluate macular involvement in cases of advanced rop . confirmation of macular detachment alters the diagnosis from stage 4a to 4b and can be used to prognosticate before surgery and also monitor postsurgical improvement . extending our observations of premature infants and structure - function correlation using the sd - oct , we have used the same principle to study older toddlers , preschool and preverbal children from our amblyopia clinic who 's vision does not improve despite seemingly compliant patching therapy and refractive correction , as well as in cases where the low vision can not be explained based on the other clinical findings alone . the clinical utility in this series is described with the following case examples . a 5-year - old girl with a refractive error of 1.25 cylinder at 10 and 170 in the right and left eye , respectively with a visual acuity of 20/50 and 20/60 despite spectacle correction for 3 years . fundus examination and color vision was within normal limits ; sd - oct images [ fig . 1 ] showed a normal inner retina with a good foveal dip and fusion of the inner retinal layers . the outer retina showed a disruption of the inner segment outer segment ( is - os ) junction at the foveal center and the loss of the foveal tent ( in comparison to the normal pediatric retina ) . an optical coherence tomography image from case 1 ( above ) with unexplained vision loss shows disruption in the ellipsoid zone at the fovea with an intact external limiting membrane . an example of normal retina is represented below ( cc : choriocapillaries ; ez : ellipsoid zone , fp : foveal pit , ft : foveal tent , gcl : ganglion cell layer , inl : inner nuclear layer , ipl : inner plexiform layer , is : inner segment of the photoreceptor , nfl : nerve fiber layer , olm : outer limiting membrane , onl : outer nuclear layer , opl : outer plexiform layer , os : outer segment of the photoreceptor , rpe : retinal pigment epithelium complex ) an 8-year - old girl showing no improvement with spectacle correction or patching with a visual acuity constant at 20/50 and 20/60 , respectively had a myopic correction of 2.25 180 and 2.50 180 , respectively . 2 ] revealed a normal inner retina , but a thickened and irregular is - os layer in both eyes more in the foveal center with the loss of the foveal tent . the os rpe layer was attenuated or near absent in the foveal center this child was unable to cooperate for multifocal erg testing . a spectral domain - optical coherence tomography image of case 2 , an 8-year - old girl with unexplained vision loss showing an abnormal ellipsoid zone , and outer segment of photoreceptors layer nystagmus and night blindness are both early signs of retinal dystrophy . in addition to an erg demonstrating abnormal retinal function , the sd - oct was able to pick up early abnormalities of the retinal microstructure in an otherwise normal looking fundus . many authors have noted the importance of sd - oct in the diagnosis of nystagmus . in our experience , sd - oct imaging was possible in most children with nystagmus in the office without anesthesia . despite the abnormal eye movement sd - oct images provide micro - structural evidence of the photoreceptor morphology in the foveal center and the retinal mid - periphery that can help diagnose , prognosticate and follow - up these cases . we were also able to investigate children with night blindness to diagnosis a rod - cone dystrophy . a 4-month - old girl presented when her parents noticed that she was unable to maintain a steady gaze . she was found to have a horizontal pendular nystagmus , with an absent foveal pit and absent foveal reflexes on fundus examination . 3 ] demonstrated an absent foveal pit , persistence of the inner retinal layers in the foveal center with normal outer retinal layers . these findings are typical of foveal hypoplasia . a spectral domain - optical coherence tomography image of case 3 demonstrating foveal hypoplasia ( box inset showing the area of the retina scanned , with * to indicate where the foveal pit was anticipated ) . there is an absence of the central foveal pit and the inner retinal layers are present throughout the scan length a 1-year - old girl presented when her parents noticed that she could not make eye contact and lacked a social smile . the fundus showed a normal retina with minimal peripheral tessellation and a normal foveal contour . a full field erg showed abnormal scotopic and photopic waveforms , with the latter affected more . 4 ] showed an absent foveal tent and a uniform hyper - reflectivity of the layer of cone os tips , with a reflectivity of the other retinal layers seeming normal . the true significance of this potentially early oct sign of abnormal photoreceptor function is not known and is being reported for the first time in this child with a cone - rod dystrophy . a spectral domain - optical coherence tomography image of case 4 with a cone - rod dystrophy demonstrating an absent foveal tent and an irregularly thickened and hyper - reflective layer of cone outer segment tips a 5-year - old boy presented to us when his parents noticed he was avoiding brightly lit areas . he was found to have a best - corrected vision of 20/120 vision in each eye , with severe loss of color perception . a full field erg performed showed a normal scotopic response , with an absent photopic response , confirming a diagnosis of complete achromatopsia . the sd - oct [ fig . 5 ] performed showed a disrupted photoreceptor os layer only at the fovea ( referred to as the hypo - reflective zone ) , which has been previously reported in complete achromatopsia . a spectral domain - optical coherence tomography image of case 5 with complete achromatopsia showing a disrupted outer segment layer at the fovea demonstrating the hypo - reflective zone seen with cone dysfunction a 2-year - old girl presented with a history of poor vision in dark and hearing loss . the child was found to have a vision of 20/2700 in the right eye and 20/960 in the left eye with tellers acuity cards . a fundus examination showed peripheral retinal pigment epithelium mottling , pale optic discs , narrow arterioles and a grossly normal macula . a full field erg showed no response from either scotopic or photopic stimuli , as expected in a severe rod - cone dystrophy . the sd - oct [ fig . 6 ] showed preserved retinal layers at the fovea , with the loss of the outer layers of the retina with increasing distance from the fovea . the photoreceptor layers were completely lost 23 mm away from the fovea and the outer plexiform layer was lost further into the mid - peripheral retina . spectral domain - optical coherence tomography images of case 6 with a rod - cone dystrophy showing the central retina ( above ) and peripheral retina ( below ) . progressive losses of the outer retinal layers are shown in these images as the retina is imaged from the fovea to the periphery the diagnosis of an intraocular tumor such as retinoblastoma is usually through fundus examination and ultrasonography and rarely requires confirmation using an sd - oct . most often in our practice , in an obviously affected eye , a small retinoblastoma lesion is almost always seen in the opposite eye , therefore making the diagnosis clear . an astrocytic hamartoma in a child with ash - leaf macules of the skin and the presence of other tumors in the myocardium and brain usually confirm a clinical diagnosis of tuberous sclerosis . it is a lesion that rarely gets referred for differentiation from a retinoblastoma , and an sd - oct can help differentiate the two . while an astrocytic hamartoma arises from the nerve fiber layer [ fig . we have reported the oct appearance of a treated retinoblastoma before and after chemotherapy [ fig . there was the disappearance of the dark streaks caused by posterior shadowing from tumor vasculature ( bar - code like appearance ) which was seen prior to chemotherapy . a spectral domain - optical coherence tomography image of an astrocytic hamartoma in a 3 years old demonstrating a smooth , regular , and well defined lesion involving the nerve fiber layer spectral domain - optical coherence tomography images of a small retinoblastoma tumor in a 3-month - old boy before ( above ) and after ( below ) the first cycle of chemotherapy ( image courtesy : mallipatna a , suren v. retinoblastoma . in : other indications of sd - oct in children with retinoblastoma have included the detection of small tumors in the screening of children who are germline predisposed to having retinoblastoma , detection of the source of vitreous seeding to help target focal laser therapy [ fig . 10 ] , follow - up of vitreo - macular traction during the course of treatment [ fig . 11 ] and the presumed involvement of the optic nerve head in children with a tumor adjacent to the optic disc [ fig . a previous paper has commented on the utility of the sd - oct in the management of retinoblastoma by assisting in the diagnosis of new lesions , monitoring response to laser therapy and identifying tumor recurrences . spectral domain - optical coherence tomography images of a retinoblastoma tumor ( indicated by an arrow ) dispersing vitreous seeds ( indicated by fnx01 ) into the vitreous . the box ( inset ) shows a single pearl - like vitreous seed with an outer layer of viable cells surrounding a core of necrotic cell debris a spectral domain - optical coherence tomography image of a recurring tumor ( indicated by an arrow ) occurring at the edge of a previously treated tumor ( irregularly hyper - reflective lesion to the right of the arrow ) serial oct images of the fovea of a 4-year - old boy treated with focal lasers , who developed a vitreo - macular traction after treatment ( above image from a tabletop sd - oct : spectral oct - slo , ophthalmic technologies inc . , toronto , canada ) , with the traction band spontaneously detaching from the fovea after four months ( below image from the hand - held sd - oct : envisue 2300 ) a spectral domain - optical coherence tomography image of a retinoblastoma tumor in a 3-year - old girl seeming to extend into the optic nerve in her only remaining eye retinal mid - peripheral and peripheral lesions such as choroidal nevi , retinitis , choroiditis , and flecked retinal syndromes can also be imaged on sd - oct . we have reported an early variant of a combined hamartoma of the retina and retinal pigment epithelium in a 6-week - old infant undergoing routine rop screening on the retcam . the lesion demonstrated a smooth contoured , homogenous lesion arising from the inner retinal layers . the foveal architecture and other retinal features in children with and without rop were studied in 368 children and have previously reported the utility of sd - oct imaging in premature infants . , , broadly these cases can be described as those with normal looking foveae ( clinically ) and those with abnormal foveae . fovea , but with subnormal visual acuity who are referred from the amblyopia clinic , those not improving visually despite compliant patching therapy , refractive errors which can not explain the full extent of visual deficit , premature infants who are undergoing longitudinal and serial imaging for correlation of visual milestones , infants who are undergoing vision rehabilitation including those with a diagnosis of delayed visual maturation and cortical vision impairment and other causes of unexplained visual deficit or suboptimal vision that can not be explained despite a there have been reports of oct changes resembling macular edema of adults in stage 2 rop in asian indian infants and subsequently in other ethnicities and in other stages of rop as well . although these changes spontaneously resolved as early as 52 weeks postmenstrual age , we found that the group with macular edema demonstrated early visual and refractive changes compared to age - matched positive and negative controls at the first year follow - up ( unpublished data ) . the retinal microstructure on oct has been correlated with visual acuity both retrospectively ( unpublished data ) and prospectively . our current understanding of foveal development in these infants suggests that the delay or absence of the inner retinal layer maturation , the growth of the photoreceptor complex and the foveal tent can influence visual acuity in these infants . the handheld oct has been used to evaluate macular involvement in cases of advanced rop . confirmation of macular detachment alters the diagnosis from stage 4a to 4b and can be used to prognosticate before surgery and also monitor postsurgical improvement . extending our observations of premature infants and structure - function correlation using the sd - oct , we have used the same principle to study older toddlers , preschool and preverbal children from our amblyopia clinic who 's vision does not improve despite seemingly compliant patching therapy and refractive correction , as well as in cases where the low vision can not be explained based on the other clinical findings alone . the clinical utility in this series is described with the following case examples . a 5-year - old girl with a refractive error of 1.25 cylinder at 10 and 170 in the right and left eye , respectively with a visual acuity of 20/50 and 20/60 despite spectacle correction for 3 years . fundus examination and color vision was within normal limits ; sd - oct images [ fig . 1 ] showed a normal inner retina with a good foveal dip and fusion of the inner retinal layers . the outer retina showed a disruption of the inner segment outer segment ( is - os ) junction at the foveal center and the loss of the foveal tent ( in comparison to the normal pediatric retina ) . an optical coherence tomography image from case 1 ( above ) with unexplained vision loss shows disruption in the ellipsoid zone at the fovea with an intact external limiting membrane . an example of normal retina is represented below ( cc : choriocapillaries ; ez : ellipsoid zone , fp : foveal pit , ft : foveal tent , gcl : ganglion cell layer , inl : inner nuclear layer , ipl : inner plexiform layer , is : inner segment of the photoreceptor , nfl : nerve fiber layer , olm : outer limiting membrane , onl : outer nuclear layer , opl : outer plexiform layer , os : outer segment of the photoreceptor , rpe : retinal pigment epithelium complex ) an 8-year - old girl showing no improvement with spectacle correction or patching with a visual acuity constant at 20/50 and 20/60 , respectively had a myopic correction of 2.25 180 and 2.50 180 , respectively . the sd - oct [ fig . 2 ] revealed a normal inner retina , but a thickened and irregular is - os layer in both eyes more in the foveal center with the loss of the foveal tent . this child was unable to cooperate for multifocal erg testing . a spectral domain - optical coherence tomography image of case 2 , an 8-year - old girl with unexplained vision loss showing an abnormal ellipsoid zone , and outer segment of photoreceptors layer a 5-year - old girl with a refractive error of 1.25 cylinder at 10 and 170 in the right and left eye , respectively with a visual acuity of 20/50 and 20/60 despite spectacle correction for 3 years . fundus examination and 1 ] showed a normal inner retina with a good foveal dip and fusion of the inner retinal layers . the outer retina showed a disruption of the inner segment outer segment ( is - os ) junction at the foveal center and the loss of the foveal tent ( in comparison to the normal pediatric retina ) . an optical coherence tomography image from case 1 ( above ) with unexplained vision loss shows disruption in the ellipsoid zone at the fovea with an intact external limiting membrane . an example of normal retina is represented below ( cc : choriocapillaries ; ez : ellipsoid zone , fp : foveal pit , ft : foveal tent , gcl : ganglion cell layer , inl : inner nuclear layer , ipl : inner plexiform layer , is : inner segment of the photoreceptor , nfl : nerve fiber layer , olm : outer limiting membrane , onl : outer nuclear layer , opl : outer plexiform layer , os : outer segment of the photoreceptor , rpe : retinal pigment epithelium complex ) an 8-year - old girl showing no improvement with spectacle correction or patching with a visual acuity constant at 20/50 and 20/60 , respectively had a myopic correction of 2.25 180 and 2.50 180 , respectively . 2 ] revealed a normal inner retina , but a thickened and irregular is - os layer in both eyes more in the foveal center with the loss of the foveal tent . this child was unable to cooperate for multifocal erg testing . a spectral domain - optical coherence tomography image of case 2 , an 8-year - old girl with unexplained vision loss showing an abnormal ellipsoid zone , and outer segment of photoreceptors layer nystagmus and night blindness are both early signs of retinal dystrophy . in addition to an erg demonstrating abnormal retinal function , the sd - oct was able to pick up early abnormalities of the retinal microstructure in an otherwise normal looking fundus . many authors have noted the importance of sd - oct in the diagnosis of nystagmus . in our experience , sd - oct imaging was possible in most children with nystagmus in the office without anesthesia . despite the abnormal eye movement sd - oct images provide micro - structural evidence of the photoreceptor morphology in the foveal center and the retinal mid - periphery that can help diagnose , prognosticate and follow - up these cases . we were also able to investigate children with night blindness to diagnosis a rod - cone dystrophy . a 4-month - old girl presented when her parents noticed that she was unable to maintain a steady gaze . she was found to have a horizontal pendular nystagmus , with an absent foveal pit and absent foveal reflexes on fundus examination . 3 ] demonstrated an absent foveal pit , persistence of the inner retinal layers in the foveal center with normal outer retinal layers . these findings are typical of foveal hypoplasia . a spectral domain - optical coherence tomography image of case 3 demonstrating foveal hypoplasia ( box inset showing the area of the retina scanned , with * to indicate where the foveal pit was anticipated ) . there is an absence of the central foveal pit and the inner retinal layers are present throughout the scan length a 1-year - old girl presented when her parents noticed that she could not make eye contact and lacked a social smile . the fundus showed a normal retina with minimal peripheral tessellation and a normal foveal contour . a full field erg showed abnormal scotopic and photopic waveforms , with the latter affected more . 4 ] showed an absent foveal tent and a uniform hyper - reflectivity of the layer of cone os tips , with a reflectivity of the other retinal layers seeming normal . the true significance of this potentially early oct sign of abnormal photoreceptor function is not known and is being reported for the first time in this child with a cone - rod dystrophy . a spectral domain - optical coherence tomography image of case 4 with a cone - rod dystrophy demonstrating an absent foveal tent and an irregularly thickened and hyper - reflective layer of cone outer segment tips a 5-year - old boy presented to us when his parents noticed he was avoiding brightly lit areas . he was found to have a best - corrected vision of 20/120 vision in each eye , with severe loss of color perception . a full field erg performed showed a normal scotopic response , with an absent photopic response , confirming a diagnosis of complete achromatopsia . the sd - oct [ fig . 5 ] performed showed a disrupted photoreceptor os layer only at the fovea ( referred to as the hypo - reflective zone ) , which has been previously reported in complete achromatopsia . a spectral domain - optical coherence tomography image of case 5 with complete achromatopsia showing a disrupted outer segment layer at the fovea demonstrating the hypo - reflective zone seen with cone dysfunction a 2-year - old girl presented with a history of poor vision in dark and hearing loss . the child was found to have a vision of 20/2700 in the right eye and 20/960 in the left eye with tellers acuity cards . a fundus examination showed peripheral retinal pigment epithelium mottling , pale optic discs , narrow arterioles and a grossly normal macula . a full field erg showed no response from either scotopic or photopic stimuli , as expected in a severe rod - cone dystrophy . 6 ] showed preserved retinal layers at the fovea , with the loss of the outer layers of the retina with increasing distance from the fovea . the photoreceptor layers were completely lost 23 mm away from the fovea and the outer plexiform layer was lost further into the mid - peripheral retina . spectral domain - optical coherence tomography images of case 6 with a rod - cone dystrophy showing the central retina ( above ) and peripheral retina ( below ) . progressive losses of the outer retinal layers are shown in these images as the retina is imaged from the fovea to the periphery a 4-month - old girl presented when her parents noticed that she was unable to maintain a steady gaze . she was found to have a horizontal pendular nystagmus , with an absent foveal pit and absent foveal reflexes on fundus examination . 3 ] demonstrated an absent foveal pit , persistence of the inner retinal layers in the foveal center with normal outer retinal layers . these findings are typical of foveal hypoplasia . a spectral domain - optical coherence tomography image of case 3 demonstrating foveal hypoplasia ( box inset showing the area of the retina scanned , with * to indicate where the foveal pit was anticipated ) . there is an absence of the central foveal pit and the inner retinal layers are present throughout the scan length a 1-year - old girl presented when her parents noticed that she could not make eye contact and lacked a social smile . the fundus showed a normal retina with minimal peripheral tessellation and a normal foveal contour . a full field erg showed abnormal scotopic and photopic waveforms , with the latter affected more . 4 ] showed an absent foveal tent and a uniform hyper - reflectivity of the layer of cone os tips , with a reflectivity of the other retinal layers seeming normal . the true significance of this potentially early oct sign of abnormal photoreceptor function is not known and is being reported for the first time in this child with a cone - rod dystrophy . a spectral domain - optical coherence tomography image of case 4 with a cone - rod dystrophy demonstrating an absent foveal tent and an irregularly thickened and hyper - reflective layer of cone outer segment tips a 5-year - old boy presented to us when his parents noticed he was avoiding brightly lit areas . he was found to have a best - corrected vision of 20/120 vision in each eye , with severe loss of color perception . a full field erg performed showed a normal scotopic response , with an absent photopic response , confirming a diagnosis of complete achromatopsia . the sd - oct [ fig . 5 ] performed showed a disrupted photoreceptor os layer only at the fovea ( referred to as the hypo - reflective zone ) , which has been previously reported in complete achromatopsia . a spectral domain - optical coherence tomography image of case 5 with complete achromatopsia showing a disrupted outer segment layer at the fovea demonstrating the hypo - reflective zone seen with cone dysfunction a 2-year - old girl presented with a history of poor vision in dark and hearing loss . the child was found to have a vision of 20/2700 in the right eye and 20/960 in the left eye with tellers acuity cards . a fundus examination showed peripheral retinal pigment epithelium mottling , pale optic discs , narrow arterioles and a grossly normal macula . a full field erg showed no response from either scotopic or photopic stimuli , as expected in a severe rod - cone dystrophy . the sd - oct [ fig . 6 ] showed preserved retinal layers at the fovea , with the loss of the outer layers of the retina with increasing distance from the fovea . the photoreceptor layers were completely lost 23 mm away from the fovea and the outer plexiform layer was lost further into the mid - peripheral retina . spectral domain - optical coherence tomography images of case 6 with a rod - cone dystrophy showing the central retina ( above ) and peripheral retina ( below ) . progressive losses of the outer retinal layers are shown in these images as the retina is imaged from the fovea to the periphery the diagnosis of an intraocular tumor such as retinoblastoma is usually through fundus examination and ultrasonography and rarely requires confirmation using an sd - oct . most often in our practice , in an obviously affected eye , a small retinoblastoma lesion is almost always seen in the opposite eye , therefore making the diagnosis clear . an astrocytic hamartoma in a child with ash - leaf macules of the skin and the presence of other tumors in the myocardium and brain usually confirm a clinical diagnosis of tuberous sclerosis . it is a lesion that rarely gets referred for differentiation from a retinoblastoma , and an sd - oct can help differentiate the two . while an astrocytic hamartoma arises from the nerve fiber layer [ fig . we have reported the oct appearance of a treated retinoblastoma before and after chemotherapy [ fig . there was the disappearance of the dark streaks caused by posterior shadowing from tumor vasculature ( bar - code like appearance ) which was seen prior to chemotherapy . a spectral domain - optical coherence tomography image of an astrocytic hamartoma in a 3 years old demonstrating a smooth , regular , and well defined lesion involving the nerve fiber layer spectral domain - optical coherence tomography images of a small retinoblastoma tumor in a 3-month - old boy before ( above ) and after ( below ) the first cycle of chemotherapy ( image courtesy : mallipatna a , suren v. retinoblastoma . in : vinekar a , new delhi : elsevier health sciences ; 2014 . ) other indications of sd - oct in children with retinoblastoma have included the detection of small tumors in the screening of children who are germline predisposed to having retinoblastoma , detection of the source of vitreous seeding to help target focal laser therapy [ fig . 10 ] , follow - up of vitreo - macular traction during the course of treatment [ fig . 11 ] and the presumed involvement of the optic nerve head in children with a tumor adjacent to the optic disc [ fig . a previous paper has commented on the utility of the sd - oct in the management of retinoblastoma by assisting in the diagnosis of new lesions , monitoring response to laser therapy and identifying tumor recurrences . spectral domain - optical coherence tomography images of a retinoblastoma tumor ( indicated by an arrow ) dispersing vitreous seeds ( indicated by fnx01 ) into the vitreous . the box ( inset ) shows a single pearl - like vitreous seed with an outer layer of viable cells surrounding a core of necrotic cell debris a spectral domain - optical coherence tomography image of a recurring tumor ( indicated by an arrow ) occurring at the edge of a previously treated tumor ( irregularly hyper - reflective lesion to the right of the arrow ) serial oct images of the fovea of a 4-year - old boy treated with focal lasers , who developed a vitreo - macular traction after treatment ( above image from a tabletop sd - oct : spectral oct - slo , ophthalmic technologies inc . , toronto , canada ) , with the traction band spontaneously detaching from the fovea after four months ( below image from the hand - held sd - oct : envisue 2300 ) a spectral domain - optical coherence tomography image of a retinoblastoma tumor in a 3-year - old girl seeming to extend into the optic nerve in her only remaining eye retinal mid - peripheral and peripheral lesions such as choroidal nevi , retinitis , choroiditis , and flecked retinal syndromes can also be imaged on sd - oct . we have reported an early variant of a combined hamartoma of the retina and retinal pigment epithelium in a 6-week - old infant undergoing routine rop screening on the retcam . the lesion demonstrated a smooth contoured , homogenous lesion arising from the inner retinal layers . with the handheld sd - oct , we now have a useful tool for the evaluation of the pediatric retina . it can assess vision development in premature children , children with unexplained vision loss and amblyopia , children with nystagmus and night blindness and children with intraocular tumors , and retinoblastoma . the list of indications is expanding , especially considering that the test can be performed with reasonable quality in some children without sedation or anesthesia . the requirement for invasive tests that sometimes need to be done under anesthesia , such as a fluorescein angiography , can be minimized with the effective use of this device . using the handheld device requires a degree of skill to obtain good quality images and is dependent on the degree of co - operation by the children . additionally , although the coverage of the peripheral retina is better than with the tabletop devices , image clarity limits how far in the periphery we can capture usable images . in this manuscript , we report our clinical experience in imaging retinal conditions in infants and children . this cohort may not represent the general prevalence of these entities because of the referral bias in the study subjects . our institute is a tertiary care referral unit and is likely to receive patients with a higher incidence of unexplained visual loss or less common retinal conditions , which will skew the outcome and make it less generalizable . hence , we described the indications under different clinical scenarios , to demonstrate the scope of handheld oct imaging in the pediatric population . we also use oct images to counsel the parents of these children by demonstrating the structural and morphological changes and correlating it with either vision or therapy at the time of diagnosis or during follow - up . though sd - oct imaging of the pediatric retina has several applications , as toth ca ( 2012 ) has summarized , some concerns include : the pediatric eye is shorter and optically differs from the adult , and measurements of the lateral extent of lesions reported by oct systems must be examined and most likely corrected based on the infant eye 's unique optical pathway the infant , and especially the premature infant , retina is notably different from the adult . inner retinal layers persist and outer retinal layers may be extremely immature . thus the sd - oct reflective bands that define adult retinal layers are very different for the premature and term infant eye sd - oct findings in infant eyes such as macular edema of prematurity and subfoveal fluid in healthy term infants at birth may represent normal development or unrecognized disease processes studies correlating sd - oct findings in infants to visual acuity are few . the pediatric eye is shorter and optically differs from the adult , and measurements of the lateral extent of lesions reported by oct systems must be examined and most likely corrected based on the infant eye 's unique optical pathway the infant , and especially the premature infant , retina is notably different from the adult . inner retinal layers persist and outer retinal layers may be extremely immature . thus the sd - oct reflective bands that define adult retinal layers are very different for the premature and term infant eye sd - oct findings in infant eyes such as macular edema of prematurity and subfoveal fluid in healthy term infants at birth may represent normal development or unrecognized disease processes studies correlating sd - oct findings in infants to visual acuity are few . nevertheless , sd - oct remains an invaluable tool for the assessment of pediatric retinal diseases . newer technologies such as the oct angiography can help take it further by providing more detailed information . intraoperative oct could help study the retinal morphological changes during surgery in ways that can help improve the surgical outcome . analyzing quantified data with image analysis and comparing it to age - specific normative data will help identify the more subtle disease in the future .

purpose : optical coherence tomography ( oct ) is an important imaging tool assessing retinal architecture . in this article , we report a single centers experience of using handheld spectral domain ( sd)-oct in a pediatric population using the envisu 2300 ( bioptigen inc . , research triangle park , nc , usa).methods : we studied sd - oct images from 975 patients imaged from january 2011 to december 2014 . the variety of cases that underwent an sd - oct was analyzed . cases examples from different case scenarios were selected to showcase unique examples of many diseases.results:three hundred and sixty - eight infants ( 37.7% ) were imaged for retinopathy of prematurity , 362 children ( 37.1% ) underwent the test for evaluation of suboptimal vision or an unexplained vision loss , 126 children ( 12.9% ) for evaluation of nystagmus or night blindness , 54 children ( 5.5% ) for an intraocular tumor or a mass lesion such as retinoblastoma , and 65 children ( 6.7% ) for other diseases of the pediatric retina . the unique findings in the retinal morphology seen with some of these diseases are discussed.conclusion:the handheld sd - oct is useful in the evaluation of the pediatric retinal diseases . the test is useful in the assessment of vision development in premature children , evaluation of unexplained vision loss and amblyopia , nystagmus and night blindness , and intraocular tumors ( including retinoblastoma ) .

Methods Results Evaluation of premature children Evaluation of children with poor vision Case 1 Case 2 Evaluation of children with nystagmus and night blindness Case 3 Case 4 Case 5 Case 6 Children with retinal tumors and other retinal lesions Discussion Financial support and sponsorship Conflicts of interest

we reviewed the image database of all consecutive pediatric patients referred to the pediatric retina department of our institute , and who underwent imaging with a handheld sd - oct device . the envisu 2300 has a maximum camera line rate of 36 khz with a typical imaging line rate of 32 khz and an axial resolution of < 4 m in tissue . during the study period ( january 2011 to december 2014 ) , 975 unique patients with good quality oct images , adequate for analysis and with complete corroborative clinical diagnoses documented were reviewed for this manuscript . of these , most of the children ( 37.7% ; 368/975 ) were examined for evaluation of premature infants with and without retinopathy of prematurity ( rop ) . clinical diagnosis of pediatric patients who underwent handheld spectral domain oct in our institute ( n=975 children ) based on the utility of the handheld oct in our series , we have categorized the indications that warrant the investigation and include : evaluation of premature childrensuboptimal vision and unexplained vision lossnystagmus and night blindnessintraocular tumors and mass lesionsmiscellaneous indications . evaluation of premature children suboptimal vision and unexplained vision loss nystagmus and night blindness intraocular tumors and mass lesions miscellaneous indications . when the 975 cases were classified according to the clinical utility of the sd - oct in our practice , we found that those that 75% ( 731 ) were clinically indicated , 20.3% ( 198 ) were performed for understanding the pathology further and 4.7% ( 46 ) did not add more information to case management than what was known through clinical examination [ table 1 ] . the foveal architecture and other retinal features in children with and without rop were studied in 368 children and have previously reported the utility of sd - oct imaging in premature infants . our current understanding of foveal development in these infants suggests that the delay or absence of the inner retinal layer maturation , the growth of the photoreceptor complex and the foveal tent can influence visual acuity in these infants . extending our observations of premature infants and structure - function correlation using the sd - oct , we have used the same principle to study older toddlers , preschool and preverbal children from our amblyopia clinic who 's vision does not improve despite seemingly compliant patching therapy and refractive correction , as well as in cases where the low vision can not be explained based on the other clinical findings alone . fundus examination and color vision was within normal limits ; sd - oct images [ fig . the outer retina showed a disruption of the inner segment outer segment ( is - os ) junction at the foveal center and the loss of the foveal tent ( in comparison to the normal pediatric retina ) . an optical coherence tomography image from case 1 ( above ) with unexplained vision loss shows disruption in the ellipsoid zone at the fovea with an intact external limiting membrane . a spectral domain - optical coherence tomography image of case 2 , an 8-year - old girl with unexplained vision loss showing an abnormal ellipsoid zone , and outer segment of photoreceptors layer nystagmus and night blindness are both early signs of retinal dystrophy . in addition to an erg demonstrating abnormal retinal function , the sd - oct was able to pick up early abnormalities of the retinal microstructure in an otherwise normal looking fundus . many authors have noted the importance of sd - oct in the diagnosis of nystagmus . in our experience , sd - oct imaging was possible in most children with nystagmus in the office without anesthesia . despite the abnormal eye movement sd - oct images provide micro - structural evidence of the photoreceptor morphology in the foveal center and the retinal mid - periphery that can help diagnose , prognosticate and follow - up these cases . 3 ] demonstrated an absent foveal pit , persistence of the inner retinal layers in the foveal center with normal outer retinal layers . a spectral domain - optical coherence tomography image of case 3 demonstrating foveal hypoplasia ( box inset showing the area of the retina scanned , with * to indicate where the foveal pit was anticipated ) . a spectral domain - optical coherence tomography image of case 4 with a cone - rod dystrophy demonstrating an absent foveal tent and an irregularly thickened and hyper - reflective layer of cone outer segment tips a 5-year - old boy presented to us when his parents noticed he was avoiding brightly lit areas . a spectral domain - optical coherence tomography image of case 5 with complete achromatopsia showing a disrupted outer segment layer at the fovea demonstrating the hypo - reflective zone seen with cone dysfunction a 2-year - old girl presented with a history of poor vision in dark and hearing loss . spectral domain - optical coherence tomography images of case 6 with a rod - cone dystrophy showing the central retina ( above ) and peripheral retina ( below ) . progressive losses of the outer retinal layers are shown in these images as the retina is imaged from the fovea to the periphery the diagnosis of an intraocular tumor such as retinoblastoma is usually through fundus examination and ultrasonography and rarely requires confirmation using an sd - oct . an astrocytic hamartoma in a child with ash - leaf macules of the skin and the presence of other tumors in the myocardium and brain usually confirm a clinical diagnosis of tuberous sclerosis . it is a lesion that rarely gets referred for differentiation from a retinoblastoma , and an sd - oct can help differentiate the two . a spectral domain - optical coherence tomography image of an astrocytic hamartoma in a 3 years old demonstrating a smooth , regular , and well defined lesion involving the nerve fiber layer spectral domain - optical coherence tomography images of a small retinoblastoma tumor in a 3-month - old boy before ( above ) and after ( below ) the first cycle of chemotherapy ( image courtesy : mallipatna a , suren v. retinoblastoma . in : other indications of sd - oct in children with retinoblastoma have included the detection of small tumors in the screening of children who are germline predisposed to having retinoblastoma , detection of the source of vitreous seeding to help target focal laser therapy [ fig . a previous paper has commented on the utility of the sd - oct in the management of retinoblastoma by assisting in the diagnosis of new lesions , monitoring response to laser therapy and identifying tumor recurrences . spectral domain - optical coherence tomography images of a retinoblastoma tumor ( indicated by an arrow ) dispersing vitreous seeds ( indicated by fnx01 ) into the vitreous . the box ( inset ) shows a single pearl - like vitreous seed with an outer layer of viable cells surrounding a core of necrotic cell debris a spectral domain - optical coherence tomography image of a recurring tumor ( indicated by an arrow ) occurring at the edge of a previously treated tumor ( irregularly hyper - reflective lesion to the right of the arrow ) serial oct images of the fovea of a 4-year - old boy treated with focal lasers , who developed a vitreo - macular traction after treatment ( above image from a tabletop sd - oct : spectral oct - slo , ophthalmic technologies inc . , toronto , canada ) , with the traction band spontaneously detaching from the fovea after four months ( below image from the hand - held sd - oct : envisue 2300 ) a spectral domain - optical coherence tomography image of a retinoblastoma tumor in a 3-year - old girl seeming to extend into the optic nerve in her only remaining eye retinal mid - peripheral and peripheral lesions such as choroidal nevi , retinitis , choroiditis , and flecked retinal syndromes can also be imaged on sd - oct . we have reported an early variant of a combined hamartoma of the retina and retinal pigment epithelium in a 6-week - old infant undergoing routine rop screening on the retcam . the foveal architecture and other retinal features in children with and without rop were studied in 368 children and have previously reported the utility of sd - oct imaging in premature infants . our current understanding of foveal development in these infants suggests that the delay or absence of the inner retinal layer maturation , the growth of the photoreceptor complex and the foveal tent can influence visual acuity in these infants . extending our observations of premature infants and structure - function correlation using the sd - oct , we have used the same principle to study older toddlers , preschool and preverbal children from our amblyopia clinic who 's vision does not improve despite seemingly compliant patching therapy and refractive correction , as well as in cases where the low vision can not be explained based on the other clinical findings alone . fundus examination and color vision was within normal limits ; sd - oct images [ fig . an optical coherence tomography image from case 1 ( above ) with unexplained vision loss shows disruption in the ellipsoid zone at the fovea with an intact external limiting membrane . a spectral domain - optical coherence tomography image of case 2 , an 8-year - old girl with unexplained vision loss showing an abnormal ellipsoid zone , and outer segment of photoreceptors layer a 5-year - old girl with a refractive error of 1.25 cylinder at 10 and 170 in the right and left eye , respectively with a visual acuity of 20/50 and 20/60 despite spectacle correction for 3 years . the outer retina showed a disruption of the inner segment outer segment ( is - os ) junction at the foveal center and the loss of the foveal tent ( in comparison to the normal pediatric retina ) . an optical coherence tomography image from case 1 ( above ) with unexplained vision loss shows disruption in the ellipsoid zone at the fovea with an intact external limiting membrane . a spectral domain - optical coherence tomography image of case 2 , an 8-year - old girl with unexplained vision loss showing an abnormal ellipsoid zone , and outer segment of photoreceptors layer nystagmus and night blindness are both early signs of retinal dystrophy . in addition to an erg demonstrating abnormal retinal function , the sd - oct was able to pick up early abnormalities of the retinal microstructure in an otherwise normal looking fundus . many authors have noted the importance of sd - oct in the diagnosis of nystagmus . in our experience , sd - oct imaging was possible in most children with nystagmus in the office without anesthesia . despite the abnormal eye movement sd - oct images provide micro - structural evidence of the photoreceptor morphology in the foveal center and the retinal mid - periphery that can help diagnose , prognosticate and follow - up these cases . 3 ] demonstrated an absent foveal pit , persistence of the inner retinal layers in the foveal center with normal outer retinal layers . a spectral domain - optical coherence tomography image of case 3 demonstrating foveal hypoplasia ( box inset showing the area of the retina scanned , with * to indicate where the foveal pit was anticipated ) . there is an absence of the central foveal pit and the inner retinal layers are present throughout the scan length a 1-year - old girl presented when her parents noticed that she could not make eye contact and lacked a social smile . a spectral domain - optical coherence tomography image of case 4 with a cone - rod dystrophy demonstrating an absent foveal tent and an irregularly thickened and hyper - reflective layer of cone outer segment tips a 5-year - old boy presented to us when his parents noticed he was avoiding brightly lit areas . the sd - oct [ fig . a spectral domain - optical coherence tomography image of case 5 with complete achromatopsia showing a disrupted outer segment layer at the fovea demonstrating the hypo - reflective zone seen with cone dysfunction a 2-year - old girl presented with a history of poor vision in dark and hearing loss . spectral domain - optical coherence tomography images of case 6 with a rod - cone dystrophy showing the central retina ( above ) and peripheral retina ( below ) . progressive losses of the outer retinal layers are shown in these images as the retina is imaged from the fovea to the periphery a 4-month - old girl presented when her parents noticed that she was unable to maintain a steady gaze . a spectral domain - optical coherence tomography image of case 3 demonstrating foveal hypoplasia ( box inset showing the area of the retina scanned , with * to indicate where the foveal pit was anticipated ) . there is an absence of the central foveal pit and the inner retinal layers are present throughout the scan length a 1-year - old girl presented when her parents noticed that she could not make eye contact and lacked a social smile . a spectral domain - optical coherence tomography image of case 4 with a cone - rod dystrophy demonstrating an absent foveal tent and an irregularly thickened and hyper - reflective layer of cone outer segment tips a 5-year - old boy presented to us when his parents noticed he was avoiding brightly lit areas . a spectral domain - optical coherence tomography image of case 5 with complete achromatopsia showing a disrupted outer segment layer at the fovea demonstrating the hypo - reflective zone seen with cone dysfunction a 2-year - old girl presented with a history of poor vision in dark and hearing loss . spectral domain - optical coherence tomography images of case 6 with a rod - cone dystrophy showing the central retina ( above ) and peripheral retina ( below ) . progressive losses of the outer retinal layers are shown in these images as the retina is imaged from the fovea to the periphery the diagnosis of an intraocular tumor such as retinoblastoma is usually through fundus examination and ultrasonography and rarely requires confirmation using an sd - oct . an astrocytic hamartoma in a child with ash - leaf macules of the skin and the presence of other tumors in the myocardium and brain usually confirm a clinical diagnosis of tuberous sclerosis . it is a lesion that rarely gets referred for differentiation from a retinoblastoma , and an sd - oct can help differentiate the two . a spectral domain - optical coherence tomography image of an astrocytic hamartoma in a 3 years old demonstrating a smooth , regular , and well defined lesion involving the nerve fiber layer spectral domain - optical coherence tomography images of a small retinoblastoma tumor in a 3-month - old boy before ( above ) and after ( below ) the first cycle of chemotherapy ( image courtesy : mallipatna a , suren v. retinoblastoma . other indications of sd - oct in children with retinoblastoma have included the detection of small tumors in the screening of children who are germline predisposed to having retinoblastoma , detection of the source of vitreous seeding to help target focal laser therapy [ fig . a previous paper has commented on the utility of the sd - oct in the management of retinoblastoma by assisting in the diagnosis of new lesions , monitoring response to laser therapy and identifying tumor recurrences . spectral domain - optical coherence tomography images of a retinoblastoma tumor ( indicated by an arrow ) dispersing vitreous seeds ( indicated by fnx01 ) into the vitreous . the box ( inset ) shows a single pearl - like vitreous seed with an outer layer of viable cells surrounding a core of necrotic cell debris a spectral domain - optical coherence tomography image of a recurring tumor ( indicated by an arrow ) occurring at the edge of a previously treated tumor ( irregularly hyper - reflective lesion to the right of the arrow ) serial oct images of the fovea of a 4-year - old boy treated with focal lasers , who developed a vitreo - macular traction after treatment ( above image from a tabletop sd - oct : spectral oct - slo , ophthalmic technologies inc . , toronto , canada ) , with the traction band spontaneously detaching from the fovea after four months ( below image from the hand - held sd - oct : envisue 2300 ) a spectral domain - optical coherence tomography image of a retinoblastoma tumor in a 3-year - old girl seeming to extend into the optic nerve in her only remaining eye retinal mid - peripheral and peripheral lesions such as choroidal nevi , retinitis , choroiditis , and flecked retinal syndromes can also be imaged on sd - oct . we have reported an early variant of a combined hamartoma of the retina and retinal pigment epithelium in a 6-week - old infant undergoing routine rop screening on the retcam . with the handheld sd - oct , we now have a useful tool for the evaluation of the pediatric retina . it can assess vision development in premature children , children with unexplained vision loss and amblyopia , children with nystagmus and night blindness and children with intraocular tumors , and retinoblastoma . additionally , although the coverage of the peripheral retina is better than with the tabletop devices , image clarity limits how far in the periphery we can capture usable images . in this manuscript , we report our clinical experience in imaging retinal conditions in infants and children . this cohort may not represent the general prevalence of these entities because of the referral bias in the study subjects . hence , we described the indications under different clinical scenarios , to demonstrate the scope of handheld oct imaging in the pediatric population . we also use oct images to counsel the parents of these children by demonstrating the structural and morphological changes and correlating it with either vision or therapy at the time of diagnosis or during follow - up . though sd - oct imaging of the pediatric retina has several applications , as toth ca ( 2012 ) has summarized , some concerns include : the pediatric eye is shorter and optically differs from the adult , and measurements of the lateral extent of lesions reported by oct systems must be examined and most likely corrected based on the infant eye 's unique optical pathway the infant , and especially the premature infant , retina is notably different from the adult . thus the sd - oct reflective bands that define adult retinal layers are very different for the premature and term infant eye sd - oct findings in infant eyes such as macular edema of prematurity and subfoveal fluid in healthy term infants at birth may represent normal development or unrecognized disease processes studies correlating sd - oct findings in infants to visual acuity are few . the pediatric eye is shorter and optically differs from the adult , and measurements of the lateral extent of lesions reported by oct systems must be examined and most likely corrected based on the infant eye 's unique optical pathway the infant , and especially the premature infant , retina is notably different from the adult . thus the sd - oct reflective bands that define adult retinal layers are very different for the premature and term infant eye sd - oct findings in infant eyes such as macular edema of prematurity and subfoveal fluid in healthy term infants at birth may represent normal development or unrecognized disease processes studies correlating sd - oct findings in infants to visual acuity are few . nevertheless , sd - oct remains an invaluable tool for the assessment of pediatric retinal diseases .

the prefrontal cortex ( pfc ) exhibits a hemispheric specialization with respect to its functional role in the integration of affective states suggesting that the right pfc is important in eliciting stress responses ( see sullivan ) . uncontrollable foot shock ( carlson et al . ) or novelty stress ( berridge et al . ) resulted in a higher dopamine turnover selectively in the right pfc . the pfc has been subdivided into three main cytoarchitectonic subareas : infralimbic ( il ) , prelimbic ( pl ) , and anterior cingulate cortex ( acx ) ( krettek and price ; ray and price ) . each of these subareas has specific cortical and subcortical connections ( vertes ) and distinct physiological functions . lesion studies have shown that after acute stress , ventral ( il / pl ) ( sullivan and gratton ) and dorsal pfc ( pl / acx ) ( diorio et al . ) specific behavioral responses such as diminished fear reactivity ( lacroix et al . ) were observed after bilateral lesions in the il ( frysztak and neafsey ) , and increased fear reactivity was detected when the region pl / acx was lesioned ( morgan and ledoux ) . anxiety - like responses were observed after lidocaine infusion into the il ( wall et al . ) or lesioning the right il ( sullivan and gratton ) . recent studies in rats showed morphological changes in pyramidal neurons in the pfc following chronic restraint stress ( radley et al . [ 14 , 15 ] ; cook and wellman ) or after chronic corticosterone treatment ( wellman ) . chronic exposure to corticosterone also reduced the volume of layer ii in all pfc subareas ( cerqueira et al . ) . chronic restraint stress for 21 days decreased the number and the length of apical dendrites in cg1cg3 ( corresponding to the region pl / acx ) ( cook and wellman ; radley et al . ) , an effect accompanied by reduced spine density in the proximal portions of the apical dendrites ( radley et al . ) . however , these studies did not investigate regional or possible hemispheric differences . in the present study , we investigated whether pyramidal neurons in the three pfc subareas have a hemisphere - specific morphology , and whether their specific dendritic architecture would be remodeled in a lateralized manner in response to chronic stress . as reference for the exact localization of the neurons prior to their morphological reconstruction , we first identified the boundaries between the pfc subareas using antibodies against parvalbumin , the neurofilament protein smi-32 , and neuronal nuclear antigen ( neun ) . to reconstruct the morphology of individual pyramidal neurons in layer iii which is known to have reciprocal connections with the mediodorsal thalamic nucleus ( groenewegen ) , we filled cells with neurobiotin using a whole - cell patch - clamp technique . intracellular neurobiotin staining is a highly sensitive method ( pyapali et al . ) for visualizing neuronal processes that are not obscured by more intensely stained portions of the neurons ( hill and oliver ; oliver et al . ) . we investigated the morphological characteristics of pyramidal cells in the three pfc subareas paying particular attention to hemispheric differences in dendritic morphology following three weeks of daily restraint stress . adult male sprague dawley rats ( harlan - winkelmann , borchen , germany ) were housed in groups of three animals per cage with food and water ad libitum . animals were maintained in temperature - controlled rooms ( 21 1c ) with a light / dark cycle of 12 hours on , 12 hours off ( lights on at 07:00 ) . all animal experiments were performed in accordance with the european communities council directive of november 24 , 1986 ( 86/eec ) , the us national institutes of health guide for the care and use of laboratory animals , and were approved by the government of lower saxony , germany . male rats ( n = 5 , weighing 220250 g ) were killed by intraper - itoneal administration of an overdose of ketamine ( 50 mg / kg body weight ; ketavet , pharmacia & upjohn , erlangen , germany ) , xylazine ( 10 mg / kg body weight ; rompun , bayer leverkusen , germany ) , and atropine ( 0.1 mg / kg body weight ; wdt , hannover , germany ) . the descending aorta was clamped and the animals were transcardially perfused with cold 0.9% nacl for five minutes , followed by cold 4% paraformaldehyde in 0.1 m phosphate buffer ( pb ) at ph 7.2 for 20 minutes . post perfusion artifacts were prevented by postfixing heads in fresh fixative at 4c ( cammermeyer ) . the following day , the brains were gently removed and stored overnight in 0.1 m pb at 4c . brains were cryoprotected by immersion in 2% dmso and 20% glycerol in 0.125 m phosphate - buffered saline ( pbs ) at 4c . a small hole in the left striatum was made with a thin needle to differentiate the left from the right hemisphere . the brains were then cut into blocks containing the entire pfc , frozen on dry ice , and stored at 80c before serial cryosectioning at a section thickness of 50 m . eight to ten complete series of coronal sections were collected and stored in 0.1 m pbs for immunocytochemistry . a stereotaxic atlas of the rat brain ( paxinos and watson ) was used during the cryosectioning procedure . free - floating sections were washed in 0.1 m pbs and then treated with 0.5% h2o2 for 30 minutes . after washing , nonspecific binding of antibodies was blocked by incubating the sections for one hour in 5% normal goat serum ( ngs ; dako , glostrup , denmark ) in 0.1 m pbs containing 0.25% triton x-100 . the sections were subsequently incubated for 48 hours at 4c with the primary antibodies , neurofilament smi-32 ( mouse - anti - smi-32 , sigma aldrich ) , parvalbumin ( mouse - anti - pv ; sigma aldrich ) , and neuronal - nuclei neun ( mouse - anti - neun , sigma aldrich ) at dilutions 1 : 1000 , 1 : 2000 , and 1 : 500 , respectively , in pbs containing triton x-100 ( 0.5% for smi-32 , 0.25% for parvalbumin and neun ) and ngs ( 3% for smi-32 and neun , 1% for parvalbumin ) . following incubation , sections were thoroughly washed with 0.1 m pbs and incubated with biotinylated goat antimouse antibody ( dako ) diluted 1 : 200 in 0.1 m pbs with 3% ngs and 0.5% triton x-100 , for 1.5 hours , followed by washing in 0.1 m pbs . the sections were then incubated with 1 : 200 horseradish peroxidase - conjugated streptavidin ( dako ) in 0.1 m pbs with 3% ngs and after washing , sections were stained with a dab kit ( vector laboratories , burlingame , calif , usa ) , which contains 3,3-diaminobenzidine ( dab ) as chromogen . staining time in dab was 810 minutes for all sections ; the reaction was stopped by washing the sections in 0.1 m pbs . sections were mounted on glass slides in 0.1% gelatin and dried overnight at 37c , after which they were cleared in xylene for 30 minutes and finally coverslipped with eukitt ( kindler , freiburg , germany ) . a series of adjacent coronal sections was also mounted on glass slides , dried overnight at room temperature , and stained with cresyl violet to obtain a clear comparison with the immunocytochemical images . coronal sections were analyzed and photographed using a zeiss axiophot ii photomicroscope ( carl zeiss , germany ) at magnifications of 2.5 x , 10 x , and 20 x. the prefrontal cortical areas were identified and their boundaries or transition zones were outlined on photomicrographs of the sections , and a contour pattern ( delineating il , pl , and acx subareas ) was drawn and stored as a coreldraw file . localization of intracellularly filled cells ( see below ) was then corroborated by overlapping a picture of a filled cell with a picture of a boundary contour pattern closest to the same region ( anterior or posterior ; see results ) . smi-32 , parvalbumin , and neun stained sections were compared to ensure that the defined areas coincided , and were treated identically for the methods and measurements described below . stereotaxic coordinates of the pfc were identified with the rat brain atlas ( paxinos and watson ) and cortical layers in the subfields were identified using the accompanying text book ( zilles and wree ) . male sprague dawley rats initially weighing 150170 g were housed in groups of three animals with ad libitum access to food and tap water . the first experimental phase ( habituation ) lasted for 14 days , during which body weight was recorded daily . the second phase of the experiment ( restraint stress ) lasted for 21 days , during which the animals of the stress group ( n = 16 ) were submitted to daily restraint stress for six hours per day ( 09:0015:00 ) . the restraint procedure was carried out according to an established paradigm ( magarios and mcewen ) . briefly , rats were placed in plastic tubes in their home cages and had no access to food or water . control rats ( n = 16 ) were not subjected to any type of stress but were handled daily . at the end of the experiment , 24 hours after the last stress exposure , animals were weighed , deeply anesthetized with a mixture of 50 mg / ml ketamine , 10 mg / ml xylazine , and 0.1 mg / ml atropine by intraperitoneal injection , and decapitated . brains were rapidly removed and processed for slice preparation ( see below ) . increased adrenal and decreased thymus weights are indicators of sustained stress . the data are expressed in milligrams per 100 grams body weight . after dissecting the pfc from the brain , a sagittal cut was made in the left temporal cortex with a razor blade to further differentiate the hemispheres . the blocks containing the left and the right pfc were rapidly submerged in ice - cold oxygenated artificial cerebrospinal fluid ( acsf ) of the following composition ( in mm ) : nacl 125.0 ; kcl 2.5 ; l - ascorbic acid 1.0 ; mgso4 2.0 ; na2hpo4 1.25 ; nahco3 26.0 ; d - glucose 14.0 ; cacl2 1.5 ( all chemicals from merck , darmstadt , germany ) . the pfc was glued to the stage of a vibratome ( vibracut 2 , ftb , bensheim , germany ) and cut in coronal , 400 m thick slices . the slices were allowed to recover for at least one hour in acsf bubbled with 95% o2 , 5% co2 at ph 7.3 , 33c , and then kept at room temperature for up to seven hours . the method for intracellular labeling previously described ( kole et al . ) was used with some modifications . neurobiotin was injected through borosilicate glass pipettes with 35 m resistances , connected to an axopatch 200b amplifier ( axon instruments , union city , ca , usa ) , using pulse software ( heka , lambrecht , germany ) . the standard pipette solution contained ( in mm ) : k meso4 120 , kcl 20 , hepes 10 , egta 0.2 , atp ( magnesium salt ) 2 , phosphocreatine ( disodium salt ) 10 , gtp ( tris - salt ) 0.3 , and 3 mg / ml neurobiotin ( vector laboratories ) . pfc slices were transferred to a submerged recording chamber , continuously oxygenated with acsf ( flow rate : 1 - 2 ml / min ) , and maintained at 33c . cell bodies were visualized by infrared - differential interference contrast ( ir - dic ) video microscopy using an upright microscope ( axioskop 2 fs , carl zeiss , germany ) equipped with a 40 x/0.80 w objective ( zeiss ir - acroplan ) . negative pressure was used to obtain tight seals ( 210 g ) onto identified pyramidal neurons . the membrane was disrupted with additional suction to form the whole - cell configuration . pyramidal neurons with membrane potentials below 55 mv were excluded from the analysis . pyramidal cells are readily identified by their specific morphology , and only pyramidal - shaped somata located in layer iii of il , pl , and acx ( readily identified under ir - dic video : dodt and zieglgnsberger ) were used for neurobiotin filling . layer iii pyramidal somata , visible by transillumination , tend to be smaller than layer v somata . the border between layers ii and iii was difficult to identify ; however , cells in layer iii were mainly found at a depth of about 400 m from the pial surface ( gabbott and bacon ) . observation of labeled neurons in relation to the pfc boundaries ( see results ) verified their location . thereafter , the patch pipette was carefully withdrawn from the membrane and the slice was fixed in 0.1 m pb with 4% paraformaldehyde ( ph 7.4 ) and stored at 4c for at least 24 hours . whole slices were processed free floating , first by blocking endogenous peroxidase activity in a 0.1 m pb solution containing 1% h2o2 . after washing , nonspecific binding of antibodies was prevented by incubating the sections for one hour with 5% ngs ( dako ) in 0.1 m pbs and 0.3% triton x-100 . subsequently , slices were incubated with avidin - biotin peroxidase ( diluted 1 : 100 ; abc , vector laboratories ) in 1% ngs ( dako ) and 0.3% triton x-100 overnight at 4c . on the following day , slices were washed and left overnight in pbs . the following day , slices were equilibrated by washing them in tbs ( ph 7.6 ) and the staining reaction was completed by incubation in a solution containing 0.04% nicl2 , 0.5 mg / ml dab , and 0.01% h2o2 ( vector laboratories ) in tbs until a dark brown color appeared , typically in less than 10 minutes . the reaction was terminated by several washes in fresh 0.1 m pbs and finally in double - distilled water . tissue sections were then dehydrated in an ascending series of ethanols ( 30%100% ) , cleared with two 10-minute incubations in xylene and flat - embedded in eukitt ( kindler ) on glass slides . slices from at least one stressed and one control animals were always processed simultaneously . labeled cells were examined by light microscopy to ensure that they fulfilled the following criteria for the three - dimensional reconstruction : ( 1 ) a clearly visible and completely stained apical dendritic tree ; ( 2 ) at least three main basilar dendritic branches , each branching at least to the third - degree branch order ; ( 3 ) soma location in layer iii of an identified pfc subarea ; and ( 4 ) visibility of the most distal apical dendrites with dense labeling of the processes ( kole et al . ; radley et al . ) . to ensure that the analysis was performed blind , each slide was coded by an independent observer prior to neuronal reconstruction , and the code was not broken until all analyses were completed . in a few cases , cell coupling was observed ( < 1% ) ; such cells were omitted from the analysis , because the dendrites could not be assigned unequivocally to a single neuron . somata of intracellularly labeled cells were located at 6070 m depth from the slice surface allowing reconstruction of almost all their main dendritic branches . compromised cells that had truncated main apical or first - order basilar branches were omitted from the analysis . in each animal , 12 neurons were filled with neurobiotin , six in the left and six in the right hemisphere , randomly distributed among the three areas of interest . complete and optimally labeled pyramidal neurons meeting the above criteria were reconstructed and morphological parameters were quantified using neurolucida software ( microbrightfield , inc . , colchester , vt , usa ) in combination with an automated stage and focus control connected to the microscope ( zeiss iii rs ) . data were collected as line drawings consisting of x , y , and z coordinates . dendritic length was measured by tracing dendrites using a 40 x ( n.a . 0.75 ) the step size of the circular cursor was 0.16 m , sufficiently below the limits of light microscopy resolution ( about 0.25 m ) . numerical analysis and graphical processing of the neurons were performed with neuroexplorer ( microbrightfield ) . sholl plots ( sholl ) were constructed by plotting the dendritic length as function of distance ( corresponding to the radius ) from the soma center , which was automatically set to zero . the length of the dendrites within each subsequent radial bin at 10 m increments was summed . ethanol dehydration and xylene clearance are known to cause tissue shrinkage ( pyapali et al . ) . however , previous analyses from our laboratory suggested that the linear shrinkage correction has no direct effect on data used for morphological comparative analysis ( kole et al . ) . body weight ( bw ) and relative organ weight ( in milligrams per 100 grams of bw ) of control and stress animals at the end of the experiment were compared using the unpaired t - test . the total number of labeled neurons that fulfilled the above criteria to be analyzed was 69 in the control and 70 in the stress group . since these labeled cells were not evenly distributed among the animals , we calculated the means of the morphometric data for each hemisphere / animal . these mean data served as analysis unit for the statistical evaluation and are indicated as n in the tables . data for the total length of dendrites , the total number of branching points , and the total number of branches were evaluated by two - way anova ( factors : hemisphere group ) ( statistica software package , release 6.0 statsoft inc . , tulsa , okla , usa ) . numbers of branches per branch order were evaluated using three - way anova ( factors : branch order hemisphere group ) . sholl analysis data were evaluated with three - way repeated measures anova ( factors : hemisphere group radius ) ( spss version 12.0 , spss inc . , bonferroni 's post hoc test was used in all cases . because the morphology of the pyramidal cells shows complex differences along the dendritic trees we restricted our post hoc analyses to distinct radii ( 10 m , 20 m , 30 m , etc . ) and single branch orders ( 1st , 2nd , 3rd order , etc . ) . data are presented as mean sem ( standard error of the mean ) . according to previous descriptions , the rat pfc can be divided into three subareas : il , pl , and acx . as a basis for the reliable localization of neurobiotin labeled pyramidal neurons in the present study , we visualized the boundaries of these subareas using specific antibodies . the three subareas that were reliably found at the same location in all investigated brains were defined as showing differential staining patterns with at least two staining methods . immunocytochemical staining with smi-32 antibody gave a staining pattern that differentiates pl from acx , and acx from the premotor cortex in dorsal regions of the pfc ( figure 1(a ) ) . in the pl , the smi-32 antibody labeled layers iii and v. this pattern became lighter and narrower in the acx , where layer iii was lightly stained whereas layer v was darker and broader . acx could be distinguished from the premotor cortex because in the latter , the deep layers were intensely labeled by the smi-32 antibody ( figure 1(a ) , lower panel ) . parvalbumin proved to be a good marker to distinguish all pfc subareas and their respective layers . in il , layer ii was only lightly stained , layer iii was slightly darker and layer v showed a pronounced staining . in the pl , layer ii was distinctly stained by the pv antibody and layer iii appeared wider than in the il . the strong staining of layer v observed in the il gradually disappeared in the pl . in the acx , all layers had more parvalbumin - immunoreactive cells compared to pl . layer ii in acx showed darker staining compared to the pl ( figure 1(b ) ) . immunoreactivity for neun provided a boundary between il and pl , and a clearly layered pattern in all pfc subareas with pronounced staining of layer ii ( figure 1(c ) ) . the il was distinguished by a wide layer i and by densely packed cells in layers ii . compared to il , the pl had a lighter layer iii , and a broader layer v. in acx , layer v was again broader than in the pl ( figure 1(c ) ) . using nissl dyes , layer i can be clearly distinguished , however , it is difficult to distinguish the other cortical layers and to detect borders between pfc subareas ( figure 1(d ) ) . by comparing the location of each neurobiotin filled layer iii neuron ( see below ) with the boundary patterns described above , we were able to accurately define its subarea - specific location . intracellular neurobiotin labeling provides a reliable and sensitive method to study dendritic morphology ( paypali et al . ) . in all experimental groups , there was complete staining of the dendritic branches with distal dendrites being reliably visualized ( figure 2 ) . the fact that during injection of the dye cells were alive and healthy and the use of relatively thick slices ( 400 m ) increased the probability to reconstruct complete dendritic arbors without compromised branches . in both groups , control and stress , we filled a total of 384 cells of which 36% ( 139 cells ) fulfilled the criteria for complete staining suitable for a quantitative analysis of dendritic morphology . since these labeled cells were not evenly distributed among the animals and to avoid any bias we calculated means / hemisphere / animal . these means served as analysis units for the statistical evaluation ( see below ) . sholl analysis ( left versus right ) for a close inspection of the dendritic trees in the left and the right hemisphere , sholl analyses were performed ( figure 3 ) . for the basilar dendrites in the il , three - way anova performed with data from both groups , control and stress , ( factors : hemisphere group radius ) revealed significant effects of hemisphere ( f(1,572 ) = 6.12 , p < .05 ) and radius ( f(29,572 ) = 22.55 , p < .001 ) . bonferroni 's post hoc test indicated a significant interhemispheric difference for basilar dendrites in controls at 10 m from soma ( df = 31 , p < .05 ) ( figure 3(a ) , left panel ) . also for apical dendrites in the il three - way anova revealed a significant effect of hemisphere ( f(1,1086 ) = 24.18 , p < .001 ) but no reliable effect of radius . bonferroni 's post hoc test showed that in the control animals , apical dendrites in the right hemisphere were longer than in the left hemisphere . the sites where these right - left differences occurred were proximal to the soma , at 10 , 20 , and 60 m ( df = 30 , p < .05 in all cases ) ( figure 3(a ) , left panel ) . in the pl , sholl analysis of the basilar dendrites displayed significant effects of hemisphere ( f(1,780 ) = 11.91 , p .001 ) and radius ( f(29,780 ) = 43.81 , p < .001 ) ( figure 3(b ) ) . however , the post hoc test depicted no reliable difference between basilar dendrites in the left and the right hemisphere of controls . apical dendrites in the pl also showed a positive effect of hemisphere ( f(1,1018 ) = 4.07 , p < .05 ) and a weak effect of radius ( f(59,1018 ) = 1.42 , p < .05 ) . bonferroni 's post hoc test revealed significant interhemispheric differences in middle and distal portions of the apical dendritic tree of controls ( at 180 and 420 m from soma , df = 25 , p < .01 ; at 160 , 170 , and 190 , df = 25 , p < for the basilar dendrites in the acx , three - way anova indicated no reliable interhemispheric difference but only an effect of radius ( f(29,399 ) = 15.55 , p < .001 ) . for the apical dendrites in acx , anova revealed a positive effect of the hemisphere ( f(1,841 ) = 7.81 , p < .01 ) and an effect of radius ( f(51,841 ) = 3.11 , p < .001 ) . the post hoc test showed no interhemispheric difference with respect to apical dendrites in the acx of controls ( figure 3(c ) , left panel ) . these data demonstrate a lateralized morphology of apical dendrites on pyramidal neurons in il and pl but not in the acx of control rats . total dendritic length ( left versus right ) the total length of basilar and apical dendrites in control rats showed no significant interhemispheric differences although apical dendrites in the right tended to be longer than in the left hemisphere ( table 1 ) . dendritic branches ( left versus right ) the complexity of the apical and basilar dendritic trees in the two hemispheres was determined by analyzing numbers of branching points and branches ( table 2 ) . for basilar dendrites , two - way anova indicated no reliable interhemispheric differences for the total number of branching points and branches in any of the subareas ( table 2 ) . numbers of branches of distinct branch orders were evaluated by three - way anova ( factors : hemisphere group branch order ) . for basilar dendrites in the il , an effect of the hemisphere ( f(1,182 ) = 10.48 , p < .05 ) and of the branch order ( f(6,182 ) = 60.82 , < .001 ) was found ( details not shown ) . for il apical dendrites , numbers of branches of distinct branch orders revealed an effect of hemisphere ( f(1,288 ) = 4.73 , p < .05 ) and of branch order ( f(11,288 ) = 13.74 , p < bonferroni 's post hoc test showed reliable interhemispheric differences for the number of branches of the orders 4 , 6 , and 11 ( df = 24 , p < .05 in all cases ) and of branch order 12 ( df = 24 , p < .01 ) . in the left il , dendritic branches of the orders 11 and 12 could not be observed , but were only present in the right il ( table 2 ) . for basilar dendrites in the pl , three - way anova depicted no effect of the hemisphere but only an effect of the branch order ( f(6,168 ) = 68.23 , p < .001 ) . also for apical dendrites in the pl there was no effect of the hemisphere but only an effect of the branch order ( f(10,253 ) = 9.86 , p < .001 ) . the post hoc test showed no significant interhemispheric difference for any branch order in the pl ( table 2 ) . in the acx of control rats , there were no significant interhemispheric differences with respect to the total number of apical and basilar branches and branching points . three - way anova depicted only effects of the branch order ( basal : f(5,118 ) = 36.11 , p < .001 ; apical : f(10,220 ) = 9.92 , p < .001 ) ( table 2 ) . total dendritic length and sholl analysis ( stress effects ) stress reduced the total length of apical dendrites on pyramidal neurons in the pl selectively in the right hemisphere ( table 1 ) . dendrites of pyramidal neurons in stressed rats are shown in figure 3 ( right panel ) and in figure 4 . for basilar dendrites in the il , three - way anova depicted no reliable effect of stress . these dendrites also displayed no significant left - right difference in stressed animals ( figure 3(a ) , right panel ) . for apical dendrites in the il , three - way anova revealed an interaction hemisphere group ( f(1,1086 ) = 5.43 , p < .05 ) , but the post hoc test depicted no significant left - right difference for apical dendrites at defined distances from the soma ( figure 3(a ) , right panel ) . however , in the right il of stressed animals , the length of proximal dendrites at several sites was significantly shorter than in controls ( at 10 , 20 , 30 , 40 , and 70 m , df = 30 , p < .05 ; at 50 and 60 m , df = 30 , p < .01 ) ( figure 4(a ) ) . in the pl of stressed rats , no reliable interhemispheric differences with respect to apical dendrites on pyramidal neurons were observed ( figure 3(b ) , right panel ) . three - way anova for apical branches in the pl , performed with data from all groups , showed an interaction hemisphere group ( f(1,1018 ) = 17.40 , p < .001 ) . the post hoc test indicated that in the right pl , stress reduced the length of apical dendrites at 160 , 170 , 190 , 420 m ( df = 25 , p < .05 ) and at 180 m ( df = 25 , p < .01 ) ( figure 4(b ) ) . for basilar dendrites in the acx , three - way anova depicted an effect of group ( f(1,399 ) = 9.23 , p < .01 ) and an interaction hemisphere group ( f(1,399 ) = 6.40 , p < .05 ) . also for the apical dendrites , three - way anova showed an effect of group ( f(1,841 ) = 9.34 , p < .01 ) but no interaction . in the left hemisphere of the acx , stress reduced the length of apical dendrites at certain distances from soma ( 210 , 220 , 240 , and 250 m ; df = 21 , p < .05 in all cases ) ( figure 4(c ) ) . also for the right acx , the post hoc test depicted a significant stress effect ( at 20 m from soma ; df = 21 , p < .05 ) . dendritic branches ( stress effects ) the effect of stress on the total number of branching points and the total number of dendritic branches was also analyzed ( table 2 ) . for apical dendrites in the il , however , the post hoc test depicted a significant stress effect on numbers of branches of the order 12 in the right il ( df = 24 , p < .01 ) . these dendritic branches could not be observed in stressed animals ( table 2 ) . in the pl , the branching pattern of basilar dendrites was apparently not affected by stress . for the total number of apical branching points , two - way anova revealed an interaction group hemisphere ( f(1,31 ) = 3.28 , p < .05 ) represented by a reliable stress induced decrease in the total number of branching points in the right hemisphere ( df = 29 , p < .05 ) . stress also reduced the number of branches of the order 3 selectively in the right hemisphere of the pl ( interaction : group hemisphere ; f(1,253 ) = 7.61 , p < .01 ; post hoc test : df = 23 , , three - way anova showed an interaction hemisphere group ( f(1,118 ) = 5.32 , p < .05 ) . for apical dendrites = 5.46 , p < .05 ) represented by a stress induced increase in the number of branches of the order 3 in the right hemisphere ( df = 20 , p < .05 ) . moreover , there was a significant left - right difference with respect to order 3 branches in the acx ; no dendritic branches of this order could be observed in stressed rats ( df = 20 , p < .05 ) ( table 2 ) . these data show that chronic restraints stress affects dendrites in the right hemisphere of il and pl . in contrast in the acx , it is the left hemisphere that is affected by stress . to assess the physiological effects of chronic stress , we measured body weight ( bw ) and weights of thymus and adrenal glands . in rats subjected to the restraint stress for 21 days , body weight at the end of the experiment was significantly lower than in controls ( control : 328.9 8.8 g ; stress : 292.3 7.0 g ; t = 3.205 , p < .05 ) . adrenal weight was significantly increased in stressed animals ( controls : 13.66 0.36 mg/100 g bw ; stress : 16.01 0.86 mg/100 g bw ; t = 2.452 , p < .05 ) and thymus weight was significantly reduced ( controls : 120.10 5.84 mg/100 g bw ; stress 100.40 4.27 mg/100 g bw ; t = 2.755 , p < .05 ) . these results agree with previous reports on physiological changes induced by chronic restraint stress ( magarios and mcewen ; watanabe et al . ; wellman ) . in the first part of this study , we identified the boundaries between the three pfc subareas . the border between pl and acx could be visualized with the smi-32 antibody which labels neurofilaments ( sternberger and sternberger ) . the parvalbumin antibody , which stains a subpopulation of cortical interneurons ( gabott and bacon ) , strongly stained layer v and was suitable for recognizing the boundaries between il and pl . the antibody against neun , a selective marker for neurons ( mullen et al . ) , proved to be better than conventional nissl staining at defining cortical layers ii and iii . delineation of the subarea boundaries and of cortical layers was a prerequisite for the exact localization of pyramidal neurons within the rat pfc . because projections from the mediodorsal thalamic nucleus principally target layer iii and v of the pfc ( uylings et al . gabbott et al . ; krettek and price ) we analyzed pyramidal cells exclusively in layer iii . the neurons that we investigated showed apical dendrites extending to the brain surface , and the distance of their somata was up to 550 m from the pia mater . therefore , their location agreed with a previous description of the pfc layers ( gabbott and bacon ) . in contrast , brown et al . reported that the somata of the pyramidal neurons they examined were located closer to the cortical surface ( distance of 250280 m ) , probably in layer ii . it is important to mention that intracellular neurobiotin labeling allows a better staining of distal dendritic branches compared to conventional methods such as golgi staining ( pyapali et al . ) . we found intrinsic morphological asymmetries in the pref - ronto - cortical pyramidal cells of control animals . in pl and il , there were interhemispheric differences in the length of apical dendrites at certain distances from the soma . these are new findings , because previous studies addressing the morphology of pyramidal neurons in the pfc did not discriminate between the hemispheres ( wellman ; cook and wellman ; radley et al . ) . intrinsic asymmetries have been observed before in several regions of the human cerebral cortex , for example , in entorhinal , auditory- and language - associated cortices ( hayes and lewis ; hutsler ) . simic et al . found larger pyramidal neurons in the human left entorhinal cortex compared to the right , and hypothesized that this asymmetry reflects a more extended dendritic arborization and enlarged neuropil volume in the left hemisphere . the present data show that in pfc subareas pl and il of the rat , the length of apical dendrites at certain distances from soma differs between the hemispheres . in pl and il , chronic stress abolished the interhemispheric differences in the length of dendrites observed at certain distances from the soma . in the right pl , stress also reduced the total length of the apical dendrites . the stress - induced factors that lead to these changes are not yet known , however , one may speculate that dopamine which is known to decrease excitability of pfc pyramidal neurons plays a role ( jedema and moghaddam ; gulledge and jaffe ) . electrophysiological recordings have shown that dopamine enhances the spatiotemporal spread of activity in the rat pfc , at least in part via layer iii pyramidal neurons ( bandyopadhyay et al . ) . as mentioned above , stress can increase dopamine turnover in the right pfc ( carlson et al . ; thiel and schwarting ) and chronic stress reduces the spontaneous activity of dopaminergic neurons in the ventral tegmental area ( vta ) which project to the pfc ( moore et al . ; benes et al . ) . in coincidence with this , it was found that repeated stress reduces dopamine ( mizoguchi et al . ) , norepinephrine ( kitayama et al . ) and serotonin in the pfc ( mangiavacchi et al . ) . although it is not known whether in the present study , the chronic restraint stress induced a deficit in dopamine and/or other monoamines , it may be hypothesized that the changes in dendrites observed here are related to maladaptive neurochemical processes . in contrast to pl and il , pyramidal neuron dendrites in the acx of control rats showed no interhemispheric differences , but the stress induced a left - right difference . previous reports described a stress induced decrease in apical dendritic length using a similar ( cook and wellman ; brown et al . ) or the same stress protocol ( radley et al . ) . while we investigated only layer iii pyramidal neurons , the former studies focused on cells more widely distributed over layers ii - iii ( cook and wellman ; radley et al . ) . sensory input or environmental enrichment has been shown to promote the formation of spines on proximal dendrites ( turner and lewis ) . in hippocampal pyramidal neurons , extensive dendritic sprouting and enhanced spine density were observed when axonal afferents were increased ( kossel et al . ) whereas the loss of afferents can lead to dendritic atrophy ( valverde ; benes et al . ; deitch and rubel ) . one may speculate that the stress - induced dendritic changes observed in the present study are related to alteration in axonal input . the observation that the dendritic alterations emerged at certain distances from soma may be related to the fact that axonal input to the pfc is site - specific and depends on the cortical layer . the stress - induced reduction in the total length of dendrites in the right pl is in line with previous findings showing lateralization of the pfc mediated stress response . right side lesions of the il / pl reduced the peak stress - induced corticosterone response ( sullivan and gratton ) and decreased anxiety ( sullivan and grattron ) , suggesting that a compromised right pfc activity results in a lack of control over the physiological and behavioral responses to stress . especially the prelimbic and the anterior cingulate cortex are important to react to environmental stimuli ( cardinal et al . ) . therefore , one may assume that alterations in the morphology of pfc pyramidal neurons have an impact on the stress response . similar stress - related processes as in the pfc have been observed in the amygdala . chronic restraint stress increased the length of apical dendrites of pyramidal cells in the basolateral amygdala ( vyas et al . ) which sends projections to and receives input from pl and acx ( vertes ) . like in the pfc , the activity of the basolateral amygdala appears to be lateralized under stress conditions ( adamec et al . ) . under normal conditions , the pfc inhibits the basolateral amygdala ( rosenkranz and grace ) ; but under stress , this inhibition might be impaired thus contributing to an over - reactivity of this nucleus . it is possible that the morphological remodeling of the pyramidal neurons in the rat pfc that we describe in the present study is related to a presumptive stress - induced change in information transfer between pfc and amygdala . studies in humans indicated that reduced lateralization correlates with pathological conditions or with aging processes as fronto - cortical activity during cognitive performance tends to be less lateralized in old compared to young adults ( dolcos et al . ) . a recent investigation on the human dorsolateral pfc demonstrated a hemispheric asymmetry in pyramidal cell density in normal subjects ( higher density left compared to right ) , which was reversed in schizophrenic patients ( cullen et al . ) . rotenberg suggested that in depressed patients , the right pfc hemisphere is over - activated , and subjects with major depression displayed a reduced size of neurons in layer iii of the right orbitofrontal cortex ( cotter et al . ) . however , studies in depressed patients that did not focus on hemispheric differences reported on decreased activity in the pfc area ventral to the genu of the corpus callosum ( drevets et al . ) , and on reduced cortical thickness , glial size , and glial densities in supragranular layers of the orbitofrontal cortex ( rajkowska et al . ) . our study in rats shows that chronic restraint stress has a strong effect on the morphology of pyramidal neurons in the right hemisphere , at least in pl and il . a shortening of even a few dendrites on ca1 hippocampal pyramidal neurons enhanced the back - propagation of action potentials ( golding et al . ; schaefer et al . ) . moreover , experiments in our laboratory revealed that a stress - induced decrease in the length of apical dendrites of ca3 pyramidal neurons in the rat hippocampus correlates with reduced onset latency of excitatory postsynaptic potentials ( kole et al . ) . however , functional studies are required to assess the physiological implications of such morphological remodeling in the rat pfc . this is the first study showing intrinsic hemispheric differences in the dendritic morphology of pyramidal neurons in subareas of the rat pfc . in pl and il of control rats , interhemispheric differences in the length of apical dendrites at certain distances from the soma chronic stress abolished these right - left differences and reduced the total length of apical dendrites in the right pl . in contrast in the acx , there was no hemispheric difference in controls but stress induced a left - right difference . these chronic stress - induced regional changes may be correlated with the specialized functions of pfc subareas in stress - related pathologies , and provide additional support for previous studies of stress - dependent activation of the right pfc .

the prefrontal cortex ( pfc ) plays an important role in the stress response . we filled pyramidal neurons in pfc layer iii with neurobiotin and analyzed dendrites in rats submitted to chronic restraint stress and in controls . in the right prelimbic cortex ( pl ) of controls , apical and distal dendrites were longer than in the left pl . stress reduced the total length of apical dendrites in right pl and abolished the hemispheric difference . in right infralimbic cortex ( il ) of controls , proximal apical dendrites were longer than in left il , and stress eliminated this hemispheric difference . no hemispheric difference was detected in anterior cingulate cortex ( acx ) of controls , but stress reduced apical dendritic length in left acx . these data demonstrate interhemispheric differences in the morphology of pyramidal neurons in pl and il of control rats and selective effects of stress on the right hemisphere . in contrast , stress reduced dendritic length in the left acx .

1. INTRODUCTION 2. MATERIALS AND METHODS 3. RESULTS 4. DISCUSSION 5. CONCLUSIONS

the prefrontal cortex ( pfc ) exhibits a hemispheric specialization with respect to its functional role in the integration of affective states suggesting that the right pfc is important in eliciting stress responses ( see sullivan ) . the pfc has been subdivided into three main cytoarchitectonic subareas : infralimbic ( il ) , prelimbic ( pl ) , and anterior cingulate cortex ( acx ) ( krettek and price ; ray and price ) . lesion studies have shown that after acute stress , ventral ( il / pl ) ( sullivan and gratton ) and dorsal pfc ( pl / acx ) ( diorio et al . ) were observed after bilateral lesions in the il ( frysztak and neafsey ) , and increased fear reactivity was detected when the region pl / acx was lesioned ( morgan and ledoux ) . recent studies in rats showed morphological changes in pyramidal neurons in the pfc following chronic restraint stress ( radley et al . chronic restraint stress for 21 days decreased the number and the length of apical dendrites in cg1cg3 ( corresponding to the region pl / acx ) ( cook and wellman ; radley et al . ) in the present study , we investigated whether pyramidal neurons in the three pfc subareas have a hemisphere - specific morphology , and whether their specific dendritic architecture would be remodeled in a lateralized manner in response to chronic stress . to reconstruct the morphology of individual pyramidal neurons in layer iii which is known to have reciprocal connections with the mediodorsal thalamic nucleus ( groenewegen ) , we filled cells with neurobiotin using a whole - cell patch - clamp technique . we investigated the morphological characteristics of pyramidal cells in the three pfc subareas paying particular attention to hemispheric differences in dendritic morphology following three weeks of daily restraint stress . a small hole in the left striatum was made with a thin needle to differentiate the left from the right hemisphere . coronal sections were analyzed and photographed using a zeiss axiophot ii photomicroscope ( carl zeiss , germany ) at magnifications of 2.5 x , 10 x , and 20 x. the prefrontal cortical areas were identified and their boundaries or transition zones were outlined on photomicrographs of the sections , and a contour pattern ( delineating il , pl , and acx subareas ) was drawn and stored as a coreldraw file . the second phase of the experiment ( restraint stress ) lasted for 21 days , during which the animals of the stress group ( n = 16 ) were submitted to daily restraint stress for six hours per day ( 09:0015:00 ) . after dissecting the pfc from the brain , a sagittal cut was made in the left temporal cortex with a razor blade to further differentiate the hemispheres . the blocks containing the left and the right pfc were rapidly submerged in ice - cold oxygenated artificial cerebrospinal fluid ( acsf ) of the following composition ( in mm ) : nacl 125.0 ; kcl 2.5 ; l - ascorbic acid 1.0 ; mgso4 2.0 ; na2hpo4 1.25 ; nahco3 26.0 ; d - glucose 14.0 ; cacl2 1.5 ( all chemicals from merck , darmstadt , germany ) . pyramidal cells are readily identified by their specific morphology , and only pyramidal - shaped somata located in layer iii of il , pl , and acx ( readily identified under ir - dic video : dodt and zieglgnsberger ) were used for neurobiotin filling . labeled cells were examined by light microscopy to ensure that they fulfilled the following criteria for the three - dimensional reconstruction : ( 1 ) a clearly visible and completely stained apical dendritic tree ; ( 2 ) at least three main basilar dendritic branches , each branching at least to the third - degree branch order ; ( 3 ) soma location in layer iii of an identified pfc subarea ; and ( 4 ) visibility of the most distal apical dendrites with dense labeling of the processes ( kole et al . in each animal , 12 neurons were filled with neurobiotin , six in the left and six in the right hemisphere , randomly distributed among the three areas of interest . body weight ( bw ) and relative organ weight ( in milligrams per 100 grams of bw ) of control and stress animals at the end of the experiment were compared using the unpaired t - test . the total number of labeled neurons that fulfilled the above criteria to be analyzed was 69 in the control and 70 in the stress group . data for the total length of dendrites , the total number of branching points , and the total number of branches were evaluated by two - way anova ( factors : hemisphere group ) ( statistica software package , release 6.0 statsoft inc . as a basis for the reliable localization of neurobiotin labeled pyramidal neurons in the present study , we visualized the boundaries of these subareas using specific antibodies . in the pl , layer ii was distinctly stained by the pv antibody and layer iii appeared wider than in the il . compared to il , the pl had a lighter layer iii , and a broader layer v. in acx , layer v was again broader than in the pl ( figure 1(c ) ) . in both groups , control and stress , we filled a total of 384 cells of which 36% ( 139 cells ) fulfilled the criteria for complete staining suitable for a quantitative analysis of dendritic morphology . sholl analysis ( left versus right ) for a close inspection of the dendritic trees in the left and the right hemisphere , sholl analyses were performed ( figure 3 ) . for the basilar dendrites in the il , three - way anova performed with data from both groups , control and stress , ( factors : hemisphere group radius ) revealed significant effects of hemisphere ( f(1,572 ) = 6.12 , p < .05 ) and radius ( f(29,572 ) = 22.55 , p < .001 ) . bonferroni 's post hoc test indicated a significant interhemispheric difference for basilar dendrites in controls at 10 m from soma ( df = 31 , p < .05 ) ( figure 3(a ) , left panel ) . also for apical dendrites in the il three - way anova revealed a significant effect of hemisphere ( f(1,1086 ) = 24.18 , p < .001 ) but no reliable effect of radius . bonferroni 's post hoc test showed that in the control animals , apical dendrites in the right hemisphere were longer than in the left hemisphere . however , the post hoc test depicted no reliable difference between basilar dendrites in the left and the right hemisphere of controls . apical dendrites in the pl also showed a positive effect of hemisphere ( f(1,1018 ) = 4.07 , p < .05 ) and a weak effect of radius ( f(59,1018 ) = 1.42 , p < .05 ) . bonferroni 's post hoc test revealed significant interhemispheric differences in middle and distal portions of the apical dendritic tree of controls ( at 180 and 420 m from soma , df = 25 , p < .01 ; at 160 , 170 , and 190 , df = 25 , p < for the basilar dendrites in the acx , three - way anova indicated no reliable interhemispheric difference but only an effect of radius ( f(29,399 ) = 15.55 , p < .001 ) . the post hoc test showed no interhemispheric difference with respect to apical dendrites in the acx of controls ( figure 3(c ) , left panel ) . these data demonstrate a lateralized morphology of apical dendrites on pyramidal neurons in il and pl but not in the acx of control rats . total dendritic length ( left versus right ) the total length of basilar and apical dendrites in control rats showed no significant interhemispheric differences although apical dendrites in the right tended to be longer than in the left hemisphere ( table 1 ) . for basilar dendrites in the il , an effect of the hemisphere ( f(1,182 ) = 10.48 , p < .05 ) and of the branch order ( f(6,182 ) = 60.82 , < .001 ) was found ( details not shown ) . for il apical dendrites , numbers of branches of distinct branch orders revealed an effect of hemisphere ( f(1,288 ) = 4.73 , p < .05 ) and of branch order ( f(11,288 ) = 13.74 , p < bonferroni 's post hoc test showed reliable interhemispheric differences for the number of branches of the orders 4 , 6 , and 11 ( df = 24 , p < .05 in all cases ) and of branch order 12 ( df = 24 , p < .01 ) . in the left il , dendritic branches of the orders 11 and 12 could not be observed , but were only present in the right il ( table 2 ) . also for apical dendrites in the pl there was no effect of the hemisphere but only an effect of the branch order ( f(10,253 ) = 9.86 , p < .001 ) . in the acx of control rats , there were no significant interhemispheric differences with respect to the total number of apical and basilar branches and branching points . total dendritic length and sholl analysis ( stress effects ) stress reduced the total length of apical dendrites on pyramidal neurons in the pl selectively in the right hemisphere ( table 1 ) . dendrites of pyramidal neurons in stressed rats are shown in figure 3 ( right panel ) and in figure 4 . for basilar dendrites in the il , three - way anova depicted no reliable effect of stress . for apical dendrites in the il , three - way anova revealed an interaction hemisphere group ( f(1,1086 ) = 5.43 , p < .05 ) , but the post hoc test depicted no significant left - right difference for apical dendrites at defined distances from the soma ( figure 3(a ) , right panel ) . however , in the right il of stressed animals , the length of proximal dendrites at several sites was significantly shorter than in controls ( at 10 , 20 , 30 , 40 , and 70 m , df = 30 , p < .05 ; at 50 and 60 m , df = 30 , p < .01 ) ( figure 4(a ) ) . in the pl of stressed rats , no reliable interhemispheric differences with respect to apical dendrites on pyramidal neurons were observed ( figure 3(b ) , right panel ) . the post hoc test indicated that in the right pl , stress reduced the length of apical dendrites at 160 , 170 , 190 , 420 m ( df = 25 , p < .05 ) and at 180 m ( df = 25 , p < .01 ) ( figure 4(b ) ) . in the left hemisphere of the acx , stress reduced the length of apical dendrites at certain distances from soma ( 210 , 220 , 240 , and 250 m ; df = 21 , p < .05 in all cases ) ( figure 4(c ) ) . dendritic branches ( stress effects ) the effect of stress on the total number of branching points and the total number of dendritic branches was also analyzed ( table 2 ) . for apical dendrites in the il , however , the post hoc test depicted a significant stress effect on numbers of branches of the order 12 in the right il ( df = 24 , p < .01 ) . for the total number of apical branching points , two - way anova revealed an interaction group hemisphere ( f(1,31 ) = 3.28 , p < .05 ) represented by a reliable stress induced decrease in the total number of branching points in the right hemisphere ( df = 29 , p < .05 ) . stress also reduced the number of branches of the order 3 selectively in the right hemisphere of the pl ( interaction : group hemisphere ; f(1,253 ) = 7.61 , p < .01 ; post hoc test : df = 23 , , three - way anova showed an interaction hemisphere group ( f(1,118 ) = 5.32 , p < .05 ) . for apical dendrites = 5.46 , p < .05 ) represented by a stress induced increase in the number of branches of the order 3 in the right hemisphere ( df = 20 , p < .05 ) . these data show that chronic restraints stress affects dendrites in the right hemisphere of il and pl . in contrast in the acx , it is the left hemisphere that is affected by stress . in rats subjected to the restraint stress for 21 days , body weight at the end of the experiment was significantly lower than in controls ( control : 328.9 8.8 g ; stress : 292.3 7.0 g ; t = 3.205 , p < .05 ) . in pl and il , there were interhemispheric differences in the length of apical dendrites at certain distances from the soma . these are new findings , because previous studies addressing the morphology of pyramidal neurons in the pfc did not discriminate between the hemispheres ( wellman ; cook and wellman ; radley et al . ) found larger pyramidal neurons in the human left entorhinal cortex compared to the right , and hypothesized that this asymmetry reflects a more extended dendritic arborization and enlarged neuropil volume in the left hemisphere . the present data show that in pfc subareas pl and il of the rat , the length of apical dendrites at certain distances from soma differs between the hemispheres . in pl and il , chronic stress abolished the interhemispheric differences in the length of dendrites observed at certain distances from the soma . in the right pl , stress also reduced the total length of the apical dendrites . electrophysiological recordings have shown that dopamine enhances the spatiotemporal spread of activity in the rat pfc , at least in part via layer iii pyramidal neurons ( bandyopadhyay et al . ) as mentioned above , stress can increase dopamine turnover in the right pfc ( carlson et al . although it is not known whether in the present study , the chronic restraint stress induced a deficit in dopamine and/or other monoamines , it may be hypothesized that the changes in dendrites observed here are related to maladaptive neurochemical processes . in contrast to pl and il , pyramidal neuron dendrites in the acx of control rats showed no interhemispheric differences , but the stress induced a left - right difference . the stress - induced reduction in the total length of dendrites in the right pl is in line with previous findings showing lateralization of the pfc mediated stress response . therefore , one may assume that alterations in the morphology of pfc pyramidal neurons have an impact on the stress response . chronic restraint stress increased the length of apical dendrites of pyramidal cells in the basolateral amygdala ( vyas et al . ) it is possible that the morphological remodeling of the pyramidal neurons in the rat pfc that we describe in the present study is related to a presumptive stress - induced change in information transfer between pfc and amygdala . rotenberg suggested that in depressed patients , the right pfc hemisphere is over - activated , and subjects with major depression displayed a reduced size of neurons in layer iii of the right orbitofrontal cortex ( cotter et al . ) , and on reduced cortical thickness , glial size , and glial densities in supragranular layers of the orbitofrontal cortex ( rajkowska et al . ) our study in rats shows that chronic restraint stress has a strong effect on the morphology of pyramidal neurons in the right hemisphere , at least in pl and il . moreover , experiments in our laboratory revealed that a stress - induced decrease in the length of apical dendrites of ca3 pyramidal neurons in the rat hippocampus correlates with reduced onset latency of excitatory postsynaptic potentials ( kole et al . ) this is the first study showing intrinsic hemispheric differences in the dendritic morphology of pyramidal neurons in subareas of the rat pfc . in pl and il of control rats , interhemispheric differences in the length of apical dendrites at certain distances from the soma chronic stress abolished these right - left differences and reduced the total length of apical dendrites in the right pl . in contrast in the acx , there was no hemispheric difference in controls but stress induced a left - right difference . these chronic stress - induced regional changes may be correlated with the specialized functions of pfc subareas in stress - related pathologies , and provide additional support for previous studies of stress - dependent activation of the right pfc .

genetic regulation of flowering is a key element of adapting plants to cultivation in different latitudes , daylength responses and/or purposes ( such as seed / grain or biomass production ) . in the semi - arid tropical habitats to which many of the world s most important seed / grain crops are native , flowering during short days of < 12.5 h of light in a 24-h period coordinates gametogenesis and reproduction with generally favorable rainfall , temperature , and solar radiation ( harper 1977 ) . photoperiod - insensitive ( day - neutral ) mutants of many seed and grain crops that flower based on accumulation of heat units have become widely used in temperate latitudes , producing higher seed / grain yields than short - day genotypes which generally flower late in the growing season . in crops grown for biomass rather than seed / grain , flowering is generally undesirable , in sugarcane , e.g. , reducing yields ( julien and soopramanien 1976 ; long 1976 ; julien et al . 1978 ; heinz 1987 ) and increasing disease susceptibility ( ricaud et al . 1980 ) . flowering is widely acknowledged as a significant obstacle in maximizing the cellulosic yield potential of temperate biomass crops such as miscanthus , a close relative of sugarcane ( jakob et al . 2009 ) . its small diploid genome with minimal gene duplication and substantial homozygosity make sorghum ( sorghum bicolor l. moench . ) an attractive genomic model ( paterson et al . 2009 ) for the andropogoneae , tropical grasses in which biochemical and morphological specializations ( c4 photosynthesis ) improve carbon assimilation at high temperatures and which include complex paleo- and/or neopolyploid genomes of important crops such as sugarcane , miscanthus , maize and pearl millet ( the latter being paniceae ) . parsimony suggests the sorghum karyotype to resemble that of the andropogoneae common ancestor ( wang et al . my of divergence from rice , sorghum has experienced only 3% differential gene loss and little rearrangement ( paterson et al . 2009 ) . with an estimated 96 my of abstinence from genome duplication ( wang et al . 2015 ) , functions of sorghum genes are likely to still resemble those of the common grass ancestor . in contrast , in only 15 my , genome duplication and associated fractionations ( gene and cis - regulator losses ) in maize ( schnable et al . 2010 ; woodhouse et al . sorghum evolution and improvement has yielded a remarkable diversity of morphologies , with genotypes of 6 m or more in height preferred in africa as an important dual - purpose ( grain + straw ) crop . in contrast , genotypes of 1 m or less are widely utilized in mechanized agriculture . introduced into usa 150 years ago , sorghum is grown on 810 million acres and has a farm - gate value of typically $1 billion / year ( usda - nass 2013 ) . its drought resistance provides an important failsafe in the agriculture of the us southern plains that often receive too little rain for other grains . likewise , in arid countries of northeast and west africa , sorghum contributes 2639% of calories in the human diet ( fao statistical database on crops 2011 ) . in addition to its importance for food , feed , and straw , sorghum is the # 2 us source of fuel ethanol and the availability of sweet sorghums , s. bicolor genotypes with stalk sugar concentrations approaching those of sugarcane , make it suitable for commercial - scale biofuel production from each of three feedstocks , starch , sugar and cellulose ( rooney et al . the requirement for qualitatively different flowering regimes hinders both use of the rich diversity of tropical crop germplasm in temperate latitudes , and of high - yielding improved temperate germplasm in the tropics . for example , wild forms of sorghum and also many tropical cultivars flower only during short day photoperiods of 12.5 h daylight or less ( quinby and karper 1945 ) , coinciding with generally favorable rainfall , temperature , and solar radiation ( harper 1977 ) . in temperate latitudes , such obligate short - day genotypes typically flower near the end of the growing season , by which time the majority of solar radiation has already been missed , and often with too little time to mature seeds before freezing temperatures arrive . much higher grain yield is generally realized in temperate regions from photoperiod - insensitive ( day - neutral ) genotypes that flower much earlier and make better use of season - long solar radiation . have sought to render rich diversity of native tropical crop germplasm more accessible to temperate breeding programs by crossing to a temperate donor genotype and converting to ( selecting for ) day - neutral flowering while backcrossing to otherwise restore the genotype of the tropical recurrent parent . to date , in sorghum alone > 700 genotypes have been converted over 50 years of effort , in most cases involving four generations of backcrossing each followed by two generations of selfing to identify recessive day - neutral flowering segregants . previously , we studied f2 progeny of a cross between s. bicolor btx623 , a day - neutral breeding line , and s. propinquum , a tropical and short - day flowering wild relative . most of the f2 population , with at least one copy of the s. propinquum short day flowering allele , flowered in mid - september or later in the latitude where the experiment was done ( 30.6n ; college station , tx ) , consistent with the short - day flowering of the tropical s. propinquum parent . we showed a single quantitative trait locus ( qtl ) , flravgd1 , to account for 85.7% of this phenotypic variation in flowering time ( lin et al . 1995 ) , and to correspond approximately to the locus of the triticeae photoperiodic genetic loci , ppd1 - 3 and ppd - h1 ( paterson , lin , et al . we show by integration of positional and association genetic evidence that the short - day flowering qtl flravgd1 locates on chromosome 6 ( formerly linkage group d ) in a 10-kb interval within the physically largest centimorgan in the sorghum genome . this interval contains a single annotated gene that we show to partly complement the day - neutral flowering phenotype . the gene accounting for flravgd1 is sb06g012260 , a member of the flowering locus t ( ft ) family of transcription factors that is highly divergent from most known floral regulators in this family . sb06g012260 is unique to panicoids , evolving as a single gene duplication shortly after the oryzoid ( rice ) panicoid ( sorghum , maize ) divergence . sb06g012260 suppresses flowering , although it is quite distant evolutionarily from other ft family members that are floral suppressors ( pin et al . we discuss its relationship to named sorghum flowering genes and implications of these findings for candidacy of other genes , as well as its evolution in an invasive sorghum species that has adapted to temperate latitudes . to conduct interval mapping of flowering time in sorghum , we previously analyzed an f2 population of s. bicolor cultivar btx623s . propinquum using 78 rflp loci spanning 935 cm with an average distance of 14 cm between markers ( lin et al . a qtl explaining 85.7% of phenotypic variation in flowering time , flravgd1 , was placed in the 21-cm interval between dna markers psb095 and psb428a . to more finely map flravgd1 , 34 f2 plants were selected that were putatively recombinant in the 21-cm interval between dna markers psb095 and psb428a on chromosome 6 . an additional 27 dna markers were applied to pooled dna from 50 to 150 selfed f3 progenies that were also grown in the field near college station , texas . about 4 of the 34 f3 families , # 10 , 187 , 191 , and 211 , were excluded because the dna marker genotypes of f2 and pooled f3 tissue were not consistent ( # 211 ) , or because the flravgd1 locus genotype of their f2 parents predicted from phenotypic segregation in f3 progenies was contradicted by both flanking markers and by virtually all other markers on the chromosome ( # 10 , 187 , 191 ) . each such inconsistency would have required a double recombination event to explain , and 3 such events among 34 progeny in a 21-cm interval are highly improbable . a few such incongruous plants were also observed in the f2 generation , and are an important example of the need for progeny testing to reliably fine - map flowering , which can be influenced by other genetic effects , temperature , and other factors such as some diseases ( quinby 1974 ) . based on progeny testing , flravgd1 was placed between dna markers psb1113 and cdsr084 , estimated to range from 0.3 to 1.1 cm apart in two different mapping populations studied ( the larger distance is indicated in fig . bac clones were identified containing each of these dna markers , but lengthy efforts to chromosome walk in this region failed due to repeatedly encountering repetitive dna near bac ends . the sorghum genome sequence revealed this 1.1 cm interval to include 34 mbp , thus having 60-fold less recombination than the genome - wide average ( paterson et al . ( a ) linkage mapping of flravgd1 to sorghum chromosome 6 ( lg d ; lin et al . 1995 ) ; ( b ) progeny testing in 30 f3 families that were recombinant in the interval containing flravgd1 delineated the locus to a 1.1-cm region including 400 genes ; ( c ) analysis of 90 diverse exotic sorghums and their corresponding converted derivatives delimited flravgd1 to a 4.1-mb region including 63 genes . ( d ) association genetics implicated sb06g012260 in short - day flowering conferred by flravgd1 . ( e ) the day - neutral haplotype of sb06g012260 contains three deletions in the 5 region , one removing a caat box essential to many eukaryotic promoters . ( a ) linkage mapping of flravgd1 to sorghum chromosome 6 ( lg d ; lin et al . 1995 ) ; ( b ) progeny testing in 30 f3 families that were recombinant in the interval containing flravgd1 delineated the locus to a 1.1-cm region including 400 genes ; ( c ) analysis of 90 diverse exotic sorghums and their corresponding converted derivatives delimited flravgd1 to a 4.1-mb region including 63 genes . ( d ) association genetics implicated sb06g012260 in short - day flowering conferred by flravgd1 . ( e ) the day - neutral haplotype of sb06g012260 contains three deletions in the 5 region , one removing a caat box essential to many eukaryotic promoters . to better circumscribe the location of flravgd1 in the 34-mbp target region , and to confirm that the s. propinquum - derived locus also accounts for intraspecific variation within s. bicolor ( the cultigen ) , we utilized recombination accumulated during conversion of diverse short - day tropical sorghums to day - neutral flowering ( stephens et al . 1967 ) . sorghum conversion involves four backcrosses each followed by two generations of selfing ( stephens et al . 1967 ) , a breeding scheme that in regions held heterozygous is expected to impart 3.5 more recombinational information to converted genotypes than f2 or recombinant inbred genotypes ( allard 1956 ) . ninety diverse exotic sorghums and their corresponding converted derivatives ( supplementary table s1 , supplementary material online ) were genotyped at nine simple sequence repeat ( ssr ) loci distributed approximately evenly through the 34 mbp target interval inferred for the qtl based on our s. bicolors . 1995 ) , and closely coincides with flravgd1 , strongly supporting the hypothesis that the s. propinquum - derived qtl locus also accounts for intraspecific variation in short - day vs. day - neutral photoperiod response in s. bicolor . in 384 diverse sorghums ( hash et al . 2008 ) phenotyped for photoperiod response index ( pri : see materials and methods ) , we identified and genotyped dna polymorphisms in annotated genes near the conversion peak . tassel was used to perform tests of association , employing population structure covariates and a kinship matrix for the sorghum germplasm panel based on published ssrs ( hash et al . the most significant association ( p < 10 ) was found with sb06g012260 ( fig . 1d , with raw data in additional data file 2 ) , a gene containing a pebp domain ( interpro ipr008914 ) shared by floral regulators in arabidopsis ( ft ) ( kardailsky et al . 1999 ) and oryza ( hd3a ) ( kojima et al . 2002 ) . by comparing s. propinquum bac sequences containing sb06g012260 to the s. bicolor draft genome and resequencing polymorphic sites in the 384 diverse sorghums , we found two major sb06g012260 haplotypes . one haplotype is widespread across tropical africa , and is shared by short - day s. propinquum . the other haplotype was most abundant in accessions from south africa , the most temperate part of the natural range of sorghum and where day - neutral flowering is common ( fig . the predominantly south african haplotype was characterized by four deletions , respectively , of 423 nt starting 5901 nt upstream of the sb06g012260 transcription - start site ; 4186 nt starting 4553 nt upstream ; 3 nt starting 219 nt upstream ; and 27 nt in the second intron of sb06g012260 ( fig . 1e ) . additional indels of 2 and 7 nt ( 5451 and 5025 nt upstream ) , and three synonymous mutations in exon 1 and two in exon 2 were not analyzed in depth . flowering of 384 diverse sorghums were compared in the 20072008 post - rainy ( short day ) , and 2008 rainy ( long day ) seasons in peninsular india to determine photoperiod response index ( as described in methods ) . hierarchical clustering of sorghum accessions was based on distances between ssr genotypes ( number of bands not shared ) . while short - day sorghums experience delayed flowering under long days ( > 12 h ) , long days accelerated flowering by 30 days for many genotypes from south africa , the most temperate part of the natural range . east asian ( asiae ) sorghums , the largest temperate - adapted group from the northern hemisphere , also had accelerated long day flowering . flowering of 384 diverse sorghums were compared in the 20072008 post - rainy ( short day ) , and 2008 rainy ( long day ) seasons in peninsular india to determine photoperiod response index ( as described in methods ) . hierarchical clustering of sorghum accessions was based on distances between ssr genotypes ( number of bands not shared ) . while short - day sorghums experience delayed flowering under long days ( > 12 h ) , long days accelerated flowering by 30 days for many genotypes from south africa , the most temperate part of the natural range . east asian ( asiae ) sorghums , the largest temperate - adapted group from the northern hemisphere , also had accelerated long day flowering . s1 , supplementary material online ) containing short - day s. propinquum sb06g012260 alleles subcloned from a bac and transformed into the day - neutral accession tx430 using published methods ( howe et al . 2006 ) each delayed flowering of transgenic f2 progeny in long days ( table 1 ) . widely used for sorghum transformation because of its high efficiency ( howe et al . 2006 ) , tx430 has day - neutral flowering but has an unusual haplotype , with deletion of seven amino acids in the 4th exon of sb06g012260 . independent t0 transformants were selfed to produce t1 segregating progenies , then 1524 plants from each t1 family were evaluated in the greenhouse under ambient long day conditions ( at 33.95 n latitude ) , planting on 17 may ( 13 h , 58 min photoperiod ) and recording the number of days to flower emergence . plants were genotyped by pcr to determine allele state for the transgene . among 13 transformation events carrying a 5 kb construct limited to sb06g012260 and its immediate upstream elements , two conferred statistically significant delays averaging 13.1 ( p = 0.03 ) and 24.8 days ( p = 0.09 ) , and one line showed unexpected accelerated flowering ( 14.1 days , p = 0.05 ) . among 10 independent events harboring a 10 kb construct spanning the entire haplotype ( from sb06g012260 through the 4,186 nt element ) , transgenic f2 progeny of only three showed significantly altered flowering , with delays of 4.1 ( p = 0.002 ) , 4.2 ( p = 0.07 ) and 5.2 ( p = 0.008 ) days . table 1regression analysis of relationship of flowering time to genotype for transgenic constructs ( transgene events with p < 0.1 are shown).constructeventwild - type*transgenic*transgene effect ( days)ppap5.21451.7564.9113.150.0310pap5.21661.0085.8224.810.0857pap5.22183.0068.8514.150.0463pap10.2348.2952.444.160.0710pap10.2647.0051.084.080.0019pap10.2749.7555.005.250.0079*least - squares means . regression analysis of relationship of flowering time to genotype for transgenic constructs ( transgene events with p < 0.1 are shown ) . the predominant day - neutral sb06g012260 haplotype includes one mutation likely to alter gene function . the 3-bp deletion located 219 nt upstream of sb06g012260 removed a predicted caat box , an invariant dna sequence in many eukaryotic promoters required for sufficient transcription ( berg et al . the 423-bp deletion removes a non - autonomous cacta transposon ; and the 4,186-nt deletion removes an open reading frame that matches a gene on chr . 7 of day - neutral sorghum ( sb07g008600 , blastp 93.01% i d , e - value 0 ) , but with a stop codon in its first exon . sb02g029725 , a family member that is more closely related to hd3a and ft than to sb06g012260 , maps near the likelihood peak of another sorghum flowering qtl , flravgb1 ( lin et al . 1995 ) . resequencing of this gene in the 384-member diversity panel used above ( hash et al . 2008 ) revealed two abundant haplotypes ( resembling s. propinquum and btx623 , respectively ) , which showed highly significant association with pri ( p = 1.5310 ) . thus , at least two members of the ft gene family are implicated in the modulation of flowering in sorghum . several other sorghum , saccharum and maize ft - containing genes occur in a clade that includes both rice hd3a ( os06g06320.1 ) ( kojima et al . indeed , sb10g003940.1 is a colinear ortholog of hd3a , which also formed a tandem duplicate in the rice lineage ( os06g06300 : fig . 3 ) . however , the sequence of rice hd3a is more similar to arabidopsis ft and another arabidopsis homolog ( at4g20370.1 ) than to sb06g012260 ( fig . six homologs were found in arabidopsis ( prefixed at , including ft ; kardailsky et al . 1999 ) , 19 in rice ( os , including hd3a ; kojima et al . 2002 ) , 19 in sorghum ( sb ) , 26 in maize ( grm or ac ) and 8 in sugarcane ( put-157a - saccharum_officinarum ) , identified by blast from http://www.plantgdb.org/prj/estcluster/progress.php>plantgdb , last accessed june 22 , 2016 . a phylogenetic tree of inferred homologous protein sequences was made at http://www.phylogeny.fr/ , last accessed june 22 , 2016 , using muscle for alignment and maximum - likelihood ( phyml ) to determine the tree . four distinct sub - families including ftl - like , ft / ftl - like , tfl1-like and mft - like proteins , are highlighted on the tree . the impact on flowering ( promoting or suppressing ) are shown for several well studied pebp genes ( reviewed in klintens et al . the panel on the right along the tree shows multiple alignments based on the arabidopsis ft protein at amino acid position 85 at exon 2 , positions 128141 ( p - loop domain ) , positions 150152 and position 164 that were shown to have critical regulatory roles . for full alignments across the entire length of these proteins , six homologs were found in arabidopsis ( prefixed at , including ft ; kardailsky et al . 1999 ) , 19 in rice ( os , including hd3a ; kojima et al . 2002 ) , 19 in sorghum ( sb ) , 26 in maize ( grm or ac ) and 8 in sugarcane ( put-157a - saccharum_officinarum ) , identified by blast from http://www.plantgdb.org/prj/estcluster/progress.php>plantgdb , last accessed june 22 , 2016 . a phylogenetic tree of inferred homologous protein sequences was made at http://www.phylogeny.fr/ , last accessed june 22 , 2016 , using muscle for alignment and maximum - likelihood ( phyml ) to determine the tree . four distinct sub - families including ftl - like , ft / ftl - like , tfl1-like and mft - like proteins , are highlighted on the tree . the impact on flowering ( promoting or suppressing ) are shown for several well studied pebp genes ( reviewed in klintens et al . the panel on the right along the tree shows multiple alignments based on the arabidopsis ft protein at amino acid position 85 at exon 2 , positions 128141 ( p - loop domain ) , positions 150152 and position 164 that were shown to have critical regulatory roles . for full alignments across the entire length of these proteins , an sb06g012260 allele found at high frequency in weedy and invasive s. halepense may have contributed to its spread . sorghum halepense ( johnson grass ) , a tetraploid derived naturally from wild s. bicolor and s. propinquum progenitors ( paterson , schertz , et al . 1995 ) , has spread across much of asia , africa , europe , north and south america , and australia . its establishment in usa is post - columbian , by introduction as a prospective forage and/or contaminant of sorghum seedlots ( mcwhorter 1971 ) . with inbreeding progenitors of tropical origin and therefore being putatively homozygous for short - day flowering , the original tetraploid s. halepense is expected to have had four copies of the short - day haplotype . among the few old world s. halepense accessions in the us national plant germplasm collection , we confirmed by pcr that pi209217 from south africa and pi271616 from india are homozygous for the short - day haplotype , and confirmed by growouts that these two genotypes do not flower in long days . with maximum seed production under 10.512 h daylengths consistent with short - day flowering , us ( temperate ) s. halepense nonetheless flowers and produces ample seed in photoperiods of up to 16 h ( warwick and black 1983 ) . we genotyped the four loci diagnostic of the short - day haplotype ( 423 nt , 4,186 nt , 3 nt , and intron indels ) in 480 s. halepense plants sampled equally from populations in ga , tx ( 2 ) , ne , and nj described previously ( morrell et al . at the two terminal loci of the haplotype , 81.6% ( 423 nt indel ) and 88.2% ( intron indel ) were homozygous for the short - day alleles , consistent with the expected genotype of their tropical progenitors . however , at the two internal loci of the haplotype ( 4,186 and 3 nt indels ) , only 1.1% and 8.0% of plants were homozygous for the short - day - associated alleles ( fig . 4 and supplementary table s2 , supplementary material online ) . only 39-bp upstream from the locus of the caat box deletion in day - neutral s. bicolor , 85.9% of s. halepense plants have at least one copy of a 4-nt insertion that disrupts a tc - rich repeat , a cis - acting motif enriched in promoters of photoperiod - responsive genes ( mongkolsiriwatana et al . we have not found this mutation in any other members of the genus ( unpublished data ) , suggesting that it is rare or novel . further , at the 4,186-nt indel , 98.9% of s. halepense also have at least one allele from the day - neutral haplotype ( fig . thus , it appears that an sb06g012260 mutation in naturalized us s. halepense independent of those in s. bicolor has resulted in 4genotype distributions of five us johnson grass populations near the sb06g012260 gene . among 480 plants sampled equally from ga , tx ( 2 ) , ne , and nj populations ( morrell et al . 2005 ) , 81.6% and 88.2% were homozygous for the short - day haplotype ( blue bars ) at two terminal loci ( 423 nt , 27 bp intron indels ) , but only 1.1% and 8.0% at two internal loci ( 4,186 and 3 nt indels ) . homozygosity for day - neutral alleles ( green ) is nearly absent from the ga sample ( from the region where johnson grass is thought to have been introduced to usa ) , but exceeds 10% in the two northerly populations ( ne , nj ) where day - neutral flowering would be most advantageous , and is intermediate in the two tx populations . genotype distributions of five us johnson grass populations near the sb06g012260 gene . among 480 plants sampled equally from ga , tx ( 2 ) , ne , and nj populations ( morrell et al . 2005 ) , 81.6% and 88.2% were homozygous for the short - day haplotype ( blue bars ) at two terminal loci ( 423 nt , 27 bp intron indels ) , but only 1.1% and 8.0% at two internal loci ( 4,186 and 3 nt indels ) . homozygosity for day - neutral alleles ( green ) is nearly absent from the ga sample ( from the region where johnson grass is thought to have been introduced to usa ) , but exceeds 10% in the two northerly populations ( ne , nj ) where day - neutral flowering would be most advantageous , and is intermediate in the two tx populations . based on several lines of evidence , sb06g012260 appears to have evolved as a single - gene duplication ( fig . 5 ) shortly after the oryzoid ( rice ) panicoid ( sorghum , maize ) divergence . first , its divergence from its nearest sorghum homolog , sb04g008320 is estimated at 40 ma ( ks = 0.43 , based on a widely - used evolutionary rate for cereal genes ; gaut et al . 1996 ) , suggesting that it evolved after oryzoid ( rice ) panicoid ( sorghum , maize ) divergence ( consistent with gene tree topography , fig . 3 ) . sb04g008320 has a colinear rice ortholog ( os02g13830.1 ) but sb06g012260 lacks colinear orthologs in both rice and brachypodium distachyon , although possessing them in setaria and maize ( fig . 5 ) . this pattern of orthology is indicative of an origin of sb06g012260 in panicoids , shortly after divergence from a common ancestor shared with oryzoids ( rice ) and pooids ( brachypodium ) . rectangles represent predicted gene models with colors showing relative orientations ( blue : same strand , green : opposite strand ) . matching gene pairs are displayed as connecting shades . microsynteny pattern around sb06g012260 across five grasses . rectangles represent predicted gene models with colors showing relative orientations ( blue : same strand , green : opposite strand ) . matching gene pairs several independent lines of evidence including fine mapping , association genetics , mutant complementation , and evolutionary analysis all implicate a single gene , sb06g012260 , as the cause of the flravgd1 qtl that accounted for 85.7% of variation in flowering time of a temperatetropical sorghum population ( lin et al . identification of this gene was complicated by its location in the physically largest single cm in the sorghum genome , containing 5% of sorghum genomic dna and 1.3% ( 400 ) of genes ( paterson et al . 2009 ) , with enrichment of retroelements and other non - genic dna . while both its panicoid - specific origin ( fig . 5 ) and 1 ) falsify the inference that ma1 corresponds to the locus of the triticeae photoperiodic genetic loci , ppd1 - 3 and ppd - h1 ( paterson , lin , et al . 10 paralog should exist at 105 mb , near the flowering qtl ( koester et al . 1993 ) . however , the nearest ft homolog to this location ( ac217051.3_fg006 : chr . 10 , 114 mb ) has a sequence so divergent from sb06g012260 that it appears non - orthologous . the wild - type sb06g012260 is the first floral suppressor discovered in the ftl clade of the ft gene family although another clade member is a floral promoter , zcn8 ( meng et al . 3 ) . despite being a floral suppressor , sb06g012260 resembles all four functionally characterized members of the tfl1 clade of the family ( hanzawa et al . 2016 ) . among the 23 transgene events that we evaluated , 5 showed statistically significant ( 0.1 or less , with 3 at 0.05 or less ) delays in flowering , whereas only one showed the acceleration that might be expected from general overexpression of a florigen . the candidate mutation that removed a caat box upstream of the day - neutral sb06g012260 allele would be expected to reduce gene expression , also consistent with the hypothesis that wild - type sb06g012260 is a floral suppressor . there is growing appreciation that floral regulation and other aspects of growth and development are regulated by antagonistic actions of multiple members of ft and other gene families ( lifschitz et al . exchange of a single amino acid between arabidopsis florigen ft and floral repressor tfl1 is sufficient to exchange the functions of these opposing genes ( hanzawa et al . 2005 ) . the finding that mutant complementation studies of sb06g012260 only partially recapitulated the short - day phenotype , underline the need for more work to understand the function and regulation of sb06g012260 . it is noteworthy that the overwhelming majority of transgenic lines with significant deviations from tx430 had delayed flowering , not the general promotion of flowering that might be explained simply by overexpression of many ft - like genes ( mcgarry and ayre 2012 ) . however , it is somewhat perplexing that only one of the 23 events reached the average 24.6 ( 3.5 ) day delay between the reference genetic stock 100 m ( murphy et al . 2011 ) that contains ma-1 ( a dominant allele conferring short - day flowering in tropical sorghums ; quinby and karper 1945 ) and tx430 under our conditions . shorter flowering delays than the ma1 reference genotype 100 m relative to putatively near - isogenic sm100 ( murphy et al . 2011 ) may indicate that some distant regulatory elements were missing from the transgene constructs and/or that its native heterochromatic chromatin environment is important to its natural function . a key element of the additional information needed is the exact timing and location of sb06g012260 gene expression . in more than 1.7 trillion reads from ( unpublished ) sorghum transcriptomic data pooled across leaves , stems , and flowers of 40 diverse genotypes grown by several different investigators in different environments , we found only four reads from sb06g012260 two each in two different genotypes . a detailed study of 19 sorghum pebp genes in roots , leaves ( four stages ) , stems , shoot apices and floral heads by semiquantitative rt - pcr also detected no expression of sb06g012260 in any tissue ( wolabu et al . further , two of the three other sorghum members of the subclade including sb06g012260 , sb04g008320 ( sbft7 ) and sb03g002500 ( sbft13 ) , also show no expression in any tissue , although the third member , sb10g021790 ( sbft9 ) shows moderate expression in leaves ( wolabu et al . finally , the nearest maize homolog of sb06g012260 , zcn21 , shows no discernible expression in a broad sampling of tissues ( danilevskaya et al . in contrast , sb02g029725 ( sbft6 ) , which we implicate above in the flravgb1 sorghum flowering qtl , showed strong expression in leaves across all developmental stages and was also detected in the floral head and florets , but does not induce flowering in arabidopsis ( wolabu et al . other members of its subclade ( sbft3 , 4 , 5 , and 11 ) all show expression but in different tissues and developmental states , and like sb06g012260 do not show expression patterns or genic interactions consistent with being florigens ( wolabu et al . where does sb06g012260 fit among the named sorghum flowering loci ? based on the high frequency at which this region has long been known ( lin et al . 1995 ) to be selected for during sorghum conversion , we inferred that the qtl in this region was ma1 , the sorghum maturity ( flowering ) locus long known to have the largest effect on phenotype ( quinby and karper 1945 ) . moreover , based on relatively coarse resolution qtl data , we previously suggested correspondence of flravgd1 to a homoeologous series of triticeae photoperiodic ( ppd1 - 3 ) loci ( paterson , lin , et al . others have suggested prr37 to be ma1 based on positional proximity to a flowering trait mapped in genetic populations different from ours , together with its expression pattern in response to photoperiodic cues ( murphy et al . , the transition from the heterochromatic surroundings of sb06g012260 to euchromatin , and an associated increase in recombination , coincides with a precipitous drop in levels of conversion at 37 mb ( fig . prr37 is in a euchromatic genomic location ( 40.3 mb ) experiencing only 15% conversion ( fig . curiously , study of 390 exotic - converted pairs genotyped at 46,062 markers ( albeit with 66% missing data before imputation ) suggested an introgression peak at 42 mb , different from both our peak and prr37 ( thurber et al . the fact that this region has 60-fold less recombination than the genome - wide average ( fig . 1 ) , suggests that limited recombination rather than multiple loci under selection ( thurber et al . 2013 ) accounts for the high frequency of introgression in this region , and indeed across much of the chromosome . evidence in support of prr37 as ma1 is further confounded by factors that were not known to its proponents ( murphy et al . 2011 ) or their reviewers . prr37 was not tested by mutant complementation , but was a positional candidate that showed striking expression differences based on comparison of near - isogenic lines called 100 m and sm100 that differ in prr37 alleles ( murphy et al . s2 , supplementary material online ) that the short - day flowering 100 m line is introgressed with not only a putatively short - day prr37 allele but also with the short - day sb06g012260 haplotype , based on genotyping at both the 423 and 4,186 nt indels that are on the distal side of the gene relative to prr37 . this indicates that the chromosome segment by which 100 m and sm100 differ contains both prr37 and sb06g012260 , as well as at least 82 intervening protein - coding genes and 6.75 mb dna ( prr37 [ sb06g014570 ] has an address of chr6 : 40,280,41440,290,602 . phenotypic differences between these lines could therefore be explained by either of these two genes , interactions between the two , or other factors . further , in the genotype that was the source of the short - day flowering allele that we mapped ( s. propinquum ) , prr37 is non - functional with a 2-nt insertion near the 5 end causing 19 nonsense mutations ( supplementary fig . s3 , supplementary material online ) , effectively ruling out that it could confer a dominant phenotype in crosses with s. bicolor . finally , the fine - scale mapping that we describe herein ( fig . in addition to ma1 , a more recently discovered flowering locus , ma6 , also maps to chromosome 6 very near the location of prr37 ( brady 2006 ) indeed , one report stated that prr37 corresponded to ma6 ( mullet et al . however , members of the same group who implicated prr37 as ma1 have more recently implicated a new positional candidate as ma6 based on apparent functional polymorphism and expression differences between long - day and short - day conditions ( murphy et al . 2014 ) whereas this new candidate corresponds to orthologs in several other cereals such as the rice ghd7 gene , once again evidence from mutant complementation is lacking . the time and cost associated with sorghum transformation is clearly a hindrance to validation of candidate genes in this promising botanical model . in partial summary , we consider the most probable scenario to be that sb06g012260 is the cause of the ma1 short - day flowering trait in sorghum , as supported by independent lines of evidence including fine mapping , association genetics , mutant complementation , and evolutionary analysis . evidence in support of another nearby gene ( prr37 ) as ma1 is now known to be confounded with previously unknown factors , specifically the presence of sb06g012260 and at least 82 additional annotated genes on the crucial introgressed chromosome segment . the non - functional prr37 allele in our population rules out the possibility that the two genes are functioning in concert to confer the trait . while this new evidence does not contradict the hypotheses that each gene independently contributes to the flowering phenotype , or that prr37 may be ma6 ( mullet et al . 2012 ) , recent data implicating the sorghum ortholog of rice grain number , plant height and heading date-7 ( ghd7 ) as ma6 is more compelling ( murphy et al . 2014 ) , albeit needing support from mutant complementation the evolution of sb06g012260 from a single gene duplicate , rather than a dosage - balanced gene from whole - genome duplication , follows a trend exemplified by key members of the c4 photosynthesis pathway ( wang et al . wgd - duplicates are a rich potential source of genetic novelty , with longer half - lives than single gene duplicates ( lynch et al . however , single - gene duplicates , often lacking their ancestral regulatory elements and in different chromatin environments from their ancestral gene(s ) , may have greater per - gene potential for the evolution of novelty as reflected by greater divergence of expression patterns than wgd - duplicates ( wang et al . an interesting hypothesis for further investigation is whether the survival of single - gene duplicates such as sb06g012260 may be favored by location in recombinationally - recalcitrant heterochromatin , where neutral or slightly - deleterious mutations tend to survive longer than in euchromatin ( bowers et al . 2009 ) and offer more opportunity for a mutation that confers new function to coincide with an environment that drives it to high frequency . recalcitrance of the ma1 region of chromosome 6 to recombination may have contributed to the evolution of a coadapted gene complex ( mayr 1954 ) . beyond flowering , qtls have been associated with this region affecting kernel weight ( paterson , lin , et al . 1995 ) , tillering , rhizomatousness and regrowth ( paterson , schertz , et al . in particular , the ma1 region also holds dw2 , the gene of largest effect on sorghum stature ( height ) ( lin et al . 1995 ) but which has long been asserted to be separable from ma1 by recombination . quinby suggested that ma1 and dw2 were different closely - linked genes , with ca . 8% crossing over ( quinby 1974 ) , but only evaluating 47 families based on phenotype . based on our observation that late flowering can occasionally be a result of factors other than allelic status at the ma1 locus and that progeny testing is necessary to validate it , such a small study must be considered tenuous . among the 30 validated f3 families in our study , three showed different segregation patterns for flowering time and plant height . since these 30 individuals comprised all confirmed recombinants in the region from a population of 370 individuals , this suggests a 0.5-cm linkage distance between ma1 and dw2 ( lin 1998 ) . while the strongest association of allelic variation with plant height in the diversity panel was at sb06g012260 itself ( p = 0.007 ) , we also found a marginally significant association at sb06g007330 ( p = 0.023 ) , a putative cation efflux family protein . further study is needed to determine if sb06g007330 is dw2 however an intriguing hypothesis for further testing is that increased height confers a competitive advantage in light interception , and alleles conferring this trait might have become abundant more quickly if co - transmitted with alleles for optimal flowering time in the native tropical environment of sorghum . moreover , further dissection of the ma1 region may reveal whether qtls for other traits that have been mapped to the region ( lin et al . 1995 ; paterson , lin , et al . 1995 ; paterson , schertz , et al . , 1995 ) are pleiotropic consequences of ma1 or dw2 , or represent additional members of a coadapted gene complex . engineering of genotypes that silence the short - day flowering trait may render obsolete the need to laboriously convert tropical grasses to day - neutral flowering by twelve ( ! ) generations of breeding ( stephens et al . 1967 ) , potentially dramatically accelerating cross - utilization of temperate and tropical germplasm for sorghum , sugarcane , and many other crops . targeted selection or engineering of strong floral repressor alleles in biomass crops may confer consistent high yields ( julien and soopramanien 1976 ; long 1976 ; julien et al . rflp and ssr mapping used published methods , with markers drawn from published maps ( bowers et al . phenotyping of f3 families was based on 50150 selfed progenies per family , grown in the field near college station , texas , at ambient daylengths described in detail and with flowering dates recorded as described ( lin et al . association genetics used a 384-member worldwide sorghum diversity panel from icrisat , previously characterized with 41 ssr markers ( hash et al . 2008 ) , evaluated in 2007 under short - day conditions ( 11.812.15 h light ) and high humidity , under which short - day sorghums are expected to initiate flowering promptly . a 2008 planting was characterized by a transition from long to short - day ( 13.111.0 h ) photoperiod and dry conditions , and short - day sorghums would be expected to delay flowering . flowering time was the number of days required for 50% of the plants in a single row to flower ( dfl50% ) . photoperiod response index ( pri ) was defined as the mean difference in dfl50% between the two planting seasons ( i.e. , pri = dfl50%2008dfl50%2007 ) . resequencing used bigdye terminator chemistry , and sequences were manually checked and aligned for single nucleotide polymorphism ( snp ) identification with sequencher 4.1 . the quantity and frequency of haplotypes , and linkage disequilibrium were determined by haplotyper 1.0 , and tassel 2.1 , respectively . tassel was used to perform tests of association , employing population structure covariates and a kinship matrix for the gcp / icrisat germplasm panel based on published ssrs ( hash et al . the bac was sequenced to confirm the integrity of sb06g012260 and identify restriction sites , then completely digested with pvuii to recover a 10.5-kb fragment that extends 5,220-bp upstream and 234-bp downstream from sb06g012260 . likewise , the bac clone was completely digested with restriction enzymes stui and bstz17i to recover a 5.2-kb fragment that extends 434-bp upstream and 234-bp downstream from sb06g012260 . both fragments were cloned into transformation vector pzp211 , and the clones ( supplementary fig . s1 , supplementary material online ) re - sequenced to confirm the integrity of the constructs . independent t0 transformants were selfed to produce t1 segregating progenies , then 1524 plants from each t1 family were evaluated in the greenhouse under ambient long day conditions ( at 33.95 n latitude ) , recording the number of days from planting on 17 may to flower emergence . genotyping of the four loci diagnostic of the ma1 haplotype ( 423 nt , 4186 nt , 3 nt , and intron indels ) in s. halepense used 480 plants sampled equally from ga , tx ( 2 ) , ne , and nj populations described previously ( morrell et al . supplementary figs . s1s4 and tables s1 and s2 are available at molecular biology and evolution online ( http://www.mbe.oxfordjournals.org/ ) .

of central importance in adapting plants of tropical origin to temperate cultivation has been selection of daylength - neutral genotypes that flower early in the temperate summer and take full advantage of its long days . a cross between tropical and temperate sorghums [ sorghum propinquum ( kunth ) hitchc.s . bicolor ( l. ) moench ] , revealed a quantitative trait locus ( qtl ) , flravgd1 , accounting for 85.7% of variation in flowering time under long days . fine - scale genetic mapping placed flravgd1 on chromosome 6 within the physically largest centimorgan in the genome . forward genetic data from converted sorghums validated the qtl . association genetic evidence from a diversity panel delineated the qtl to a 10-kb interval containing only one annotated gene , sb06g012260 , that was shown by reverse genetics to complement a recessive allele . sb06g012260 ( sbft12 ) contains a phosphatidylethanolamine - binding ( pebp ) protein domain characteristic of members of the ft family of flowering genes acting as a floral suppressor . sb06g012260 appears to have evolved 40 ma in a panicoid ancestor after divergence from oryzoid and pooid lineages . a species - specific sb06g012260 mutation may have contributed to spread to temperate regions by s. halepense ( johnsongrass ) , one of the world s most widespread invasives . alternative alleles for another family member , sb02g029725 ( sbft6 ) , mapping near another flowering qtl , also showed highly significant association with photoperiod response index ( p = 1.5310 6 ) . the evolution of sb06g012260 adds to evidence that single gene duplicates play large roles in important environmental adaptations . increased knowledge of sb06g012260 opens new doors to improvement of sorghum and other grain and cellulosic biomass crops .

Introduction Results Discussion Materials and Methods Supplementary Material

in the semi - arid tropical habitats to which many of the world s most important seed / grain crops are native , flowering during short days of < 12.5 h of light in a 24-h period coordinates gametogenesis and reproduction with generally favorable rainfall , temperature , and solar radiation ( harper 1977 ) . photoperiod - insensitive ( day - neutral ) mutants of many seed and grain crops that flower based on accumulation of heat units have become widely used in temperate latitudes , producing higher seed / grain yields than short - day genotypes which generally flower late in the growing season . 2015 ) , functions of sorghum genes are likely to still resemble those of the common grass ancestor . the requirement for qualitatively different flowering regimes hinders both use of the rich diversity of tropical crop germplasm in temperate latitudes , and of high - yielding improved temperate germplasm in the tropics . for example , wild forms of sorghum and also many tropical cultivars flower only during short day photoperiods of 12.5 h daylight or less ( quinby and karper 1945 ) , coinciding with generally favorable rainfall , temperature , and solar radiation ( harper 1977 ) . much higher grain yield is generally realized in temperate regions from photoperiod - insensitive ( day - neutral ) genotypes that flower much earlier and make better use of season - long solar radiation . have sought to render rich diversity of native tropical crop germplasm more accessible to temperate breeding programs by crossing to a temperate donor genotype and converting to ( selecting for ) day - neutral flowering while backcrossing to otherwise restore the genotype of the tropical recurrent parent . previously , we studied f2 progeny of a cross between s. bicolor btx623 , a day - neutral breeding line , and s. propinquum , a tropical and short - day flowering wild relative . most of the f2 population , with at least one copy of the s. propinquum short day flowering allele , flowered in mid - september or later in the latitude where the experiment was done ( 30.6n ; college station , tx ) , consistent with the short - day flowering of the tropical s. propinquum parent . we showed a single quantitative trait locus ( qtl ) , flravgd1 , to account for 85.7% of this phenotypic variation in flowering time ( lin et al . we show by integration of positional and association genetic evidence that the short - day flowering qtl flravgd1 locates on chromosome 6 ( formerly linkage group d ) in a 10-kb interval within the physically largest centimorgan in the sorghum genome . this interval contains a single annotated gene that we show to partly complement the day - neutral flowering phenotype . the gene accounting for flravgd1 is sb06g012260 , a member of the flowering locus t ( ft ) family of transcription factors that is highly divergent from most known floral regulators in this family . to conduct interval mapping of flowering time in sorghum , we previously analyzed an f2 population of s. bicolor cultivar btx623s . a qtl explaining 85.7% of phenotypic variation in flowering time , flravgd1 , was placed in the 21-cm interval between dna markers psb095 and psb428a . to more finely map flravgd1 , 34 f2 plants were selected that were putatively recombinant in the 21-cm interval between dna markers psb095 and psb428a on chromosome 6 . a few such incongruous plants were also observed in the f2 generation , and are an important example of the need for progeny testing to reliably fine - map flowering , which can be influenced by other genetic effects , temperature , and other factors such as some diseases ( quinby 1974 ) . 1995 ) ; ( b ) progeny testing in 30 f3 families that were recombinant in the interval containing flravgd1 delineated the locus to a 1.1-cm region including 400 genes ; ( c ) analysis of 90 diverse exotic sorghums and their corresponding converted derivatives delimited flravgd1 to a 4.1-mb region including 63 genes . ( e ) the day - neutral haplotype of sb06g012260 contains three deletions in the 5 region , one removing a caat box essential to many eukaryotic promoters . 1995 ) ; ( b ) progeny testing in 30 f3 families that were recombinant in the interval containing flravgd1 delineated the locus to a 1.1-cm region including 400 genes ; ( c ) analysis of 90 diverse exotic sorghums and their corresponding converted derivatives delimited flravgd1 to a 4.1-mb region including 63 genes . ( e ) the day - neutral haplotype of sb06g012260 contains three deletions in the 5 region , one removing a caat box essential to many eukaryotic promoters . to better circumscribe the location of flravgd1 in the 34-mbp target region , and to confirm that the s. propinquum - derived locus also accounts for intraspecific variation within s. bicolor ( the cultigen ) , we utilized recombination accumulated during conversion of diverse short - day tropical sorghums to day - neutral flowering ( stephens et al . 1995 ) , and closely coincides with flravgd1 , strongly supporting the hypothesis that the s. propinquum - derived qtl locus also accounts for intraspecific variation in short - day vs. day - neutral photoperiod response in s. bicolor . 2008 ) phenotyped for photoperiod response index ( pri : see materials and methods ) , we identified and genotyped dna polymorphisms in annotated genes near the conversion peak . the most significant association ( p < 10 ) was found with sb06g012260 ( fig . the other haplotype was most abundant in accessions from south africa , the most temperate part of the natural range of sorghum and where day - neutral flowering is common ( fig . the predominantly south african haplotype was characterized by four deletions , respectively , of 423 nt starting 5901 nt upstream of the sb06g012260 transcription - start site ; 4186 nt starting 4553 nt upstream ; 3 nt starting 219 nt upstream ; and 27 nt in the second intron of sb06g012260 ( fig . flowering of 384 diverse sorghums were compared in the 20072008 post - rainy ( short day ) , and 2008 rainy ( long day ) seasons in peninsular india to determine photoperiod response index ( as described in methods ) . while short - day sorghums experience delayed flowering under long days ( > 12 h ) , long days accelerated flowering by 30 days for many genotypes from south africa , the most temperate part of the natural range . flowering of 384 diverse sorghums were compared in the 20072008 post - rainy ( short day ) , and 2008 rainy ( long day ) seasons in peninsular india to determine photoperiod response index ( as described in methods ) . while short - day sorghums experience delayed flowering under long days ( > 12 h ) , long days accelerated flowering by 30 days for many genotypes from south africa , the most temperate part of the natural range . 2006 ) , tx430 has day - neutral flowering but has an unusual haplotype , with deletion of seven amino acids in the 4th exon of sb06g012260 . among 13 transformation events carrying a 5 kb construct limited to sb06g012260 and its immediate upstream elements , two conferred statistically significant delays averaging 13.1 ( p = 0.03 ) and 24.8 days ( p = 0.09 ) , and one line showed unexpected accelerated flowering ( 14.1 days , p = 0.05 ) . among 10 independent events harboring a 10 kb construct spanning the entire haplotype ( from sb06g012260 through the 4,186 nt element ) , transgenic f2 progeny of only three showed significantly altered flowering , with delays of 4.1 ( p = 0.002 ) , 4.2 ( p = 0.07 ) and 5.2 ( p = 0.008 ) days . sb02g029725 , a family member that is more closely related to hd3a and ft than to sb06g012260 , maps near the likelihood peak of another sorghum flowering qtl , flravgb1 ( lin et al . 2008 ) revealed two abundant haplotypes ( resembling s. propinquum and btx623 , respectively ) , which showed highly significant association with pri ( p = 1.5310 ) . thus , at least two members of the ft gene family are implicated in the modulation of flowering in sorghum . the panel on the right along the tree shows multiple alignments based on the arabidopsis ft protein at amino acid position 85 at exon 2 , positions 128141 ( p - loop domain ) , positions 150152 and position 164 that were shown to have critical regulatory roles . the panel on the right along the tree shows multiple alignments based on the arabidopsis ft protein at amino acid position 85 at exon 2 , positions 128141 ( p - loop domain ) , positions 150152 and position 164 that were shown to have critical regulatory roles . for full alignments across the entire length of these proteins , an sb06g012260 allele found at high frequency in weedy and invasive s. halepense may have contributed to its spread . sorghum halepense ( johnson grass ) , a tetraploid derived naturally from wild s. bicolor and s. propinquum progenitors ( paterson , schertz , et al . its establishment in usa is post - columbian , by introduction as a prospective forage and/or contaminant of sorghum seedlots ( mcwhorter 1971 ) . with inbreeding progenitors of tropical origin and therefore being putatively homozygous for short - day flowering , the original tetraploid s. halepense is expected to have had four copies of the short - day haplotype . among the few old world s. halepense accessions in the us national plant germplasm collection , we confirmed by pcr that pi209217 from south africa and pi271616 from india are homozygous for the short - day haplotype , and confirmed by growouts that these two genotypes do not flower in long days . we genotyped the four loci diagnostic of the short - day haplotype ( 423 nt , 4,186 nt , 3 nt , and intron indels ) in 480 s. halepense plants sampled equally from populations in ga , tx ( 2 ) , ne , and nj described previously ( morrell et al . however , at the two internal loci of the haplotype ( 4,186 and 3 nt indels ) , only 1.1% and 8.0% of plants were homozygous for the short - day - associated alleles ( fig . only 39-bp upstream from the locus of the caat box deletion in day - neutral s. bicolor , 85.9% of s. halepense plants have at least one copy of a 4-nt insertion that disrupts a tc - rich repeat , a cis - acting motif enriched in promoters of photoperiod - responsive genes ( mongkolsiriwatana et al . we have not found this mutation in any other members of the genus ( unpublished data ) , suggesting that it is rare or novel . further , at the 4,186-nt indel , 98.9% of s. halepense also have at least one allele from the day - neutral haplotype ( fig . thus , it appears that an sb06g012260 mutation in naturalized us s. halepense independent of those in s. bicolor has resulted in 4genotype distributions of five us johnson grass populations near the sb06g012260 gene . homozygosity for day - neutral alleles ( green ) is nearly absent from the ga sample ( from the region where johnson grass is thought to have been introduced to usa ) , but exceeds 10% in the two northerly populations ( ne , nj ) where day - neutral flowering would be most advantageous , and is intermediate in the two tx populations . homozygosity for day - neutral alleles ( green ) is nearly absent from the ga sample ( from the region where johnson grass is thought to have been introduced to usa ) , but exceeds 10% in the two northerly populations ( ne , nj ) where day - neutral flowering would be most advantageous , and is intermediate in the two tx populations . based on several lines of evidence , sb06g012260 appears to have evolved as a single - gene duplication ( fig . first , its divergence from its nearest sorghum homolog , sb04g008320 is estimated at 40 ma ( ks = 0.43 , based on a widely - used evolutionary rate for cereal genes ; gaut et al . this pattern of orthology is indicative of an origin of sb06g012260 in panicoids , shortly after divergence from a common ancestor shared with oryzoids ( rice ) and pooids ( brachypodium ) . matching gene pairs several independent lines of evidence including fine mapping , association genetics , mutant complementation , and evolutionary analysis all implicate a single gene , sb06g012260 , as the cause of the flravgd1 qtl that accounted for 85.7% of variation in flowering time of a temperatetropical sorghum population ( lin et al . identification of this gene was complicated by its location in the physically largest single cm in the sorghum genome , containing 5% of sorghum genomic dna and 1.3% ( 400 ) of genes ( paterson et al . 2009 ) , with enrichment of retroelements and other non - genic dna . the wild - type sb06g012260 is the first floral suppressor discovered in the ftl clade of the ft gene family although another clade member is a floral promoter , zcn8 ( meng et al . despite being a floral suppressor , sb06g012260 resembles all four functionally characterized members of the tfl1 clade of the family ( hanzawa et al . the candidate mutation that removed a caat box upstream of the day - neutral sb06g012260 allele would be expected to reduce gene expression , also consistent with the hypothesis that wild - type sb06g012260 is a floral suppressor . there is growing appreciation that floral regulation and other aspects of growth and development are regulated by antagonistic actions of multiple members of ft and other gene families ( lifschitz et al . however , it is somewhat perplexing that only one of the 23 events reached the average 24.6 ( 3.5 ) day delay between the reference genetic stock 100 m ( murphy et al . a detailed study of 19 sorghum pebp genes in roots , leaves ( four stages ) , stems , shoot apices and floral heads by semiquantitative rt - pcr also detected no expression of sb06g012260 in any tissue ( wolabu et al . further , two of the three other sorghum members of the subclade including sb06g012260 , sb04g008320 ( sbft7 ) and sb03g002500 ( sbft13 ) , also show no expression in any tissue , although the third member , sb10g021790 ( sbft9 ) shows moderate expression in leaves ( wolabu et al . finally , the nearest maize homolog of sb06g012260 , zcn21 , shows no discernible expression in a broad sampling of tissues ( danilevskaya et al . in contrast , sb02g029725 ( sbft6 ) , which we implicate above in the flravgb1 sorghum flowering qtl , showed strong expression in leaves across all developmental stages and was also detected in the floral head and florets , but does not induce flowering in arabidopsis ( wolabu et al . further , in the genotype that was the source of the short - day flowering allele that we mapped ( s. propinquum ) , prr37 is non - functional with a 2-nt insertion near the 5 end causing 19 nonsense mutations ( supplementary fig . in addition to ma1 , a more recently discovered flowering locus , ma6 , also maps to chromosome 6 very near the location of prr37 ( brady 2006 ) indeed , one report stated that prr37 corresponded to ma6 ( mullet et al . 2014 ) , albeit needing support from mutant complementation the evolution of sb06g012260 from a single gene duplicate , rather than a dosage - balanced gene from whole - genome duplication , follows a trend exemplified by key members of the c4 photosynthesis pathway ( wang et al . however , single - gene duplicates , often lacking their ancestral regulatory elements and in different chromatin environments from their ancestral gene(s ) , may have greater per - gene potential for the evolution of novelty as reflected by greater divergence of expression patterns than wgd - duplicates ( wang et al . recalcitrance of the ma1 region of chromosome 6 to recombination may have contributed to the evolution of a coadapted gene complex ( mayr 1954 ) . while the strongest association of allelic variation with plant height in the diversity panel was at sb06g012260 itself ( p = 0.007 ) , we also found a marginally significant association at sb06g007330 ( p = 0.023 ) , a putative cation efflux family protein . further study is needed to determine if sb06g007330 is dw2 however an intriguing hypothesis for further testing is that increased height confers a competitive advantage in light interception , and alleles conferring this trait might have become abundant more quickly if co - transmitted with alleles for optimal flowering time in the native tropical environment of sorghum . engineering of genotypes that silence the short - day flowering trait may render obsolete the need to laboriously convert tropical grasses to day - neutral flowering by twelve ( ! ) flowering time was the number of days required for 50% of the plants in a single row to flower ( dfl50% ) . photoperiod response index ( pri ) was defined as the mean difference in dfl50% between the two planting seasons ( i.e. genotyping of the four loci diagnostic of the ma1 haplotype ( 423 nt , 4186 nt , 3 nt , and intron indels ) in s. halepense used 480 plants sampled equally from ga , tx ( 2 ) , ne , and nj populations described previously ( morrell et al .

microarray analysis is exploratory and very high dimensional , and the primary purpose is to generate a list of differentially regulated genes that can provide insight into the biological phenomena under investigation . however , analysis should not stop with a list , it should be the starting point for secondary analyses that aim at deciphering the molecular mechanisms underlying the biological phenotypes analyzed . combining microarray data with prior biological knowledge is a fundamental key to the interpretation of the list of genes . this prior knowledge is stored in various public and private databases and covers several aspects of genes functions and biological information such as regulatory sequence analysis , gene ontology , and pathway information . in this review , we will describe the tools and places where to find prior accurate biological information and how to incorporate them into the analysis of microarray data . the plant genome outreach portal ( http://www.plantgdb.org/pgrop/pgrop.php?app=pgrop ) list many of these resources and other tools and resources such as est resources and blast that are not covered in this review . we also address some theoretical aspects and methodological issues of the algorithms implemented in the tools that have been recently developed for bioinformatic and what needs to be considered when selecting a tool for use . the goal of these class level functional annotation tools is to relate the expression data to other attributes such as cellular localization , biological process , and molecular function for groups of related genes . the most common way to functionally analyze a gene list is to gather information from the literature or from databases covering the whole genome . the recent developments in technologies and instrumentation enabled a rapid accumulation of large amount of in silico data in the area of genomics , transcriptomics , and proteomics as well . the gene ontology ( go ) consortium was created to develop consistent descriptions of gene products in different databases . the go provides researchers with a powerful way to query and analyze this information in a way that is independent of species . go allows for the annotation of genes at different levels of abstraction due to the directed acyclic graph ( dag ) structure of the go . in this particular hierarchical structure , each term can have one or more child terms as well as one or more parent terms . for instance , the same gene list is annotated with a more general go term such as cell communication at a higher level of abstraction , whereas the lowest level provides a more specific ontology term such as intracellular signaling cascade . in recent years , various tools have been developed to assess the statistical significance of association of a list of genes with go annotations terms , and new ones are being regularly released . there has been extensive discussion of the most appropriate methods for the class level analysis of microarray data [ 46 ] . there are two key points to consider : ( 1 ) whether the method uses gene sampling or subject sampling and ( 2 ) whether the method uses competitive or self - contained procedures . the subject sampling methods are preferred and the competitive versus self - contained debate continues . gene sampling methods base their calculation of the p - value for the geneset on a distribution in which the gene is the unit of sampling , while the subject sampling methods take the subject as the sampling unit . the latter is more valid for the unit of randomization is the subjects not the genes [ 79 ] . competitive tests , which encompass most of the existing tools , test whether a gene class , defined by a specific go term or pathway or similar , is overrepresented in the list of genes differentially expressed compared to a reference set of genes . a self - contained test compares the gene set toa fixed standard that does not depend on the measurements of genes outside the gene set . [ 10 , 11 ] , mansmann and meister , and tomfohr et al . applied the self - contained methods . another important aspect of ontological analysis regardless of the tool or statistical method is the choice of the reference gene list against which the list of differentially regulated genes is compared . inappropriate choice of reference genes may lead to false functional characterization of the differentiated gene list . khatri and drghici pointed out that only the genes represented on the array , although quite incomplete , should be used as reference list instead of the whole genome as it is a common practice . in addition correct , up to date , and complete annotation of genes with go terms is critical . the competitive and gene sample - based procedures tend to have better and more complete databases . go allows for the annotation of genes at different levels of abstraction due to the directed acyclic graph ( dag ) structure of the go . in this particular hierarchical structure , each term can have one or more child terms as well as one or more parent terms . for instance , the same gene list is annotated with a more general go term such as cell communication at a higher level of abstraction , whereas the lowest level provides a more specific ontology term such as intracellular signaling cascade . it is important to integrate the hierarchical structure of the go in the analysis since various levels of abstraction usually give different p - values . the large number ( hundreds or thousands ) of tests performed during ontological analysis may lead to spurious associations just by chance , thus correction for multiple testing is a necessary step to take . we present here a nonexhaustive list of tools available that can be used to perform functional annotation of gene list and attempt to compare their functionalities ( table 1 ) . all tools accept input data from arabidopsis thaliana , the most used model organism in plant studies , as well as many animal organism models . onto - express ( oe ) : http://vortex.cs.wayne.edu/projects.htm#onto-express onto - express is a software application used to translate a list of differentially regulated genes into a functional profile [ 12 , 13 ] . onto - express constructs a profile for each of the go categories : cellular component , biological process , molecular function , and chromosome location as well . onto - express implements hypergeometric , binomial , and fisher 's exact tests . the results are displayed in a graphical form that allows the user to collapse or expand go node and visualize the p - values associated with each level of go abstraction . onto - express performs bonferroni , holm , sidak , and fdr corrections to adjust for multiple testing . users have an option of either providing their own reference gene list or selecting a microarray platform as reference gene list . an extensive list of up to date annotations is provided for many arrays . onto - express is a software application used to translate a list of differentially regulated genes into a functional profile [ 12 , 13 ] . onto - express constructs a profile for each of the go categories : cellular component , biological process , molecular function , and chromosome location as well . onto - express implements hypergeometric , binomial , and fisher 's exact tests . the results are displayed in a graphical form that allows the user to collapse or expand go node and visualize the p - values associated with each level of go abstraction . onto - express performs bonferroni , holm , sidak , and fdr corrections to adjust for multiple testing . users have an option of either providing their own reference gene list or selecting a microarray platform as reference gene list . an extensive list of up to date annotations is provided for many arrays . funcassociate : http://llama.med.harvard.edu/cgi/func/funcassociate funcassociate is a web - based tool that characterizes large sets of genes with go terms using the fisher 's exact test . among all annotation tools funcassociate stands out in that it implements a monte carlo simulation to correct for multiple testing . although funcassociate supports 10 organisms , it does not provide visualization or level information for the go annotation . funcassociate is a web - based tool that characterizes large sets of genes with go terms using the fisher 's exact test . among all annotation tools funcassociate stands out in that it implements a monte carlo simulation to correct for multiple testing . although funcassociate supports 10 organisms , it does not provide visualization or level information for the go annotation . safe ( significance analysis of function and expression)safe is a bioconductor / r algorithm that first computes gene - specific statistics in order to test for association between gene expression and the phenotype of interest . gene - specific statistics are used to estimate global statistics that detects shifts in the local statistics within a gene category . the significance of the global statistics is assessed by repeatedly permuting the response values . safe implements a rank - based global statistics that enables a better use of marginally significant genes than those based on a p - value cutoff . safe is a bioconductor / r algorithm that first computes gene - specific statistics in order to test for association between gene expression and the phenotype of interest . gene - specific statistics are used to estimate global statistics that detects shifts in the local statistics within a gene category . the significance of the global statistics is assessed by repeatedly permuting the response values . safe implements a rank - based global statistics that enables a better use of marginally significant genes than those based on a p - value cutoff . global testglobal test is a bioconductor / r package that tests the association of expression pattern of a group of genes with selected phenotypes of interest using self - contained methods . the method is based on a penalized regression model that shrinks regression coefficient between gene expression and phenotype toward a common mean . the algorithm allows the users to test biological hypothesis or to search go databases for potential pathways . global test is a bioconductor / r package that tests the association of expression pattern of a group of genes with selected phenotypes of interest using self - contained methods . the method is based on a penalized regression model that shrinks regression coefficient between gene expression and phenotype toward a common mean . the algorithm allows the users to test biological hypothesis or to search go databases for potential pathways . fatigo+ ( fast assignment and transference of information):http://babelomics2.bioinfo.cipf.es / fatigoplus / cgi - bin / fatigoplus.cgi fatigo+ tests for significant difference in distribution of go terms between any two groups of genes ( ideally a group of interest and a reference set of genes ) using a fisher 's exact test for 2 by 2 contingency table . fatigo+ implements an inclusive analysis in which at a given level in the go dag hierarchy , genes annotated with child go terms take the annotation from the parent . fatigo+ tests for significant difference in distribution of go terms between any two groups of genes ( ideally a group of interest and a reference set of genes ) using a fisher 's exact test for 2 by 2 contingency table . fatigo+ implements an inclusive analysis in which at a given level in the go dag hierarchy , genes annotated with child go terms take the annotation from the parent . gotoolbox : http://burgundy.cmmt.ubc.ca/gotoolbox/ gotoolbox identifies over - or under - represented go terms in a gene set using either hypergeometric distribution - based tests or binomial test . the user has the option of choosing between the total set of genes in the genome as reference or provides his own list of reference genes . its also allows the user to select a specific level of go abstraction prior to the analysis . gotoolbox identifies over - or under - represented go terms in a gene set using either hypergeometric distribution - based tests or binomial test . the user has the option of choosing between the total set of genes in the genome as reference or provides his own list of reference genes . its also allows the user to select a specific level of go abstraction prior to the analysis . clench2 ( cluster enrichment ) : http://www.stanford.edu/~nigam/cgi-bin/dokuwiki/doku.php?id=clench clench is used to calculate cluster enrichment for go terms . the program accepts two lists of genes : a reference set of genes and the list of changed genes . clench performs hypergeometric , binomial and tests to estimate go terms enrichment . the program allows the user to choose an fdr cutoff in order to account for multiple testing . the program accepts two lists of genes : a reference set of genes and the list of changed genes . clench performs hypergeometric , binomial and tests to estimate go terms enrichment . the program allows the user to choose an fdr cutoff in order to account for multiple testing . bingo ( biological network gene ontology tool ) : http://www.psb.ugent.be/cbd/papers/bingo/ bingo is a java - based tool to determine which gene ontology ( go ) categories are statistically overrepresented in a set of genes or a subgraph of a biological network . bingo is implemented as a plugin for cytoscape , which is an open source bioinformatics software platform for visualizing and integrating molecular interaction networks . the program implements hypergeometric test and binomial test and performs fdr to control multiple testing . one limitation is that the user can only choose between the whole genome or the network under study as reference set of gene for the enrichment test . bingo is a java - based tool to determine which gene ontology ( go ) categories are statistically overrepresented in a set of genes or a subgraph of a biological network . bingo is implemented as a plugin for cytoscape , which is an open source bioinformatics software platform for visualizing and integrating molecular interaction networks . the program implements hypergeometric test and binomial test and performs fdr to control multiple testing . one limitation is that the user can only choose between the whole genome or the network under study as reference set of gene for the enrichment test . gosurfer : http://bioinformatics.bioen.uiuc.edu/gosurfer/ gosurfer is used to visualize and compare gene sets by mapping them onto gene ontology ( go ) information in the form of a hierarchical tree . users can manipulate the tree output by various means , like setting heuristic thresholds or using statistical tests . gosurfer is used to visualize and compare gene sets by mapping them onto gene ontology ( go ) information in the form of a hierarchical tree . users can manipulate the tree output by various means , like setting heuristic thresholds or using statistical tests . in most microarray studies , gene expressions are measured on a small number of arrays or samples ; however , large collections of arrays are available in microarray database that contain transcript levels data from thousands of genes across a wide variety of experiments and samples . these tools provide scientists with the opportunity to analyze the transcriptome by pooling gene expression information from multiple data sets . this meta - analytic approach allows biologists to test the consistency of gene expression patterns across different studies . most importantly , the analysis of concerted changes in transcript levels between genes can lead to biological function discovery . it has been demonstrated that genes which protein products cooperate in the same pathway or are in a multimeric protein complex display similar expression patterns across a variety of experimental conditions [ 22 , 23 ] . using the guilt - by - association principle , investigators can functionally characterize a previously uncharacterized gene when it displays expression pattern similar to that of known genes . the coexpression relationship between two genes is usually assessed by computing the pearson 's correlation coefficient or other distance measures . then the genes which expression is significantly correlated with bait - genes expression are analyzed to identify new potential actors in a given pathway or biological process . however , coexpression between two genes does not necessarily translate into similar function between both genes . some authors suggest that to be sustainable the gene coexpressions observed in one species should be confirmed in other evolutionary close species . tools have been developed that make use of the large sample size available in these databases to identify more reliable concerted changes in transcripts levels as well as to examine the coordinated change of gene expression levels . cress - express : http://www.cressexpress.org/ cress - express estimates the coexpression between a user - provided list of genes and all genes from affymetrix ath1 platform using up to 1779 arrays . plc identifies and ranks genes based on their coexpression with a group of genes . cress - express also delivers results in bulk formats suitable for downstream data mining via web services . the tool has the data processed with a variety of image processing methods : rma , mas5 , and gcrma . cress - express estimates the coexpression between a user - provided list of genes and all genes from affymetrix ath1 platform using up to 1779 arrays . plc identifies and ranks genes based on their coexpression with a group of genes . the tool has the data processed with a variety of image processing methods : rma , mas5 , and gcrma . investigators can select which of over 100 experiments to include in coexpression analysis . atted - ii ( arabidopsis thaliana transfactor and cis - element prediction database ) : http://www.atted.bio.titech.ac.jp/ atted - ii provides coregulated gene relationships in arabidopsis thaliana to estimate gene functions . in addition , it can predict overrepresented cis - elements based upon all possible heptamers . there is also several visualization tools and databases of annotations attached to the coexpression . atted - ii provides coregulated gene relationships in arabidopsis thaliana to estimate gene functions . in addition , it can predict overrepresented cis - elements based upon all possible heptamers . there is also several visualization tools and databases of annotations attached to the coexpression . genevestigator : http://www.genevestigator.ethz.ch/ genevestigator is a web - based discovery tool to study the expression and regulation of genes , pathways , and networks [ 26 , 27 ] . among other applications , the software allows the user to look at individual gene expression or group of genes coexpression in many different tissues , at multiple developmental stages , or in response to large sets of stimuli , diseases , drug treatments , or mutations . in addition genevestigator is a web - based discovery tool to study the expression and regulation of genes , pathways , and networks [ 26 , 27 ] . among other applications , the software allows the user to look at individual gene expression or group of genes coexpression in many different tissues , at multiple developmental stages , or in response to large sets of stimuli , diseases , drug treatments , or mutations . in addition bar ( the botany array resource ) expression angler : http://www.bar.utoronto.ca/ the expression anger allows the user to identify genes with similar expression profile with the user provided gene across multiple samples . the user can specify the pearson correlation coefficient threshold and the array database to use for the coexpression analysis . the expression anger allows the user to identify genes with similar expression profile with the user provided gene across multiple samples . the user can specify the pearson correlation coefficient threshold and the array database to use for the coexpression analysis . athcor@csb.db ( a. thaliana coresponse database ) : http://csbdb.mpimp-golm.mpg.de/csbdb/dbcor/ath.html athcor is a coexpression tool that allows the use of functional ontology filter to identify genes coexpressed with a gene of interest filtering the search by functional ontologies . athcor is a coexpression tool that allows the use of functional ontology filter to identify genes coexpressed with a gene of interest filtering the search by functional ontologies . plexdb ( plant expression database ) : http://www.plexdb.org/ plexdb serves as a comprehensive public repository for gene expression for plants and plant pathogens . the integrated tools of plexdb allow plant investigators to perform comparative and functional genomics analyses using large - scale expression data sets . plexdb serves as a comprehensive public repository for gene expression for plants and plant pathogens . the integrated tools of plexdb allow plant investigators to perform comparative and functional genomics analyses using large - scale expression data sets . act ( arabidopsis coexpression data mining tool ) : http://www.arabidopsis.leeds.ac.uk/act/index.php act estimates the coexpression of 21 891 arabidopsis genes based on affymetrix ath1 platform using a simple correlation test . the web server includes a database that stores precalculated correlation results from over 300 arrays of the nasc / garnet dataset . tool allows the user to identify groups of consistently coexpressed genes within a user - defined list of genes . the identification of genes with a known function within a cluster allows inference to be made about the other genes . users can also visualize the coexpression scatter plots of all genes against a group of genes . act estimates the coexpression of 21 891 arabidopsis genes based on affymetrix ath1 platform using a simple correlation test . the web server includes a database that stores precalculated correlation results from over 300 arrays of the nasc / garnet dataset . tool allows the user to identify groups of consistently coexpressed genes within a user - defined list of genes . the identification of genes with a known function within a cluster allows inference to be made about the other genes . users can also visualize the coexpression scatter plots of all genes against a group of genes . genes and their protein products are related to each other through a complex network of interactions . in higher metazoa , on average each gene is estimated to interact with five other genes , and to be involved in ten different biological functions during development . on a molecular level , the function of a gene depends on its cellular context , and the activity of a cell is determined by which genes are being expressed and which are not and how they interact with each other . in such high interconnectedness , analyzing a network as a whole is essential to understanding the complex molecular processes underlying biological systems . the traditional reductionist approach that investigates biological phenomena by analyzing one gene at a time can not address this complexity . by using systems biology approach and network theories , investigators can analyze the behavior and relationships of all of the elements in a particular biological system to arrive at a more complete description of how the system functions . high - throughput gene expression profiling offers the opportunity to analyze gene interrelationships at the genome scale . clustering analysis on microarray expression data only extracts lists of coregulated genes out of a large - scale expression data . however , the task of modeling dynamic systems with large number of variables can be computationally challenging . in gene regulatory networks , genes , mrna , or proteins correspond to the network nodes and the links among the nodes stand for the regulatory interactions ( activations or inhibitions ) . in this section , we will describe some of the methods and tools used to reconstruct , visualize , and explore gene networks . one method is based on bayesian inference theory which seeks to find the most probable network given the observed expression patterns of the genes to be included in the network . the regulatory interactions among genes and their directions are derived from expression data . several network structures are proposed and scored on the basis of how well they explain the data as it has been successfully implemented in yeast . the second approach is based on mutual information as a measure of correlation between gene expression patterns . a regulatory interaction between two genes is established if the mutual information on their expression patterns is significantly larger than a p - threshold value calculated from the mutual information between random permutations of the same patterns . unlike the bayesian theory , which tries out whole networks and selects the one that best explains the observed data , the mutual information method constructs a network by selecting or rejecting regulatory interactions between pairs of genes . we present below selected tools that implement either of the aforementioned approaches to reverse - engineer gene regulatory networks . bnarray ( bayesian network array ) : http://www.cls.zju.edu.cn/binfo/bnarray/ bnarray is a tool developed in r for inferring gene regulatory networks from dna microarray data by using a bayesian network . it allows the reconstruction of significant submodules within regulatory networks using an extended subnetwork mining algorithm . bnarray is a tool developed in r for inferring gene regulatory networks from dna microarray data by using a bayesian network . it allows the reconstruction of significant submodules within regulatory networks using an extended subnetwork mining algorithm . banjo implements bayesian and dynamic bayesian networks to infer networks from both steady - state and time - series expression data . component of banjo uses heuristic approaches to search the network space for potential network structures . each network structure is explored and an overall network 's score is computed based on the parameters of the conditional probability density distribution . the network with the best overall score is accepted by a decider component of the software . the network retained is processed by banjo to compute influence scores on the edges indicating the direction of the regulation between genes . gna is a freely available software used for modeling and simulating genetic regulatory networks from gene expression data and regulatory interaction information . in gna , the dynamics of a regulatory network is modeled by a class of piecewise - linear differential equations . thus the software uses qualitative constraints in the form of algebraic inequalities instead of numerical values . pathwayassist allows the users to create their own pathways by combining the user - submitted microarray expression data with knowledge from biological databases such as bind , kegg , dip . the software provides a graphical user interface and publication quality figures . as a result of the explosion and advances in experimental technologies that allow genome - wide characterization of molecular states and interactions among thousands of genes , researchers are often faced with the need for tools for the visualization , display , and evaluation of large structure data . the main aim of these tools is to provide a summarized yet understandable view of large amount of data while integrating additional information regarding the biological processes and functions . several network visualization tools have been developed of which we will describe some of the most popular . cytoscape is a general - purpose , open - source software environment for the large scale integration of molecular interaction network data . dynamic states on molecules and molecular interactions are handled as attributes on nodes and edges , whereas static hierarchical data , such as protein - functional ontologies , are supported by use of annotations . the cytoscape core handles basic features such as network layout and mapping of data attributes to visual display properties . cytoscape is a general - purpose , open - source software environment for the large scale integration of molecular interaction network data . dynamic states on molecules and molecular interactions are handled as attributes on nodes and edges , whereas static hierarchical data , such as protein - functional ontologies , are supported by use of annotations . the cytoscape core handles basic features such as network layout and mapping of data attributes to visual display properties . celldesigner is a structured diagram editor for drawing gene - regulatory and biochemical networks based on standardized technologies and with wide transportability to other systems biology markup language ( sbml ) compliant applications and systems biology workbench ( sbw ) . the user can browse and modify existing sbml models with references to existing databases , simulate and view the dynamics through an intuitive graphical interface . vanted is a freely available tool for network visualization that allows users to map their own experimental data on networks drawn in the tool , downloaded from kegg pathway database , or imported using standard imported formats . statistical methods implemented in vanted allow the comparison between treatments or groups of genes , the generation of correlation matrix , or the clustering of genes based on expression pattern . osprey allows user defined colors to indicate gene function , experimental systems , and data sources . genes are colored by their biological process as defined by standardized gene ontology ( go ) annotations . as a network complexity increases , osprey simplifies network layouts through user - implemented node relaxation , which disperses nodes and edges according to anyone of a number of layout options . visant is a freely available open - source tool for integrating biomolecular interaction data into a cohesive , graphical interface [ 4547 ] . visant offers an online interface for a large range of published datasets on biomolecular interactions , as well as databases for organized annotation , including genbank , kegg , and swissprot . one of the main focuses in the postgenomic era is to study the network of molecular interactions in order to reveal the complex roles played by genes , gene products , and the cellular environments in different biological processes . the nodes ( genes ) of a network can be associated with additional information regarding the gene products , gene positions in the chromosome , or the gene functional annotation . the edges in the network symbolize specific interaction that can be associated with a transcription factor - promoter bond for instance . this information can be automatically retrieved in a number of specialized and publicly accessible databases containing data about the nodes and the interactions . network exploration tools enable the user to perform analysis on single genes , gene families , patterns of molecular interactions , as well as on the global structure of the network . the user usually has an option to construct interaction networks either by curation or by computation and to associate microarray expression data with known metabolic pathways . here metnet ( metabolic networking database ) : http://www.metnetdb.org/ metnet is a publicly available software for analysis of genome - wide mrna , protein , and metabolite profiling data . the software is designed to enable the biologist to visualize , statistically analyze , and model a metabolic and regulatory network map of arabidopsis , combined with gene expression profiling data . metnet provides a framework for the formulation of testable hypotheses regarding the function of specific genes . the tools within metnet allow the user to map metabolic and regulatory networks ; to integrate and visualize data together ; to explore and model the metabolic and regulatory flow in the network . metnet is a publicly available software for analysis of genome - wide mrna , protein , and metabolite profiling data . the software is designed to enable the biologist to visualize , statistically analyze , and model a metabolic and regulatory network map of arabidopsis , combined with gene expression profiling data . metnet provides a framework for the formulation of testable hypotheses regarding the function of specific genes . the tools within metnet allow the user to map metabolic and regulatory networks ; to integrate and visualize data together ; to explore and model the metabolic and regulatory flow in the network . biologicalnetworks is a bioinformatics and systems biology software platform for visualizing molecular interaction networks , sequence and 3d structure information . the tool performs easy retrieval , construction , and visualization of complex biological networks , including genome - scale integrated networks of protein - protein , protein - dna , and genetic interactions . biologicalnetworks also allow the analysis and the mapping of expression profiles of genes or proteins onto regulatory , metabolic , and cellular networks . the main component of pavesy is a relational sql database system that stores biological objects , such as metabolites , proteins , genes , and their interrelationships . the user can annotate the biological objects with specific attributes that are integrated in the database . the specific roles of the objects are derived from these attributes in the context of user - defined interactions . pavesy can display an individualized view on the database content that facilitates user customization . it combines automatically produced or user - created graphical representations of networks ( e.g. , gene modules or pathways ) for the exploratory analysis of a large compendium of gene expression profiles . effects on gene expression can be projected onto these networks for the following ontologies : anatomy , development , stimulus , and mutation , in form of comparison sets . one of the downstream applications of the reconstruction of a gene regulatory networks or the identification of clusters of functionally related genes is to associate the genes and their interconnections with known metabolic pathways . biochemists summarized the sequence of enzyme - catalyzed metabolic reactions between biomolecules as a network of interactions that results from the conversion of one organic substance ( substrate ) to another ( product ) . depending on the type of interactions analyzed , several types of biochemical networks are identified . these biochemical networks represent the potential mechanistic associations between genes and gene products that are involved in specific biological processes . because of the curse of dimensionality that sometimes hampers the whole network analysis , investigators often focus on pathway rather than many specialized databases are available that store and summarize large amount of information on metabolic reactions . increasingly , identifying and searching . this task can be tedious due to the large number of databases available . for a more comprehensive list of biological pathways resources on the web , kegg ( kyoto encyclopedia of genes and genomes ) : http://www.genome.jp/kegg kegg aims to link lower - level information ( genes , proteins , enzymes , reaction molecules , etc . ) with higher - level information ( interactions , enzymatic reactions , pathways , etc . ) . kegg aims to link lower - level information ( genes , proteins , enzymes , reaction molecules , etc . ) with higher - level information ( interactions , enzymatic reactions , pathways , etc . ) . its goal is to serve as a metabolic encyclopedia , containing a collection of nonredundant pathways , enzymatic reactions , enzymes , chemical compounds , genes and review - level comments . enzyme information includes substrate specificity , kinetic properties , activators , inhibitors , cofactor requirements and links to sequence and structure databases . araccyc ( http://www.arabidopsis.org/biocyc/index.jsp ) uses metacyc as reference database for visualization of arabidopsis thaliana biochemical pathways . biocarta catalogs pathways , regulation and interaction information for over 120,000 genes covering most model organisms . data in biocarta are constantly updated , and new pathways are suggested by the life science research community . the genenet system is designed for formalized description and automated visualization of gene networks . the genenet system includes database on gene network components , java program for the data visualization . genenet allows the users to select entities that are involved in the functioning of a particular gene network , to describe the regulatory relations for a particular gene network , and to search for potential transcription factors . most organisms encode a large number of dna - binding proteins that act as transcription factors . in arabidopsis , transcription factors bind to short conserved dna motifs ( cis - acting regulatory elements care ) located at the 5end of the gene ( in a region called promoter ) to initiate mrna transcription . thus dna - binding proteins play a key role in all aspects of genetic activity within an organism . they participate in promoting or repressing the transcription of specific genes . elucidating the mechanisms that underlie the expression of genomes is one of the major challenges in bioinformatics . an interesting hypothesis one might formulate after a successful microarray study is that the genes that are coexpressed may also be coregulated at the transcriptional level . one way to test this hypothesis is to identify overrepresented oligonucleotides sequences as potential binding sites for transcriptions factors in promoter regions of genes clustered in the same group . the statistical test for overrepresentation of regulatory motifs in intergenic regions is the general principle implemented in most algorithms for regulatory motif detection . cares can also be predicted through phylogenetic footprinting that is based on sequence similarity between orthologous promoters . some other approaches have been proposed that integrates comparative , structural , and functional genomics to identify conserved motifs in coregulated genes . following is a list of transcription factors database and tools ( table 3 ) . plant promoter database ( plantprom db ) : http://mendel.cs.rhul.ac.uk or http://www.softberry.com/ plantprom is a plant promoter database . the database represents a collection of annotated , nonredundant proximal promoter sequences for rna polymerase ii with experimentally determined transcription start site from various plant species . the database represents a collection of annotated , nonredundant proximal promoter sequences for rna polymerase ii with experimentally determined transcription start site from various plant species . the motif analysis tool of the tair compares the frequency of 6-mer motif in promoter regions of query set of genes with the frequency of the 6-mer motif in the whole a. thaliana genome . the user can specify the size of the genes 5upstream region to 500 bp or 1 kb . the database contains data on transcription factors , their target genes and their experimental - proven binding sites in genes . tools within transfac allow the users to automatically visualize gene - regulatory networks based on interlinked factor and gene entries in the database . athamap : http://www.athamap.de/index.php athamap is a database that organizes a genome - wide map of potential transcription factor binding sites in arabidopsis thaliana . athamap allows the user to test for the overrepresentation of transcription factors in a set of query genes . a colocalization tool performs combinatorial analysis to identify synchronized binding of pairs of transcription factors . athamap is a database that organizes a genome - wide map of potential transcription factor binding sites in arabidopsis thaliana . athamap allows the user to test for the overrepresentation of transcription factors in a set of query genes . a colocalization tool performs combinatorial analysis to identify synchronized binding of pairs of transcription factors . plantcare ( plant cis - acting regulatory elements ) : http://bioinformatics.psb.ugent.be/webtools/plantcare/html/ plantcare is a database of plant cis - acting regulatory elements and a portal to tools for in silico analysis of promoter sequences . the database can be queried on names of tf binding sites , function , species , cell type , genes , and reference literatures . the program returns a list of entries with links to other information within the database or beyond through accession to transfac , embl , genbank , or medline . plantcare is a database of plant cis - acting regulatory elements and a portal to tools for in silico analysis of promoter sequences . the database can be queried on names of tf binding sites , function , species , cell type , genes , and reference literatures . the program returns a list of entries with links to other information within the database or beyond through accession to transfac , embl , genbank , or medline . place ( plant cis - acting regulatory dna elements ) : http://www.dna.affrc.go.jp/place/ place is a database of motifs found in plant cis - acting regulatory dna elements , all from previously published reports . in addition to the motifs originally reported their variations in other genes or in other plant species reported later are also compiled . the place database also contains a brief description of each motif and relevant literature with pubmed i d numbers . place is a database of motifs found in plant cis - acting regulatory dna elements , all from previously published reports . in addition to the motifs originally reported their variations in other genes or in other plant species reported later are also compiled . the place database also contains a brief description of each motif and relevant literature with pubmed i d numbers . athena : http://www.bioinformatics2.wsu.edu/cgi-bin/athena/cgi/home.pl athena is a database which contains over 30 000 predicted arabidopsis promoters sequences and consensus sequences for 105 previously characterized tf binding sites . athena enables the user to visualize and rapidly inspect key regulatory elements in multiple promoters . the software includes tools for testing the overrepresentation of tf sites among subset of promoters . a data - mining tool allows the selection of promoter sequences containing specific combination of tf binding sites . athena is a database which contains over 30 000 predicted arabidopsis promoters sequences and consensus sequences for 105 previously characterized tf binding sites . athena enables the user to visualize and rapidly inspect key regulatory elements in multiple promoters . the software includes tools for testing the overrepresentation of tf sites among subset of promoters . a data - mining tool allows the selection of promoter sequences containing specific combination of tf binding sites . agris ( arabidopsis gene regulatory information server ) : http://arabidopsis.med.ohio-state.edu/ agris is an information resource for retrieving arabidopsis promoter sequences , transcription factors and their target genes . users can query the database with a gene name , gene symbol to retrieve its promoter along with other genes regulated by the same transcription factor . agris is an information resource for retrieving arabidopsis promoter sequences , transcription factors and their target genes . users can query the database with a gene name , gene symbol to retrieve its promoter along with other genes regulated by the same transcription factor . various innovative and advanced technologies have allowed scientists to rapidly generate genome - scale or omics . however , it is only by integrating genomics , transcriptomics proteomics , metabolomics , and other recent omics types of data such as interactomics , localizomics , lipidomics , and phenomics that biologists can gain access to a more complete picture of living organisms and unexplored areas of biology . this challenging task requires a systems level approach to perform systematic data mining , cross - knowledge validation , and cross - species interpolation . some investigators attempted the integration of genomic data and transcriptomic data , and the integration of protein - protein interation data and transcriptomic data to analyze the dynamics of biological networks in yeast . the approach commonly used comprises three steps : ( 1 ) identification of the network that describes all interactions between cellular components from integrating various genome scale data ; ( 2 ) decomposing the network into its constituent parts or network modules ; ( 3 ) building a mathematical model that simulates biological systems for the purpose of simulation or prediction . we describe below proteomics and metabolomics , and the potential of their integration with transcritomic data . gene mrna expression profiling on a global scale in response to specific conditions is not sufficient to render the complexities and dynamics of systems biology . furthermore , mrna levels are not always well correlated with the levels of the corresponding protein and one gene can produce several protein species . indeed , proteins undergo a series of posttranslational molecular modifications such as glycosylation , phosphorylation , cleavage or complex formation may also occur that overall influence their function . proteomics is the systematic large - scale study of proteins of an organism or a specific type of tissue , particularly their structure , function , and spatiotemporal distribution . thus proteomics is an essential component of any functional genomics study aiming at understanding biological processes . the integration of transcriptome and proteome data has not always resulted in consistent results . the methods and techniques used to measure the transcript level and the protein level may affect the results concordance . nonetheless , the interpretation of the data in terms of biological pathways or functional groups gives better correlation of transcriptome with proteome in yeast . as the plant model organism of choice , arabidopsis proteome database contains more data compared to other species . protein amino acid sequence databases and repositories for two - dimensional polyacrylamide gel electrophoresis as reference maps of proteomes are becoming popular as tools for analyzing and comparing the plant proteome . phytoprot ( http://urgi.versailles.inra.fr/phytoprot ) is a database of clusters of all the plants full - length protein sequences retrieved from swissprot / trembl . proteins are grouped into clusters based on their peptide sequence similarity in order to track erroneous annotations made at the genome level . the database can be searched for any protein or group of proteins using protein i d or words appearing in protein description . metabolomics is the study of all low molecular weight chemicals in a plant as the end products of the cellular processes . metabolic profiling provides an instantaneous snapshot of the chemistry of a sample and defines the biochemical phenotype of a cell or a tissue . similar to transcript level and protein level , the level of metabolites in an organism or a tissue is influenced by the biological context . thus measure of mrna gene expression and protein content of a sample do not tell the whole story of biological phenomena unfolding in that sample . although plant metabolomics is still in its infancy , recent advances in mass spectrometry have enabled the accumulation of metabolites data on a large scale for some species . metabolomics provide scientist with the ability ( 1 ) to characterize genotypes , ecotypes , or phenotypes with metabolites levels ; ( 2 ) to identify sites within a genetic network where metabolites levels are regulated ; ( 3 ) to analyze genes functions at the light of metabolites levels . currently , one of the most pressing needs in the fields of metabolomics for bioinformatics application is the creation of specific databases and biochemical ontologies . such tools would help clearly describe the function , localization , and interaction of metabolites . however , databases imbedded in kegg and aracyc can be useful at least in part for the purpose of metabolites referencing . the deluge of large - scale biological data in the recent years has made the development of computational tools critical to biological investigation . a great variety of tools have been developed for the specific task of drawing biological meaning from microarray data . most of the tools available exploit prior biological knowledge accumulated in numerous publicly available databases in an attempt to provide a comprehensive view of biological phenomena . these tools differ in many respects and the guidance provided in this review will help biologists with little knowledge in statistics understand some of the key concepts . the integration of transcriptomics data with all other omics data is a challenging task that can be addressed by a systems - level approach .

while it is possible to interpret microarray experiments a single gene at a time , most studies generate long lists of differentially expressed genes whose interpretation requires the integration of prior biological knowledge . this prior knowledge is stored in various public and private databases and covers several aspects of gene function and biological information . in this review , we will describe the tools and places where to find prior accurate biological information and how to process and incorporate them to interpret microarray data analyses . here , we highlight selected tools and resources for gene class level ontology analysis ( section 2 ) , gene coexpression analysis ( section 3 ) , gene network analysis ( section 4 ) , biological pathway analysis ( section 5 ) , analysis of transcriptional regulation ( section 6 ) , and omics data integration ( section 7 ) . the overall goal of this review is to provide researchers with tools and information to facilitate the interpretation of microarray data .

1. INTRODUCTION 2. CLASS LEVEL FUNCTIONAL ANNOTATION TOOLS 3. GENE COEXPRESSION ANALYSIS TOOLS 4. GENE NETWORK ANALYSIS 5. BIOLOGICAL PATHWAY RESOURCES 6. TRANSCRIPTION REGULATION ANALYSIS TOOLS 7. OMICS DATA INTEGRATION TOOLS 8. CONCLUSION

microarray analysis is exploratory and very high dimensional , and the primary purpose is to generate a list of differentially regulated genes that can provide insight into the biological phenomena under investigation . however , analysis should not stop with a list , it should be the starting point for secondary analyses that aim at deciphering the molecular mechanisms underlying the biological phenotypes analyzed . combining microarray data with prior biological knowledge is a fundamental key to the interpretation of the list of genes . this prior knowledge is stored in various public and private databases and covers several aspects of genes functions and biological information such as regulatory sequence analysis , gene ontology , and pathway information . in this review , we will describe the tools and places where to find prior accurate biological information and how to incorporate them into the analysis of microarray data . the plant genome outreach portal ( http://www.plantgdb.org/pgrop/pgrop.php?app=pgrop ) list many of these resources and other tools and resources such as est resources and blast that are not covered in this review . we also address some theoretical aspects and methodological issues of the algorithms implemented in the tools that have been recently developed for bioinformatic and what needs to be considered when selecting a tool for use . the goal of these class level functional annotation tools is to relate the expression data to other attributes such as cellular localization , biological process , and molecular function for groups of related genes . the most common way to functionally analyze a gene list is to gather information from the literature or from databases covering the whole genome . the recent developments in technologies and instrumentation enabled a rapid accumulation of large amount of in silico data in the area of genomics , transcriptomics , and proteomics as well . the gene ontology ( go ) consortium was created to develop consistent descriptions of gene products in different databases . the go provides researchers with a powerful way to query and analyze this information in a way that is independent of species . in this particular hierarchical structure , each term can have one or more child terms as well as one or more parent terms . for instance , the same gene list is annotated with a more general go term such as cell communication at a higher level of abstraction , whereas the lowest level provides a more specific ontology term such as intracellular signaling cascade . in recent years , various tools have been developed to assess the statistical significance of association of a list of genes with go annotations terms , and new ones are being regularly released . there has been extensive discussion of the most appropriate methods for the class level analysis of microarray data [ 46 ] . there are two key points to consider : ( 1 ) whether the method uses gene sampling or subject sampling and ( 2 ) whether the method uses competitive or self - contained procedures . competitive tests , which encompass most of the existing tools , test whether a gene class , defined by a specific go term or pathway or similar , is overrepresented in the list of genes differentially expressed compared to a reference set of genes . [ 10 , 11 ] , mansmann and meister , and tomfohr et al . another important aspect of ontological analysis regardless of the tool or statistical method is the choice of the reference gene list against which the list of differentially regulated genes is compared . khatri and drghici pointed out that only the genes represented on the array , although quite incomplete , should be used as reference list instead of the whole genome as it is a common practice . in addition correct , up to date , and complete annotation of genes with go terms is critical . in this particular hierarchical structure , each term can have one or more child terms as well as one or more parent terms . for instance , the same gene list is annotated with a more general go term such as cell communication at a higher level of abstraction , whereas the lowest level provides a more specific ontology term such as intracellular signaling cascade . it is important to integrate the hierarchical structure of the go in the analysis since various levels of abstraction usually give different p - values . we present here a nonexhaustive list of tools available that can be used to perform functional annotation of gene list and attempt to compare their functionalities ( table 1 ) . onto - express ( oe ) : http://vortex.cs.wayne.edu/projects.htm#onto-express onto - express is a software application used to translate a list of differentially regulated genes into a functional profile [ 12 , 13 ] . onto - express constructs a profile for each of the go categories : cellular component , biological process , molecular function , and chromosome location as well . onto - express implements hypergeometric , binomial , and fisher 's exact tests . onto - express performs bonferroni , holm , sidak , and fdr corrections to adjust for multiple testing . onto - express is a software application used to translate a list of differentially regulated genes into a functional profile [ 12 , 13 ] . onto - express constructs a profile for each of the go categories : cellular component , biological process , molecular function , and chromosome location as well . onto - express implements hypergeometric , binomial , and fisher 's exact tests . onto - express performs bonferroni , holm , sidak , and fdr corrections to adjust for multiple testing . safe ( significance analysis of function and expression)safe is a bioconductor / r algorithm that first computes gene - specific statistics in order to test for association between gene expression and the phenotype of interest . fatigo+ implements an inclusive analysis in which at a given level in the go dag hierarchy , genes annotated with child go terms take the annotation from the parent . fatigo+ implements an inclusive analysis in which at a given level in the go dag hierarchy , genes annotated with child go terms take the annotation from the parent . the program accepts two lists of genes : a reference set of genes and the list of changed genes . the program accepts two lists of genes : a reference set of genes and the list of changed genes . one limitation is that the user can only choose between the whole genome or the network under study as reference set of gene for the enrichment test . one limitation is that the user can only choose between the whole genome or the network under study as reference set of gene for the enrichment test . gosurfer : http://bioinformatics.bioen.uiuc.edu/gosurfer/ gosurfer is used to visualize and compare gene sets by mapping them onto gene ontology ( go ) information in the form of a hierarchical tree . gosurfer is used to visualize and compare gene sets by mapping them onto gene ontology ( go ) information in the form of a hierarchical tree . in most microarray studies , gene expressions are measured on a small number of arrays or samples ; however , large collections of arrays are available in microarray database that contain transcript levels data from thousands of genes across a wide variety of experiments and samples . this meta - analytic approach allows biologists to test the consistency of gene expression patterns across different studies . most importantly , the analysis of concerted changes in transcript levels between genes can lead to biological function discovery . tools have been developed that make use of the large sample size available in these databases to identify more reliable concerted changes in transcripts levels as well as to examine the coordinated change of gene expression levels . the tool has the data processed with a variety of image processing methods : rma , mas5 , and gcrma . the tool has the data processed with a variety of image processing methods : rma , mas5 , and gcrma . investigators can select which of over 100 experiments to include in coexpression analysis . there is also several visualization tools and databases of annotations attached to the coexpression . there is also several visualization tools and databases of annotations attached to the coexpression . genevestigator : http://www.genevestigator.ethz.ch/ genevestigator is a web - based discovery tool to study the expression and regulation of genes , pathways , and networks [ 26 , 27 ] . in addition genevestigator is a web - based discovery tool to study the expression and regulation of genes , pathways , and networks [ 26 , 27 ] . the user can specify the pearson correlation coefficient threshold and the array database to use for the coexpression analysis . the user can specify the pearson correlation coefficient threshold and the array database to use for the coexpression analysis . plexdb ( plant expression database ) : http://www.plexdb.org/ plexdb serves as a comprehensive public repository for gene expression for plants and plant pathogens . in higher metazoa , on average each gene is estimated to interact with five other genes , and to be involved in ten different biological functions during development . on a molecular level , the function of a gene depends on its cellular context , and the activity of a cell is determined by which genes are being expressed and which are not and how they interact with each other . the traditional reductionist approach that investigates biological phenomena by analyzing one gene at a time can not address this complexity . by using systems biology approach and network theories , investigators can analyze the behavior and relationships of all of the elements in a particular biological system to arrive at a more complete description of how the system functions . clustering analysis on microarray expression data only extracts lists of coregulated genes out of a large - scale expression data . in this section , we will describe some of the methods and tools used to reconstruct , visualize , and explore gene networks . one method is based on bayesian inference theory which seeks to find the most probable network given the observed expression patterns of the genes to be included in the network . we present below selected tools that implement either of the aforementioned approaches to reverse - engineer gene regulatory networks . bnarray ( bayesian network array ) : http://www.cls.zju.edu.cn/binfo/bnarray/ bnarray is a tool developed in r for inferring gene regulatory networks from dna microarray data by using a bayesian network . bnarray is a tool developed in r for inferring gene regulatory networks from dna microarray data by using a bayesian network . as a result of the explosion and advances in experimental technologies that allow genome - wide characterization of molecular states and interactions among thousands of genes , researchers are often faced with the need for tools for the visualization , display , and evaluation of large structure data . the main aim of these tools is to provide a summarized yet understandable view of large amount of data while integrating additional information regarding the biological processes and functions . several network visualization tools have been developed of which we will describe some of the most popular . cytoscape is a general - purpose , open - source software environment for the large scale integration of molecular interaction network data . cytoscape is a general - purpose , open - source software environment for the large scale integration of molecular interaction network data . osprey allows user defined colors to indicate gene function , experimental systems , and data sources . visant offers an online interface for a large range of published datasets on biomolecular interactions , as well as databases for organized annotation , including genbank , kegg , and swissprot . one of the main focuses in the postgenomic era is to study the network of molecular interactions in order to reveal the complex roles played by genes , gene products , and the cellular environments in different biological processes . the nodes ( genes ) of a network can be associated with additional information regarding the gene products , gene positions in the chromosome , or the gene functional annotation . network exploration tools enable the user to perform analysis on single genes , gene families , patterns of molecular interactions , as well as on the global structure of the network . here metnet ( metabolic networking database ) : http://www.metnetdb.org/ metnet is a publicly available software for analysis of genome - wide mrna , protein , and metabolite profiling data . the software is designed to enable the biologist to visualize , statistically analyze , and model a metabolic and regulatory network map of arabidopsis , combined with gene expression profiling data . the tools within metnet allow the user to map metabolic and regulatory networks ; to integrate and visualize data together ; to explore and model the metabolic and regulatory flow in the network . metnet is a publicly available software for analysis of genome - wide mrna , protein , and metabolite profiling data . the software is designed to enable the biologist to visualize , statistically analyze , and model a metabolic and regulatory network map of arabidopsis , combined with gene expression profiling data . the tools within metnet allow the user to map metabolic and regulatory networks ; to integrate and visualize data together ; to explore and model the metabolic and regulatory flow in the network . the tool performs easy retrieval , construction , and visualization of complex biological networks , including genome - scale integrated networks of protein - protein , protein - dna , and genetic interactions . biologicalnetworks also allow the analysis and the mapping of expression profiles of genes or proteins onto regulatory , metabolic , and cellular networks . the main component of pavesy is a relational sql database system that stores biological objects , such as metabolites , proteins , genes , and their interrelationships . , gene modules or pathways ) for the exploratory analysis of a large compendium of gene expression profiles . effects on gene expression can be projected onto these networks for the following ontologies : anatomy , development , stimulus , and mutation , in form of comparison sets . one of the downstream applications of the reconstruction of a gene regulatory networks or the identification of clusters of functionally related genes is to associate the genes and their interconnections with known metabolic pathways . because of the curse of dimensionality that sometimes hampers the whole network analysis , investigators often focus on pathway rather than many specialized databases are available that store and summarize large amount of information on metabolic reactions . its goal is to serve as a metabolic encyclopedia , containing a collection of nonredundant pathways , enzymatic reactions , enzymes , chemical compounds , genes and review - level comments . data in biocarta are constantly updated , and new pathways are suggested by the life science research community . the genenet system is designed for formalized description and automated visualization of gene networks . the genenet system includes database on gene network components , java program for the data visualization . genenet allows the users to select entities that are involved in the functioning of a particular gene network , to describe the regulatory relations for a particular gene network , and to search for potential transcription factors . thus dna - binding proteins play a key role in all aspects of genetic activity within an organism . some other approaches have been proposed that integrates comparative , structural , and functional genomics to identify conserved motifs in coregulated genes . following is a list of transcription factors database and tools ( table 3 ) . plantcare is a database of plant cis - acting regulatory elements and a portal to tools for in silico analysis of promoter sequences . the database can be queried on names of tf binding sites , function , species , cell type , genes , and reference literatures . users can query the database with a gene name , gene symbol to retrieve its promoter along with other genes regulated by the same transcription factor . however , it is only by integrating genomics , transcriptomics proteomics , metabolomics , and other recent omics types of data such as interactomics , localizomics , lipidomics , and phenomics that biologists can gain access to a more complete picture of living organisms and unexplored areas of biology . this challenging task requires a systems level approach to perform systematic data mining , cross - knowledge validation , and cross - species interpolation . some investigators attempted the integration of genomic data and transcriptomic data , and the integration of protein - protein interation data and transcriptomic data to analyze the dynamics of biological networks in yeast . the approach commonly used comprises three steps : ( 1 ) identification of the network that describes all interactions between cellular components from integrating various genome scale data ; ( 2 ) decomposing the network into its constituent parts or network modules ; ( 3 ) building a mathematical model that simulates biological systems for the purpose of simulation or prediction . we describe below proteomics and metabolomics , and the potential of their integration with transcritomic data . proteomics is the systematic large - scale study of proteins of an organism or a specific type of tissue , particularly their structure , function , and spatiotemporal distribution . the integration of transcriptome and proteome data has not always resulted in consistent results . nonetheless , the interpretation of the data in terms of biological pathways or functional groups gives better correlation of transcriptome with proteome in yeast . protein amino acid sequence databases and repositories for two - dimensional polyacrylamide gel electrophoresis as reference maps of proteomes are becoming popular as tools for analyzing and comparing the plant proteome . metabolomics provide scientist with the ability ( 1 ) to characterize genotypes , ecotypes , or phenotypes with metabolites levels ; ( 2 ) to identify sites within a genetic network where metabolites levels are regulated ; ( 3 ) to analyze genes functions at the light of metabolites levels . currently , one of the most pressing needs in the fields of metabolomics for bioinformatics application is the creation of specific databases and biochemical ontologies . such tools would help clearly describe the function , localization , and interaction of metabolites . most of the tools available exploit prior biological knowledge accumulated in numerous publicly available databases in an attempt to provide a comprehensive view of biological phenomena . these tools differ in many respects and the guidance provided in this review will help biologists with little knowledge in statistics understand some of the key concepts . the integration of transcriptomics data with all other omics data is a challenging task that can be addressed by a systems - level approach .

the immune response must be highly regulated to eliminate infections but prevent attacks on self - tissue that lead to autoimmunity . helper t cells play an essential role in regulating the immune response , and a great deal of effort has been undertaken to understand their development and function ( illustrated in several recent reviews ) . however , many questions remain , and many approaches have been utilized to study the mechanisms controlling helper t cell development and function . these have ranged from the use of in vitro cell culture systems to whole animals . cell culture systems , especially those using cell lines , offer the benefit of ease of use and the ability to generate large amount of material to do sophisticated biochemical analyses . however , they suffer from their limited ability to reproduce the actual conditions occurring in an immune response . in contrast , whole animal experiments offer the benefit of relevance , but they can suffer from difficulties in manipulation and the ability to perform precise controls in addition to their large costs and ethical implications . nevertheless , the vast majority of helper t cells studies today still require the use of whole animal experiments involving primary t cells because of the inability of cell lines to duplicate the exact steps occurring in the whole animal . therefore , it is essential to utilize cost effective approaches that are highly informative . genetics is one powerful tool to study helper t cell development and function , yet traditional methods involving gene knockouts or transgenes are time consuming and expensive so they are often out of reach of small labs . however , retroviral transduction offers a powerful , rapid and , cost effective genetic approach to study the mechanisms of specific gene products . therefore , it is commonly used in papers studying helper t cell development and function . it utilizes the pmig ( murine stem cell virus - internal ribosomal entry site - green fluorescent protein ) retroviral expression vector , in which the gene of interest can be cloned and thereby expressed from the retrovirus long terminal repeat ( ltr ) . in addition , downstream of the inserted gene of interest is an internal ribosome entry sequence ( ires ) followed by the green fluorescent protein ( gfp ) gene so transduced cells can easily be followed by their expression of gfp . the vector was originally derived from the murine stem cell virus ( mscv ) vectors , which contain mutations in repressor binding sites in the ltrs making them resistant to silencing and thus , giving high expression in many cell types including helper t cells . production of high titer retrovirus requires a simple transient transfection protocol of human embryonic kidney ( hek ) 293 t cells with the mig vector and a helper virus vector that expresses the retroviral gag , pol , and env genes . for this the pcl - eco helper virus vector works well in producing high titer replication incompetent retroviruses . here these protocols for retroviral production and transduction of primary murine t cells are described in addition to some of our results using this approach to study mirna regulation of gene expression controlling helper t cell differentiation . mirnas are small rnas of approximately 22 nucleotides in length that post - transcriptionally regulate gene expression by targeting homologous sequences in protein encoding messenger rnas and suppressing translation and inducing message instability . they are essential in the earliest stages of development , as embryos that can not produce mirnas die at a very early stage . they are thought to function by fine - tuning the expression of genes required for developmental programs . in helper t cells mirnas play multiple roles and are required for regulatory t cell ( treg ) development . we used retroviral transduction as a means to dissect the mechanisms of mirna regulation of treg differentiation . through such studies important subsequently , relevant genes regulated by these mirnas were identified in order to understand the molecular pathways regulated by mirnas in helper t cell differentiation . all mouse work performed in these protocols was undertaken according to the animals scientific procedures act , uk under the animal project license 70/6965 . prior to proceeding obtain all required approvals for producing genetically modified organisms and the use of retroviruses in mammalian cells . growth of hek 293 t cells grow hek 293 t cells on 10 cm tissue culture dishes in hek 293 t medium ( dulbecco 's modified eagle 's medium ( dmem ) with 10% fetal bovine serum ( fbs ) , 100 units / ml penicillin 0.1 mg / ml streptomycin , and 2 mm l - glutamine ) . for general cell passage , split cells 1:10 when they reach confluence by removing medium and pipetting the cells off the plate with fresh hek 293 t medium . 293 t hek cells attach loosely to the plate so trypsinization is not required.24 hr prior to transfection , split a confluent plate of cells 1:10 to a 10 cm plate ( one plate per transfection ) so that the next day the cells are ~50% confluent . grow hek 293 t cells on 10 cm tissue culture dishes in hek 293 t medium ( dulbecco 's modified eagle 's medium ( dmem ) with 10% fetal bovine serum ( fbs ) , 100 units / ml penicillin 0.1 mg / ml streptomycin , and 2 mm l - glutamine ) . for general cell passage , split cells 1:10 when they reach confluence by removing medium and pipetting the cells off the plate with fresh hek 293 t medium . note : cells should reach confluence in 2 - 3 days . 24 hr prior to transfection , split a confluent plate of cells 1:10 to a 10 cm plate ( one plate per transfection ) so that the next day the cells are ~50% confluent . transfection by calcium phosphate precipitation approximately 1 hr prior to transfection , remove medium from cells and carefully add 9 ml of fresh hek 293 t medium to prevent cells detaching from the plate.prepare 2x hepes buffered saline ( 2x hbs ) and a fresh 2.5 m cacl2 solution as described in table 1 . thaw dna stocks . for the most efficient transfections use plasmid prep quality dna.in a 6 ml round bottom or 15 ml conical tube add 5 g of retroviral dna ( pmig ) , 5 g of helper virus dna ( pcl - eco ) , and h2o to 420 l . note : this along with the next step ( addition of 2x hbs ) can be done on a regular bench as long as general sterile technique is used.slowly add 500 l of 2x hbs drop by drop to the above dna mixture while gently vortexing so that the solution is continuously mixed and the capo4 precipitates in even - sized crystals.transfer to a tissue culture hood and slowly add the capo4 dna mixture ( 1 ml ) dropwise to the 9 ml of medium covering the cells while constantly and gently swirling the plate . once again be careful not to detach cells from the plate . note : at this point the capo4 precipitate will be readily visible under a microscope as small crystals about the size of bacteria . these are easily seen after 30 - 60 min when the crystals have had a chance to settle to the bottom of the plate . approximately 1 hr prior to transfection , remove medium from cells and carefully add 9 ml of fresh hek 293 t medium to prevent cells detaching from the plate . prepare 2x hepes buffered saline ( 2x hbs ) and a fresh 2.5 m cacl2 solution as described in table 1 . thaw dna stocks . for the most efficient transfections use plasmid prep quality dna . in a 6 ml round bottom or 15 ml conical tube add 5 g of retroviral dna ( pmig ) , 5 g of helper virus dna ( pcl - eco ) , and h2o to 420 l . note : this along with the next step ( addition of 2x hbs ) can be done on a regular bench as long as general sterile technique is used . slowly add 500 l of 2x hbs drop by drop to the above dna mixture while gently vortexing so that the solution is continuously mixed and the capo4 precipitates in even - sized crystals . transfer to a tissue culture hood and slowly add the capo4 dna mixture ( 1 ml ) dropwise to the 9 ml of medium covering the cells while constantly and gently swirling the plate . place the cells back into the incubator . note : at this point the capo4 precipitate will be readily visible under a microscope as small crystals about the size of bacteria . these are easily seen after 30 - 60 min when the crystals have had a chance to settle to the bottom of the plate . the following day ( anywhere from 12 - 24 hr after transfection ) remove the medium and feed the cells with 10 ml of fresh hek 293 t medium once again being careful not to dislodge the cells from the plate . note : this dilutes out lymphocyte inhibitory substances that the hek 293 t cells secrete into the medium during the transfection.in the evening of the second day ( ~24 hr post transfection ) feed cells with 3.5 ml of fresh hek 293 t medium . be careful that the plate is placed exactly level in the incubator so that one side is not left dry.collect medium ~12 hr later ( store at 4 c ) and feed with 3.5 ml of fresh hek 293 t medium . repeat collection 2 more times at roughly 12 hr intervals so that about 10 ml of medium is collected . this is the virus stock.spin out any residual hek 293 t cells and cellular debris by centrifuging at 600 x g for 5 min . store virus at 4 c , and for best titers , use within a day or two , as the titer will slowly diminish at 4 c . do not freeze , as this significantly decreases the titer . the following day ( anywhere from 12 - 24 hr after transfection ) remove the medium and feed the cells with 10 ml of fresh hek 293 t medium once again being careful not to dislodge the cells from the plate . note : this dilutes out lymphocyte inhibitory substances that the hek 293 t cells secrete into the medium during the transfection . in the evening of the second day ( ~24 hr post transfection ) feed cells with 3.5 ml of fresh hek 293 t medium . be careful that the plate is placed exactly level in the incubator so that one side is not left dry . collect medium ~12 hr later ( store at 4 c ) and feed with 3.5 ml of fresh hek 293 t medium . repeat collection 2 more times at roughly 12 hr intervals so that about 10 ml of medium is collected . spin out any residual hek 293 t cells and cellular debris by centrifuging at 600 x g for 5 min . store virus at 4 c , and for best titers , use within a day or two , as the titer will slowly diminish at 4 c . isolation of leukocyte cells euthanize mice by cervical dislocation , co2 asphyxiation , or other humane protocol appropriate to the animal handling rules of the institution.dissect freshly euthanized mice and remove spleen and desired lymph nodes . place the organs in wells of a 6 well tissue culture plate containing r10 medium ( rpmi medium with 10% heat inactivated fbs , 100 units / ml penicillin , 0.1 mg / ml streptomycin , 2 mm l - glutamine , and 0.1% -mercaptoethanol ) . use a cell culture hood for dissection of mice and all subsequent manipulations to minimize chance of contamination in cultures . note : keep the r10 medium cold and perform all the downstream purification steps at 4 c.place a 70 m cell culture strainer into a 50 ml conical tube containing approximately 5 ml of r10 medium . use one strainer for the spleen and lymph nodes of up to three mice.add spleen and lymph nodes to the cell strainer and macerate using the end of a 5 ml syringe plunger . rinse with 1 - 2 ml of r10 medium.transfer cells to a 15 ml conical tube and bring the volume up to 14 ml with r10 . discard supernatant by pouring it off with one clean movement to prevent disturbing the cell pellet.add 2 ml of cold ( 4 c ) red cell lysis buffer ( table 1 ) to each tube . note : the timing of red cell lysis is critical to prevent death of lymphocytes . therefore , the exact timing of each batch of red cell lysis buffer will need to be optimized.add 12 - 13 ml of r10 medium . immediately centrifuge and discard supernatant as in step 2.1.5 . perform the washing steps 2x more with r10 medium to remove any residual red cell lysis buffer from the cells and avoid death of lymphocytes.count cells in a hemocytometer diluting 1:20 in trypan blue to determine the yield of viable cells . euthanize mice by cervical dislocation , co2 asphyxiation , or other humane protocol appropriate to the animal handling rules of the institution . place the organs in wells of a 6 well tissue culture plate containing r10 medium ( rpmi medium with 10% heat inactivated fbs , 100 units / ml penicillin , 0.1 mg / ml streptomycin , 2 mm l - glutamine , and 0.1% -mercaptoethanol ) . use a cell culture hood for dissection of mice and all subsequent manipulations to minimize chance of contamination in cultures . note : keep the r10 medium cold and perform all the downstream purification steps at 4 c . place a 70 m cell culture strainer into a 50 ml conical tube containing approximately 5 ml of r10 medium . use one strainer for the spleen and lymph nodes of up to three mice . add spleen and lymph nodes to the cell strainer and macerate using the end of a 5 ml syringe plunger . transfer cells to a 15 ml conical tube and bring the volume up to 14 ml with r10 . discard supernatant by pouring it off with one clean movement to prevent disturbing the cell pellet . add 2 ml of cold ( 4 c ) red cell lysis buffer ( table 1 ) to each tube . note : the timing of red cell lysis is critical to prevent death of lymphocytes . therefore , the exact timing of each batch of red cell lysis buffer will need to be optimized . add 12 - 13 ml of r10 medium . immediately centrifuge and discard supernatant as in step 2.1.5 . perform the washing steps 2x more with r10 medium to remove any residual red cell lysis buffer from the cells and avoid death of lymphocytes . count cells in a hemocytometer diluting 1:20 in trypan blue to determine the yield of viable cells . isolation of cd4 t cells using magnetic beads kit to remove cd8 , cd11b , cd16/32 , cd45r , mhc class ii and ter-119 cells . aliquot 8 - 10 x 10 cells from step 2.1.8 per 15 ml conical tube and centrifuge at 600 x g for 5 min at 4 c . resuspend the cells in each tube in 100 l of heat inactivated fbs then add 100 l of antibody mix from the kit . incubate for 30 min at 4 c with gentle mixing on a roller.while the above cells are incubating , prepare the beads . resuspend beads in the vial by vortexing for > 30 sec then transfer 1 ml of beads per 8 - 10 x 10 prepared cells to a 15 ml conical tube . add 2 ml of r10 medium per ml of beads and gently mix.place the tube in the magnet for 1 min then discard the supernatant . remove tube from the magnet and resuspend beads in 4 ml of r10 medium . wash the cells at the end of the antibody incubation ( step 2.2.1 ) by adding 10 ml of r10 medium per tube . gently mix well with swirling of the tube and centrifuge cells at 600 x g for 5 min at 4 c . resuspend cells in 1 ml of r10 medium.add 2 ml of the washed beads from step 2.2.2.2 ( the amount prepared for each tube ) to each tube of antibody treated cells and incubate for 30 min at 4 c with gentle mixing on a roller.resuspend the cell - bead mixture by gently pipetting 5 times with a pipet containing a narrow tip opening . place the tube in the beads magnet for 2 min then transfer the supernatant containing the negatively selected cells to a new tube . keep these cells , as they are the cd4 population.repeat negative selection one more time . spin cells from step 2.2.5 at 600 x g for 5 min at 4 c . pour off the supernatant as in step 2.2.3 and resuspend in 1 ml of r10 medium . after the final magnetic bead selection , count the cells to determine recovery ( typical yields are 15 - 20 x 10 cells per 10 leukocytes ) . aliquot 8 - 10 x 10 cells from step 2.1.8 per 15 ml conical tube and centrifuge at 600 x g for 5 min at 4 c . resuspend the cells in each tube in 100 l of heat inactivated fbs then add 100 l of antibody mix from the kit . resuspend beads in the vial by vortexing for > 30 sec then transfer 1 ml of beads per 8 - 10 x 10 prepared cells to a 15 ml conical tube . add 2 ml of r10 medium per ml of beads and gently mix.place the tube in the magnet for 1 min then discard the supernatant . remove tube from the magnet and resuspend beads in 4 ml of r10 medium . resuspend beads in the vial by vortexing for > 30 sec then transfer 1 ml of beads per 8 - 10 x 10 prepared cells to a 15 ml conical tube . place the tube in the magnet for 1 min then discard the supernatant . remove tube from the magnet and resuspend beads in 4 ml of r10 medium . wash the cells at the end of the antibody incubation ( step 2.2.1 ) by adding 10 ml of r10 medium per tube . gently mix well with swirling of the tube and centrifuge cells at 600 x g for 5 min at 4 c . add 2 ml of the washed beads from step 2.2.2.2 ( the amount prepared for each tube ) to each tube of antibody treated cells and incubate for 30 min at 4 c with gentle mixing on a roller . resuspend the cell - bead mixture by gently pipetting 5 times with a pipet containing a narrow tip opening . place the tube in the beads magnet for 2 min then transfer the supernatant containing the negatively selected cells to a new tube . keep these cells , as they are the cd4 population . spin cells from step 2.2.5 at 600 x g for 5 min at 4 c . pour off the supernatant as in step 2.2.3 and resuspend in 1 ml of r10 medium . after the final magnetic bead selection , count the cells to determine recovery ( typical yields are 15 - 20 x 10 cells per 10 leukocytes ) . isolation of nave cd4 t cells ( cd25 and cd62l ) using cell separation columns centrifuge cd4 t cells at 600 x g for 5 min at 4 c and resuspend in 150 - 200 l of r10 medium per 10 cells . add 2 l of stock biotinylated cd25 monoclonal antibody ( clone 7d4 ) . incubate for 30 min at 4 c with gentle mixing on a roller.wash cells by adding 10 ml of cell separation column buffer ( table 1 ) . remove as much supernatant as possible and estimate the volume of buffer / cells remaining . resuspend to a final volume of approximately 90 l per 10 cells.add 20 l of streptavidin beads per 10 cells and mix with gentle flicks . incubate for 15 min at 4 c.while cells are incubating , prepare the medium cell separation ( ms ) columns for the manual approach . each ms column retains 10 cells , and since cd25 cells typically represent about 10% of total cd4 cells , use 1 column per 10 cd4 t cells . add 500 l of cell separation column buffer to the column and let flow through . repeat 2 more times.at end of cell / bead incubation , wash cells by adding 10 ml of cell separation column buffer and centrifuging at 600 x g for 5 min at 4 c . resuspend cells in 500 l of cell separation column buffer per 10 cells , but still use 500 l if there are fewer cells.apply 500 l of cell / bead suspension to the column and collect the flow through . pass this over the column 1 more time and collect the flow through once again . rinse the column 3 times with 500 l of cell separation column buffer , adding each flow through from these rinses to the collected cells . count cells to determine yield.if tregs ( cd25 cells ) are desired , isolate as follows . remove the column from the separator and hold it above an open universal tube taking care to maintain sterility . quickly pipette 500 l of cell separation buffer onto the column and firmly flush out the positive fraction , using the plunger supplied with the column.repeat the flushing procedure 2 more times . pass the positive fraction over a fresh ms cell separation column and discard this flow - through . firmly flush out the positive cells three times , as before and centrifuge cells at 600 x g for 5 min at 4 c. resuspend cells in 1 ml of r10 medium and count to determine the yield . to obtain cd25 cd62l t cells , centrifuge the cd25 t cells isolated above in step 2.3.6 at 600 x g for 5 min at 4 and resuspend in 150 - 200 l of r10 medium per 10 cells . add 5 l of stock biotinylated cd62l monoclonal antibody ( clone mel-14 ) and incubate for 30 min at 4 c with gentle mixing on a roller as before.perform wash , streptavidin bead binding , and cell separation column preparation as described for treg isolation protocol ( 2.3.7 ) . this time use the large cell separation ( ls ) column , which has a capacity of 10 cells . since cd62l t cells typically represent about 60 - 70% of cd25 cells , use 1 ls cell separation column per 10 cells . add cell / bead mixture to ls cell separation column as described in step 2.3.6 this time discarding the final flow through and column rinses . collect the cd62l t cells as described in step 2.3.7 for the cd25 cell collection.centrifuge cells at 600 x g for 5 min at 4 c. resuspend cells in 1 ml of r10 and count to determine yield . centrifuge cd4 t cells at 600 x g for 5 min at 4 c and resuspend in 150 - 200 l of r10 medium per 10 cells . wash cells by adding 10 ml of cell separation column buffer ( table 1 ) . remove as much supernatant as possible and estimate the volume of buffer / cells remaining . while cells are incubating , prepare the medium cell separation ( ms ) columns for the manual approach . each ms column retains 10 cells , and since cd25 cells typically represent about 10% of total cd4 cells , use 1 column per 10 cd4 t cells . add 500 l of cell separation column buffer to the column and let flow through . / bead incubation , wash cells by adding 10 ml of cell separation column buffer and centrifuging at 600 x g for 5 min at 4 c . resuspend cells in 500 l of cell separation column buffer per 10 cells , but still use 500 l if there are fewer cells . apply 500 l of cell / bead suspension to the column and collect the flow through . pass this over the column 1 more time and collect the flow through once again . rinse the column 3 times with 500 l of cell separation column buffer , adding each flow through from these rinses to the collected cells . remove the column from the separator and hold it above an open universal tube taking care to maintain sterility . quickly pipette 500 l of cell separation buffer onto the column and firmly flush out the positive fraction , using the plunger supplied with the column.repeat the flushing procedure 2 more times . pass the positive fraction over a fresh ms cell separation column and discard this flow - through . firmly flush out the positive cells three times , as before and centrifuge cells at 600 x g for 5 min at 4 c. resuspend cells in 1 ml of r10 medium and count to determine the yield . remove the column from the separator and hold it above an open universal tube taking care to maintain sterility . quickly pipette 500 l of cell separation buffer onto the column and firmly flush out the positive fraction , using the plunger supplied with the column . pass the positive fraction over a fresh ms cell separation column and discard this flow - through . firmly flush out the positive cells three times , as before and centrifuge cells at 600 x g for 5 min at 4 c. resuspend cells in 1 ml of r10 medium and count to determine the yield . to obtain cd25 cd62l t cells , centrifuge the cd25 t cells isolated above in step 2.3.6 at 600 x g for 5 min at 4 and resuspend in 150 - 200 l of r10 medium per 10 cells . add 5 l of stock biotinylated cd62l monoclonal antibody ( clone mel-14 ) and incubate for 30 min at 4 c with gentle mixing on a roller as before . perform wash , streptavidin bead binding , and cell separation column preparation as described for treg isolation protocol ( 2.3.7 ) . this time use the large cell separation ( ls ) column , which has a capacity of 10 cells . since cd62l t cells typically represent about 60 - 70% of cd25 cells , use 1 ls cell separation column per 10 cells . add cell / bead mixture to ls cell separation column as described in step 2.3.6 collect the cd62l t cells as described in step 2.3.7 for the cd25 cell collection.centrifuge cells at 600 x g for 5 min at 4 c. resuspend cells in 1 ml of r10 and count to determine yield . dilute to 0.75 - 1 x10 cells per ml in r10 medium . since cd62l t cells typically represent about 60 - 70% of cd25 cells , use 1 ls cell separation column per 10 cells . add cell / bead mixture to ls cell separation column as described in step 2.3.6 this time discarding the final flow through and column rinses . collect the cd62l t cells as described in step 2.3.7 for the cd25 cell collection . centrifuge cells at 600 x g for 5 min at 4 c. resuspend cells in 1 ml of r10 and count to determine yield . dilute to 0.75 - 1 x10 cells per ml in r10 medium . facs staining strategy for cells pre and post all stages of isolation , stain with cd4-fitc , cd8a - percp - cy5.5 ( helper and cytotoxic t cells ) , and mhcii - pe ( mhc class ii expressing cells).for cells pre and post cd25 isolation , stain with cd4-fitc and cd25-pe ( tregs versus other helper t cells).for cells pre and post cd62l isolation stain with cd4-fitc , cd62l - pe and cd44-apc ( nave versus memory and effector helper t cells ) . for each analysis place 10 cells in a well of a 96 well u - bottom plate . centrifuge as above and pour off dpbs.add 50 l of dpbs per well and 0.5 l of each antibody ( as indicated in 2.4.1 ) and incubate at rt for 30 min in the dark to avoid bleaching of fluorescent label.wash cells 2x with dpbs as in 2.4.2 and immediately analyze on a flow cytometer using protocols and guidelines specific to the instrument at hand . note : after the final isolation step of a good preparation , 90 - 95% of cells will be cd4 cd25 cd62l t cells . facs staining strategy for cells pre and post all stages of isolation , stain with cd4-fitc , cd8a - percp - cy5.5 ( helper and cytotoxic t cells ) , and mhcii - pe ( mhc class ii expressing cells).for cells pre and post cd25 isolation , stain with cd4-fitc and cd25-pe ( tregs versus other helper t cells).for cells pre and post cd62l isolation stain with cd4-fitc , cd62l - pe and cd44-apc ( nave versus memory and effector helper t cells ) . for cells pre and post all stages of isolation , stain with cd4-fitc , cd8a - percp - cy5.5 ( helper and cytotoxic t cells ) , and mhcii - pe ( mhc class ii expressing cells ) . for cells pre and post cd25 isolation , stain with cd4-fitc and cd25-pe ( tregs versus other helper t cells ) . for cells pre and post cd62l isolation stain with cd4-fitc , cd62l - pe and cd44-apc ( nave versus memory and effector helper t cells ) . for each analysis place 10 cells in a well of a 96 well u - bottom plate . centrifuge as above and pour off dpbs . add 50 l of dpbs per well and 0.5 l of each antibody ( as indicated in 2.4.1 ) and incubate at rt for 30 min in the dark to avoid bleaching of fluorescent label . wash cells 2x with dpbs as in 2.4.2 and immediately analyze on a flow cytometer using protocols and guidelines specific to the instrument at hand . note : after the final isolation step of a good preparation , 90 - 95% of cells will be cd4 cd25 cd62l t cells . retroviral transduction note : retroviral integration and expression of genes requires cell division . therefore , t cells must be activated o / n . while isolating the nave t cells , coat a 24 well tissue culture plate with anti - cd3 and anti - cd28 antibodies diluted in dpbs . use anti anti - cd3 at 1g / ml . use anti - cd28 at 2 g / ml if cells will ultimately be differentiated under th0 , th1 , th2 and itregs conditions . incubate ~2 hr at 37 c in a tissue culture incubator.just prior to culturing the cells , remove the antibody solution and wash the plate with ~250 l of dpbs per well to remove unbound antibody . be careful not to let the plate dry out so process a maximum of 6 wells at a time . remove dpbs wash and add 1 ml of nave cd4 t cells in r10 medium at 0.75 - 1 x 10 cells per ml . activate cells o / n ( 14 - 16 hr).the following day , centrifuge cells in the plate at 900 x g for 5 min at 30 c to attach cells to the bottom of the wells.collect and save the medium being careful not to displace the cells , and replace with 1 ml virus culture supernatant prepared from the virus production protocol . do not let the cells dry out so process a maximum of 4 wells at a time . to each well add 1 l of 8 mg / ml polybrene and 10 l of 1 m hepes ph 7.5 to aid virus uptake and prevent significant alkalization in the ambient co2 during the following centrifugation . centrifuge cells in plate at 900 x g for 90 min at 30 c. while isolating the nave t cells , coat a 24 well tissue culture plate with anti - cd3 and anti - cd28 antibodies diluted in dpbs . use anti anti - cd3 at 1g / ml . use anti - cd28 at 2 g / ml if cells will ultimately be differentiated under th0 , th1 , th2 and itregs conditions . use anti - cd28 at 10 g / ml for th9 and th17 conditions . incubate ~2 hr at 37 c in a tissue culture incubator . just prior to culturing the cells , remove the antibody solution and wash the plate with ~250 l of dpbs per well to remove unbound antibody . be careful not to let the plate dry out so process a maximum of 6 wells at a time . remove dpbs wash and add 1 ml of nave cd4 t cells in r10 medium at 0.75 - 1 x 10 cells per ml . activate cells o / n ( 14 - 16 hr ) . the following day , centrifuge cells in the plate at 900 x g for 5 min at 30 c to attach cells to the bottom of the wells . collect and save the medium being careful not to displace the cells , and replace with 1 ml virus culture supernatant prepared from the virus production protocol . do not let the cells dry out so process a maximum of 4 wells at a time . to each well add 1 l of 8 mg / ml polybrene and 10 l of 1 m hepes ph 7.5 to aid virus uptake and prevent significant alkalization in the ambient co2 during the following centrifugation . centrifuge cells in plate at 900 x g for 90 min at 30 c. to each well add 1 l of 8 mg / ml polybrene and 10 l of 1 m hepes ph 7.5 to aid virus uptake and prevent significant alkalization in the ambient co2 during the following centrifugation . centrifuge cells in plate at 900 x g for 90 min at 30 c. differentiation of cells into specific subsets carefully remove the virus culture supernatant and replace with 1 ml of the medium collected above in step 3.1.4 , as it contains il-2 and other t cell growth factors . add reagents indicated in table 2 and culture cells for 3 - 4 days to differentiate cells into specific subsets . carefully remove the virus culture supernatant and replace with 1 ml of the medium collected above in step 3.1.4 , as it contains il-2 and other t cell growth factors . add reagents indicated in table 2 and culture cells for 3 - 4 days to differentiate cells into specific subsets . analysis of cells for intracellular staining of cytokines , treat cells with 1 g / ml each of phorbol 12-myristate 13-acetate ( pma ) and ionomycin for 4 hr . during the last 2 hr of stimulation , add 1 g / ml of brefeldin a.resuspend cells from the bottom of each well by pipetting up and down several times . transfer approximately 10 cells to a 96 well u bottom plate ( usually 100 l from the 1 ml of culture of th cells ) for fixation and staining.for gfp staining , fix cells with 100 l of 2% paraformaldehyde at rt for exactly 5 min , as a longer incubation time will bleach the gfp and interfere with foxp3 staining . wash cells by adding 100 l of dpbs to each well and centrifuge at 600 x g at 22 c for 5 min . caution : paraformaldehyde is toxic . handle it in a fume hood with skin and eye protection.fix the cells again using 100 l 1x fixation buffer made from the foxp3 staining kit ( 1 volume of fixation buffer to 3 volumes of diluent ) . incubate the cells for 45 min at rt or alternatively incubate at 4 c for 16 hr.after fixation , permeabilize cells by adding 100 l of permeabilization buffer from the same kit . incubate at rt for 30 - 45 min then centrifuge at 600 x g at 22 c for 5 min.for antibody staining , remove fixation / permeabilization buffer and add 50 l of fresh permeabilization buffer . add the required antibody diluted in permeabilization buffer at 0.5 l per 50 l buffer per sample . incubate for 30 - 45 min at rt in dark to avoid bleaching of fluorescent label.add 150 l permeabilization buffer and centrifuge at 600 x g at 22 c for 5 min . analyze on a flow cytometer using protocols and guidelines specific to the instrument at hand . note : be sure to include proper controls for cytokine staining such as staining with isotype control antibodies , analyzing non - activated or th0 activated cells , etc . for intracellular staining of cytokines , treat cells with 1 g / ml each of phorbol 12-myristate 13-acetate ( pma ) and ionomycin for 4 hr . during the last 2 hr of stimulation , add 1 g / ml of brefeldin a. resuspend cells from the bottom of each well by pipetting up and down several times . transfer approximately 10 cells to a 96 well u bottom plate ( usually 100 l from the 1 ml of culture of th cells ) for fixation and staining . for gfp staining , fix cells with 100 l of 2% paraformaldehyde at rt for exactly 5 min , as a longer incubation time will bleach the gfp and interfere with foxp3 staining . wash cells by adding 100 l of dpbs to each well and centrifuge at 600 x g at 22 c for 5 min . caution : paraformaldehyde is toxic . handle it in a fume hood with skin and eye protection . fix the cells again using 100 l 1x fixation buffer made from the foxp3 staining kit ( 1 volume of fixation buffer to 3 volumes of diluent ) . incubate the cells for 45 min at rt or alternatively incubate at 4 c for 16 hr . after fixation , permeabilize cells by adding 100 l of permeabilization buffer from the same kit . incubate at rt for 30 - 45 min then centrifuge at 600 x g at 22 c for 5 min . for antibody staining , remove fixation / permeabilization buffer and add 50 l of fresh permeabilization buffer . add the required antibody diluted in permeabilization buffer at 0.5 l per 50 l buffer per sample . incubate for 30 - 45 min at rt in dark to avoid bleaching of fluorescent label . add 150 l permeabilization buffer and centrifuge at 600 x g at 22 c for 5 min . analyze on a flow cytometer using protocols and guidelines specific to the instrument at hand . note : be sure to include proper controls for cytokine staining such as staining with isotype control antibodies , analyzing non - activated or th0 activated cells , etc . prior to proceeding obtain all required approvals for producing genetically modified organisms and the use of retroviruses in mammalian cells . growth of hek 293 t cells grow hek 293 t cells on 10 cm tissue culture dishes in hek 293 t medium ( dulbecco 's modified eagle 's medium ( dmem ) with 10% fetal bovine serum ( fbs ) , 100 units / ml penicillin 0.1 mg / ml streptomycin , and 2 mm l - glutamine ) . for general cell passage , split cells 1:10 when they reach confluence by removing medium and pipetting the cells off the plate with fresh hek 293 t medium . 293 t hek cells attach loosely to the plate so trypsinization is not required.24 hr prior to transfection , split a confluent plate of cells 1:10 to a 10 cm plate ( one plate per transfection ) so that the next day the cells are ~50% confluent . grow hek 293 t cells on 10 cm tissue culture dishes in hek 293 t medium ( dulbecco 's modified eagle 's medium ( dmem ) with 10% fetal bovine serum ( fbs ) , 100 units / ml penicillin 0.1 mg / ml streptomycin , and 2 mm l - glutamine ) . for general cell passage , split cells 1:10 when they reach confluence by removing medium and pipetting the cells off the plate with fresh hek 293 t medium . 24 hr prior to transfection , split a confluent plate of cells 1:10 to a 10 cm plate ( one plate per transfection ) so that the next day the cells are ~50% confluent . transfection by calcium phosphate precipitation approximately 1 hr prior to transfection , remove medium from cells and carefully add 9 ml of fresh hek 293 t medium to prevent cells detaching from the plate.prepare 2x hepes buffered saline ( 2x hbs ) and a fresh 2.5 m cacl2 solution as described in table 1 . thaw dna stocks . for the most efficient transfections use plasmid prep quality dna.in a 6 ml round bottom or 15 ml conical tube add 5 g of retroviral dna ( pmig ) , 5 g of helper virus dna ( pcl - eco ) , and h2o to 420 l . note : this along with the next step ( addition of 2x hbs ) can be done on a regular bench as long as general sterile technique is used.slowly add 500 l of 2x hbs drop by drop to the above dna mixture while gently vortexing so that the solution is continuously mixed and the capo4 precipitates in even - sized crystals.transfer to a tissue culture hood and slowly add the capo4 dna mixture ( 1 ml ) dropwise to the 9 ml of medium covering the cells while constantly and gently swirling the plate . once again place the cells back into the incubator . note : at this point the capo4 precipitate will be readily visible under a microscope as small crystals about the size of bacteria . these are easily seen after 30 - 60 min when the crystals have had a chance to settle to the bottom of the plate . approximately 1 hr prior to transfection , remove medium from cells and carefully add 9 ml of fresh hek 293 t medium to prevent cells detaching from the plate . prepare 2x hepes buffered saline ( 2x hbs ) and a fresh 2.5 m cacl2 solution as described in table 1 . thaw dna stocks . for the most efficient transfections use plasmid prep quality dna . in a 6 ml round bottom or 15 ml conical tube add 5 g of retroviral dna ( pmig ) , 5 g of helper virus dna ( pcl - eco ) , and h2o to 420 l . add 80 l of 2.5 m cacl2 bringing the final volume to 500 l . note : this along with the next step ( addition of 2x hbs ) can be done on a regular bench as long as general sterile technique is used . slowly add 500 l of 2x hbs drop by drop to the above dna mixture while gently vortexing so that the solution is continuously mixed and the capo4 precipitates in even - sized crystals . transfer to a tissue culture hood and slowly add the capo4 dna mixture ( 1 ml ) dropwise to the 9 ml of medium covering the cells while constantly and gently swirling the plate . place the cells back into the incubator . note : at this point the capo4 precipitate will be readily visible under a microscope as small crystals about the size of bacteria . these are easily seen after 30 - 60 min when the crystals have had a chance to settle to the bottom of the plate . the following day ( anywhere from 12 - 24 hr after transfection ) remove the medium and feed the cells with 10 ml of fresh hek 293 t medium once again being careful not to dislodge the cells from the plate . note : this dilutes out lymphocyte inhibitory substances that the hek 293 t cells secrete into the medium during the transfection.in the evening of the second day ( ~24 hr post transfection ) feed cells with 3.5 ml of fresh hek 293 t medium . be careful that the plate is placed exactly level in the incubator so that one side is not left dry.collect medium ~12 hr later ( store at 4 c ) and feed with 3.5 ml of fresh hek 293 t medium . repeat collection 2 more times at roughly 12 hr intervals so that about 10 ml of medium is collected . this is the virus stock.spin out any residual hek 293 t cells and cellular debris by centrifuging at 600 x g for 5 min . store virus at 4 c , and for best titers , use within a day or two , as the titer will slowly diminish at 4 c . the following day ( anywhere from 12 - 24 hr after transfection ) remove the medium and feed the cells with 10 ml of fresh hek 293 t medium once again being careful not to dislodge the cells from the plate . note : this dilutes out lymphocyte inhibitory substances that the hek 293 t cells secrete into the medium during the transfection . in the evening of the second day ( ~24 hr post transfection ) feed cells with 3.5 ml of fresh hek 293 t medium . be careful that the plate is placed exactly level in the incubator so that one side is not left dry . collect medium ~12 hr later ( store at 4 c ) and feed with 3.5 ml of fresh hek 293 t medium . repeat collection 2 more times at roughly 12 hr intervals so that about 10 ml of medium is collected . spin out any residual hek 293 t cells and cellular debris by centrifuging at 600 x g for 5 min . alternatively , filter using a 0.45 m , low protein binding , syringe filter . store virus at 4 c , and for best titers , use within a day or two , as the titer will slowly diminish at 4 c . isolation of leukocyte cells euthanize mice by cervical dislocation , co2 asphyxiation , or other humane protocol appropriate to the animal handling rules of the institution.dissect freshly euthanized mice and remove spleen and desired lymph nodes . place the organs in wells of a 6 well tissue culture plate containing r10 medium ( rpmi medium with 10% heat inactivated fbs , 100 units / ml penicillin , 0.1 mg / ml streptomycin , 2 mm l - glutamine , and 0.1% -mercaptoethanol ) . use a cell culture hood for dissection of mice and all subsequent manipulations to minimize chance of contamination in cultures . note : keep the r10 medium cold and perform all the downstream purification steps at 4 c.place a 70 m cell culture strainer into a 50 ml conical tube containing approximately 5 ml of r10 medium . use one strainer for the spleen and lymph nodes of up to three mice.add spleen and lymph nodes to the cell strainer and macerate using the end of a 5 ml syringe plunger . rinse with 1 - 2 ml of r10 medium.transfer cells to a 15 ml conical tube and bring the volume up to 14 ml with r10 . discard supernatant by pouring it off with one clean movement to prevent disturbing the cell pellet.add 2 ml of cold ( 4 c ) red cell lysis buffer ( table 1 ) to each tube . note : the timing of red cell lysis is critical to prevent death of lymphocytes . therefore , the exact timing of each batch of red cell lysis buffer will need to be optimized.add 12 - 13 ml of r10 medium . immediately centrifuge and discard supernatant as in step 2.1.5 . perform the washing steps 2x more with r10 medium to remove any residual red cell lysis buffer from the cells and avoid death of lymphocytes.count cells in a hemocytometer diluting 1:20 in trypan blue to determine the yield of viable cells . euthanize mice by cervical dislocation , co2 asphyxiation , or other humane protocol appropriate to the animal handling rules of the institution . place the organs in wells of a 6 well tissue culture plate containing r10 medium ( rpmi medium with 10% heat inactivated fbs , 100 units / ml penicillin , 0.1 mg / ml streptomycin , 2 mm l - glutamine , and 0.1% -mercaptoethanol ) . use a cell culture hood for dissection of mice and all subsequent manipulations to minimize chance of contamination in cultures . note : keep the r10 medium cold and perform all the downstream purification steps at 4 c . place a 70 m cell culture strainer into a 50 ml conical tube containing approximately 5 ml of r10 medium . add spleen and lymph nodes to the cell strainer and macerate using the end of a 5 ml syringe plunger . transfer cells to a 15 ml conical tube and bring the volume up to 14 ml with r10 . discard supernatant by pouring it off with one clean movement to prevent disturbing the cell pellet . add 2 ml of cold ( 4 c ) red cell lysis buffer ( table 1 ) to each tube . gently mix for 3.5 min , keeping the tube on ice . note : the timing of red cell lysis is critical to prevent death of lymphocytes . therefore , the exact timing of each batch of red cell lysis buffer will need to be optimized . centrifuge and discard supernatant as in step 2.1.5 . perform the washing steps 2x more with r10 medium to remove any residual red cell lysis buffer from the cells and avoid death of lymphocytes . count cells in a hemocytometer diluting 1:20 in trypan blue to determine the yield of viable cells . isolation of cd4 t cells using magnetic beads kit to remove cd8 , cd11b , cd16/32 , cd45r , mhc class ii and ter-119 cells . aliquot 8 - 10 x 10 cells from step 2.1.8 per 15 ml conical tube and centrifuge at 600 x g for 5 min at 4 c . resuspend the cells in each tube in 100 l of heat inactivated fbs then add 100 l of antibody mix from the kit . incubate for 30 min at 4 c with gentle mixing on a roller.while the above cells are incubating , prepare the beads . resuspend beads in the vial by vortexing for > 30 sec then transfer 1 ml of beads per 8 - 10 x 10 prepared cells to a 15 ml conical tube . add 2 ml of r10 medium per ml of beads and gently mix.place the tube in the magnet for 1 min then discard the supernatant . remove tube from the magnet and resuspend beads in 4 ml of r10 medium . wash the cells at the end of the antibody incubation ( step 2.2.1 ) by adding 10 ml of r10 medium per tube . gently mix well with swirling of the tube and centrifuge cells at 600 x g for 5 min at 4 c . resuspend cells in 1 ml of r10 medium.add 2 ml of the washed beads from step 2.2.2.2 ( the amount prepared for each tube ) to each tube of antibody treated cells and incubate for 30 min at 4 c with gentle mixing on a roller.resuspend the cell - bead mixture by gently pipetting 5 times with a pipet containing a narrow tip opening . avoid foaming . place the tube in the beads magnet for 2 min then transfer the supernatant containing the negatively selected cells to a new tube . keep these cells , as they are the cd4 population.repeat negative selection one more time . spin cells from step 2.2.5 at 600 x g for 5 min at 4 c . pour off the supernatant as in step 2.2.3 and resuspend in 1 ml of r10 medium . after the final magnetic bead selection , count the cells to determine recovery ( typical yields are 15 - 20 x 10 cells per 10 leukocytes ) . aliquot 8 - 10 x 10 cells from step 2.1.8 per 15 ml conical tube and centrifuge at 600 x g for 5 min at 4 c . resuspend the cells in each tube in 100 l of heat inactivated fbs then add 100 l of antibody mix from the kit . resuspend beads in the vial by vortexing for > 30 sec then transfer 1 ml of beads per 8 - 10 x 10 prepared cells to a 15 ml conical tube . add 2 ml of r10 medium per ml of beads and gently mix.place the tube in the magnet for 1 min then discard the supernatant . remove tube from the magnet and resuspend beads in 4 ml of r10 medium . resuspend beads in the vial by vortexing for > 30 sec then transfer 1 ml of beads per 8 - 10 x 10 prepared cells to a 15 ml conical tube . wash the cells at the end of the antibody incubation ( step 2.2.1 ) by adding 10 ml of r10 medium per tube . gently mix well with swirling of the tube and centrifuge cells at 600 x g for 5 min at 4 c . add 2 ml of the washed beads from step 2.2.2.2 ( the amount prepared for each tube ) to each tube of antibody treated cells and incubate for 30 min at 4 c with gentle mixing on a roller . resuspend the cell - bead mixture by gently pipetting 5 times with a pipet containing a narrow tip opening . place the tube in the beads magnet for 2 min then transfer the supernatant containing the negatively selected cells to a new tube . keep these cells , as they are the cd4 population . spin cells from step 2.2.5 at 600 x g for 5 min at 4 c . pour off the supernatant as in step 2.2.3 and resuspend in 1 ml of r10 medium . after the final magnetic bead selection , count the cells to determine recovery ( typical yields are 15 - 20 x 10 cells per 10 leukocytes ) . isolation of nave cd4 t cells ( cd25 and cd62l ) using cell separation columns centrifuge cd4 t cells at 600 x g for 5 min at 4 c and resuspend in 150 - 200 l of r10 medium per 10 cells . incubate for 30 min at 4 c with gentle mixing on a roller.wash cells by adding 10 ml of cell separation column buffer ( table 1 ) . remove as much supernatant as possible and estimate the volume of buffer / cells remaining . resuspend to a final volume of approximately 90 l per 10 cells.add 20 l of streptavidin beads per 10 cells and mix with gentle flicks . incubate for 15 min at 4 c.while cells are incubating , prepare the medium cell separation ( ms ) columns for the manual approach . each ms column retains 10 cells , and since cd25 cells typically represent about 10% of total cd4 cells , use 1 column per 10 cd4 t cells . add 500 l of cell separation column buffer to the column and let flow through . repeat 2 more times.at end of cell / bead incubation , wash cells by adding 10 ml of cell separation column buffer and centrifuging at 600 x g for 5 min at 4 c . resuspend cells in 500 l of cell separation column buffer per 10 cells , but still use 500 l if there are fewer cells.apply 500 l of cell / bead suspension to the column and collect the flow through . pass this over the column 1 more time and collect the flow through once again . rinse the column 3 times with 500 l of cell separation column buffer , adding each flow through from these rinses to the collected cells . count cells to determine yield.if tregs ( cd25 cells ) are desired , isolate as follows . remove the column from the separator and hold it above an open universal tube taking care to maintain sterility . quickly pipette 500 l of cell separation buffer onto the column and firmly flush out the positive fraction , using the plunger supplied with the column.repeat the flushing procedure 2 more times . pass the positive fraction over a fresh ms cell separation column and discard this flow - through . firmly flush out the positive cells three times , as before and centrifuge cells at 600 x g for 5 min at 4 c. resuspend cells in 1 ml of r10 medium and count to determine the yield . to obtain cd25 cd62l t cells , centrifuge the cd25 t cells isolated above in step 2.3.6 at 600 x g for 5 min at 4 and resuspend in 150 - 200 l of r10 medium per 10 cells . add 5 l of stock biotinylated cd62l monoclonal antibody ( clone mel-14 ) and incubate for 30 min at 4 c with gentle mixing on a roller as before.perform wash , streptavidin bead binding , and cell separation column preparation as described for treg isolation protocol ( 2.3.7 ) . this time use the large cell separation ( ls ) column , which has a capacity of 10 cells . since cd62l t cells typically represent about 60 - 70% of cd25 cells , use 1 ls cell separation column per 10 cells . add cell / bead mixture to ls cell separation column as described in step 2.3.6 this time discarding the final flow through and column rinses . collect the cd62l t cells as described in step 2.3.7 for the cd25 cell collection.centrifuge cells at 600 x g for 5 min at 4 c. resuspend cells in 1 ml of r10 and count to determine yield . centrifuge cd4 t cells at 600 x g for 5 min at 4 c and resuspend in 150 - 200 l of r10 medium per 10 cells . wash cells by adding 10 ml of cell separation column buffer ( table 1 ) . remove as much supernatant as possible and estimate the volume of buffer / cells remaining . while cells are incubating , prepare the medium cell separation ( ms ) columns for the manual approach . each ms column retains 10 cells , and since cd25 cells typically represent about 10% of total cd4 cells , use 1 column per 10 cd4 t cells . add 500 l of cell separation column buffer to the column and let flow through . incubation , wash cells by adding 10 ml of cell separation column buffer and centrifuging at 600 x g for 5 min at 4 c . resuspend cells in 500 l of cell separation column buffer per 10 cells , but still use 500 l if there are fewer cells . apply 500 l of cell / bead suspension to the column and collect the flow through . pass this over the column 1 more time and collect the flow through once again . rinse the column 3 times with 500 l of cell separation column buffer , adding each flow through from these rinses to the collected cells . remove the column from the separator and hold it above an open universal tube taking care to maintain sterility . quickly pipette 500 l of cell separation buffer onto the column and firmly flush out the positive fraction , using the plunger supplied with the column.repeat the flushing procedure 2 more times . pass the positive fraction over a fresh ms cell separation column and discard this flow - through . firmly flush out the positive cells three times , as before and centrifuge cells at 600 x g for 5 min at 4 c. resuspend cells in 1 ml of r10 medium and count to determine the yield . remove the column from the separator and hold it above an open universal tube taking care to maintain sterility . quickly pipette 500 l of cell separation buffer onto the column and firmly flush out the positive fraction , using the plunger supplied with the column . pass the positive fraction over a fresh ms cell separation column and discard this flow - through . firmly flush out the positive cells three times , as before and centrifuge cells at 600 x g for 5 min at 4 c. resuspend cells in 1 ml of r10 medium and count to determine the yield . to obtain cd25 cd62l t cells , centrifuge the cd25 t cells isolated above in step 2.3.6 at 600 x g for 5 min at 4 and resuspend in 150 - 200 l of r10 medium per 10 cells . add 5 l of stock biotinylated cd62l monoclonal antibody ( clone mel-14 ) and incubate for 30 min at 4 c with gentle mixing on a roller as before . perform wash , streptavidin bead binding , and cell separation column preparation as described for treg isolation protocol ( 2.3.7 ) . this time use the large cell separation ( ls ) column , which has a capacity of 10 cells . since cd62l t cells typically represent about 60 - 70% of cd25 cells , use 1 ls cell separation column per 10 cells . add cell / bead mixture to ls cell separation column as described in step 2.3.6 collect the cd62l t cells as described in step 2.3.7 for the cd25 cell collection.centrifuge cells at 600 x g for 5 min at 4 c. resuspend cells in 1 ml of r10 and count to determine yield . dilute to 0.75 - 1 x10 cells per ml in r10 medium . since cd62l t cells typically represent about 60 - 70% of cd25 cells , use 1 ls cell separation column per 10 cells . add cell / bead mixture to ls cell separation column as described in step 2.3.6 this time discarding the final flow through and column rinses . collect the cd62l t cells as described in step 2.3.7 for the cd25 cell collection . centrifuge cells at 600 x g for 5 min at 4 c. resuspend cells in 1 ml of r10 and count to determine yield . dilute to 0.75 - 1 x10 cells per ml in r10 medium . analyze purity of cells by fluorescent - activated cell sorting ( facs ) . facs staining strategy for cells pre and post all stages of isolation , stain with cd4-fitc , cd8a - percp - cy5.5 ( helper and cytotoxic t cells ) , and mhcii - pe ( mhc class ii expressing cells).for cells pre and post cd25 isolation , stain with cd4-fitc and cd25-pe ( tregs versus other helper t cells).for cells pre and post cd62l isolation stain with cd4-fitc , cd62l - pe and cd44-apc ( nave versus memory and effector helper t cells ) . for each analysis place 10 cells in a well of a 96 well u - bottom plate . centrifuge as above and pour off dpbs.add 50 l of dpbs per well and 0.5 l of each antibody ( as indicated in 2.4.1 ) and incubate at rt for 30 min in the dark to avoid bleaching of fluorescent label.wash cells 2x with dpbs as in 2.4.2 and immediately analyze on a flow cytometer using protocols and guidelines specific to the instrument at hand . note : after the final isolation step of a good preparation , 90 - 95% of cells will be cd4 cd25 cd62l t cells . facs staining strategy for cells pre and post all stages of isolation , stain with cd4-fitc , cd8a - percp - cy5.5 ( helper and cytotoxic t cells ) , and mhcii - pe ( mhc class ii expressing cells).for cells pre and post cd25 isolation , stain with cd4-fitc and cd25-pe ( tregs versus other helper t cells).for cells pre and post cd62l isolation stain with cd4-fitc , cd62l - pe and cd44-apc ( nave versus memory and effector helper t cells ) . for cells pre and post all stages of isolation , stain with cd4-fitc , cd8a - percp - cy5.5 ( helper and cytotoxic t cells ) , and mhcii - pe ( mhc class ii expressing cells ) . for cells pre and post cd25 isolation , stain with cd4-fitc and cd25-pe ( tregs versus other helper t cells ) . for cells pre and post cd62l isolation stain with cd4-fitc , cd62l - pe and cd44-apc ( nave versus memory and effector helper t cells ) . for each analysis place 10 cells in a well of a 96 well u - bottom plate . centrifuge as above and pour off dpbs . add 50 l of dpbs per well and 0.5 l of each antibody ( as indicated in 2.4.1 ) and incubate at rt for 30 min in the dark to avoid bleaching of fluorescent label . wash cells 2x with dpbs as in 2.4.2 and immediately analyze on a flow cytometer using protocols and guidelines specific to the instrument at hand . note : after the final isolation step of a good preparation , 90 - 95% of cells will be cd4 cd25 cd62l t cells . retroviral transduction note : retroviral integration and expression of genes requires cell division . therefore , t cells must be activated o / n . while isolating the nave t cells , coat a 24 well tissue culture plate with anti - cd3 and anti - cd28 antibodies diluted in dpbs . use anti anti - cd3 at 1g / ml . use anti - cd28 at 2 g / ml if cells will ultimately be differentiated under th0 , th1 , th2 and itregs conditions . incubate ~2 hr at 37 c in a tissue culture incubator.just prior to culturing the cells , remove the antibody solution and wash the plate with ~250 l of dpbs per well to remove unbound antibody . be careful not to let the plate dry out so process a maximum of 6 wells at a time . remove dpbs wash and add 1 ml of nave cd4 t cells in r10 medium at 0.75 - 1 x 10 cells per ml . activate cells o / n ( 14 - 16 hr).the following day , centrifuge cells in the plate at 900 x g for 5 min at 30 c to attach cells to the bottom of the wells.collect and save the medium being careful not to displace the cells , and replace with 1 ml virus culture supernatant prepared from the virus production protocol . do not let the cells dry out so process a maximum of 4 wells at a time . to each well add 1 l of 8 mg / ml polybrene and 10 l of 1 m hepes ph 7.5 to aid virus uptake and prevent significant alkalization in the ambient co2 during the following centrifugation . centrifuge cells in plate at 900 x g for 90 min at 30 c. while isolating the nave t cells , coat a 24 well tissue culture plate with anti - cd3 and anti - cd28 antibodies diluted in dpbs . use anti - cd28 at 2 g / ml if cells will ultimately be differentiated under th0 , th1 , th2 and itregs conditions . use anti - cd28 at 10 g / ml for th9 and th17 conditions . incubate ~2 hr at 37 c in a tissue culture incubator . just prior to culturing the cells , remove the antibody solution and wash the plate with ~250 l of dpbs per well to remove unbound antibody . be careful not to let the plate dry out so process a maximum of 6 wells at a time . remove dpbs wash and add 1 ml of nave cd4 t cells in r10 medium at 0.75 - 1 x 10 cells per ml . activate cells o / n ( 14 - 16 hr ) . the following day , centrifuge cells in the plate at 900 x g for 5 min at 30 c to attach cells to the bottom of the wells . collect and save the medium being careful not to displace the cells , and replace with 1 ml virus culture supernatant prepared from the virus production protocol . do not let the cells dry out so process a maximum of 4 wells at a time . to each well add 1 l of 8 mg / ml polybrene and 10 l of 1 m hepes ph 7.5 to aid virus uptake and prevent significant alkalization in the ambient co2 during the following centrifugation . centrifuge cells in plate at 900 x g for 90 min at 30 c. to each well add 1 l of 8 mg / ml polybrene and 10 l of 1 m hepes ph 7.5 to aid virus uptake and prevent significant alkalization in the ambient co2 during the following centrifugation . centrifuge cells in plate at 900 x g for 90 min at 30 c. differentiation of cells into specific subsets carefully remove the virus culture supernatant and replace with 1 ml of the medium collected above in step 3.1.4 , as it contains il-2 and other t cell growth factors . add reagents indicated in table 2 and culture cells for 3 - 4 days to differentiate cells into specific subsets . carefully remove the virus culture supernatant and replace with 1 ml of the medium collected above in step 3.1.4 , as it contains il-2 and other t cell growth factors . add reagents indicated in table 2 and culture cells for 3 - 4 days to differentiate cells into specific subsets . analysis of cells for intracellular staining of cytokines , treat cells with 1 g / ml each of phorbol 12-myristate 13-acetate ( pma ) and ionomycin for 4 hr . during the last 2 hr of stimulation , add 1 g / ml of brefeldin a.resuspend cells from the bottom of each well by pipetting up and down several times . transfer approximately 10 cells to a 96 well u bottom plate ( usually 100 l from the 1 ml of culture of th cells ) for fixation and staining.for gfp staining , fix cells with 100 l of 2% paraformaldehyde at rt for exactly 5 min , as a longer incubation time will bleach the gfp and interfere with foxp3 staining . wash cells by adding 100 l of dpbs to each well and centrifuge at 600 x g at 22 c for 5 min . caution : paraformaldehyde is toxic . handle it in a fume hood with skin and eye protection.fix the cells again using 100 l 1x fixation buffer made from the foxp3 staining kit ( 1 volume of fixation buffer to 3 volumes of diluent ) . incubate the cells for 45 min at rt or alternatively incubate at 4 c for 16 hr.after fixation , permeabilize cells by adding 100 l of permeabilization buffer from the same kit . incubate at rt for 30 - 45 min then centrifuge at 600 x g at 22 c for 5 min.for antibody staining , remove fixation / permeabilization buffer and add 50 l of fresh permeabilization buffer . add the required antibody diluted in permeabilization buffer at 0.5 l per 50 l buffer per sample . incubate for 30 - 45 min at rt in dark to avoid bleaching of fluorescent label.add 150 l permeabilization buffer and centrifuge at 600 x g at 22 c for 5 min . discard the permeabilization buffer and add 200 l dpbs . analyze on a flow cytometer using protocols and guidelines specific to the instrument at hand . note : be sure to include proper controls for cytokine staining such as staining with isotype control antibodies , analyzing non - activated or th0 activated cells , etc . for intracellular staining of cytokines , treat cells with 1 g / ml each of phorbol 12-myristate 13-acetate ( pma ) and ionomycin for 4 hr . during the last 2 hr of stimulation , add 1 g / ml of brefeldin a. resuspend cells from the bottom of each well by pipetting up and down several times . transfer approximately 10 cells to a 96 well u bottom plate ( usually 100 l from the 1 ml of culture of th cells ) for fixation and staining . for gfp staining , fix cells with 100 l of 2% paraformaldehyde at rt for exactly 5 min , as a longer incubation time will bleach the gfp and interfere with foxp3 staining . wash cells by adding 100 l of dpbs to each well and centrifuge at 600 x g at 22 c for 5 min . caution : paraformaldehyde is toxic . handle it in a fume hood with skin and eye protection . fix the cells again using 100 l 1x fixation buffer made from the foxp3 staining kit ( 1 volume of fixation buffer to 3 volumes of diluent ) . incubate the cells for 45 min at rt or alternatively incubate at 4 c for 16 hr . after fixation , permeabilize cells by adding 100 l of permeabilization buffer from the same kit . incubate at rt for 30 - 45 min then centrifuge at 600 x g at 22 c for 5 min . for antibody staining , remove fixation / permeabilization buffer and add 50 l of fresh permeabilization buffer . add the required antibody diluted in permeabilization buffer at 0.5 l per 50 l buffer per sample . incubate for 30 - 45 min at rt in dark to avoid bleaching of fluorescent label . add 150 l permeabilization buffer and centrifuge at 600 x g at 22 c for 5 min . analyze on a flow cytometer using protocols and guidelines specific to the instrument at hand . note : be sure to include proper controls for cytokine staining such as staining with isotype control antibodies , analyzing non - activated or th0 activated cells , etc . the success of this experimental system requires highly pure populations of t cells and high titer retrovirus preparations . figure 1 shows the typical purity of pre- and post - selected populations at each stage of the nave helper t cell isolation protocol . figure 2 and 3 illustrate the analysis of retrovirus production through gfp expression in the transfected hek 293 t cells ( figure 2 ) and transduced t cells ( figure 3 ) . transfection efficiencies of the hek 293 t cells can vary significantly with different retroviral constructs , but this often does n't correlate with the level of retrovirus production observed with the number of gfp t cells . also , the number of gfp t cells can vary depending on the polarization conditions . furthermore , the mean expression level of gfp and the inserted gene can vary depending on the number of virus copies integrated , the effect of the integration site on transcription , and post - transcriptional regulatory mechanisms affecting the viral transcript . finally , figure 4 shows some typical results we have observed with helper t cell differentiation when the mirnas mir-15b/16 are overexpressed . these results show some of the variability that can occur within an individual experiment so true effects must be substantiated by statistical analysis of multiple repeat experiments using different preparations of helper t cells . in these experiments th2 responses can be difficult to observe in the c57bl/6 line used here because they are prone to th1 responses . therefore , it is imperative to do isotype controls and set up proper compensation to ensure correct gating of cytokine expression . in our results we have found that mir-15b/16 enhances itreg induction by inhibiting the mtor signaling pathway through suppressing the expression of the components rictor and mtor . mir-15b/16 can sometimes influence th0 , th1 , and th17 differentiation in individual experiments , but there is no significant effect when examined in multiple repeat experiments . representative flow cytometry results of the indicated antigens are shown from the gate of live cells designated in the forward scatter ( fsc ) and side scatter ( ssc ) plots . these illustrate the enrichment of helper t cells and the loss of cytotoxic t cells and mhc class ii expressing cells . are the expression profiles of cd4 , cd8a , and mhcii , and on the right are the cd4 and cd25 expression profiles pre and post selection . at this point on the right the expression profiles for cd62l and cd44 are shown for pre- and post - cd62l selected cells along with cd4 and cd62l expression profile of post selected cells . after cd62l selection virtually all memory cells ( cd44 ) are removed leaving a highly enriched population of nave helper t cells that contains 10 - 15% effector cells ( cd62l ) . for all facs profiles , equivalent settings and scales for a specific parameter were maintained throughout . numbers represent the percentage of cells within a gated population . the slight decrease in size of the cells after the initial selection is presumably due to mechanical stress during the protocol . gfp expression is shown in hek 293 t cells that were either untransfected or transfected and analyzed after collection of viral culture supernatants . gfp analysis was done on live cells from the gate on the fsc and ssc plot in the first panel . gfp expression is shown in retroviral - transduced helper t cells after differentiation in th0 , th1 , th2 , th9 , th17 , and treg polarization conditions for three days . analysis was gated on live and activated cells indicated in the fsc / ssc panel . transduction efficiencies can vary between 10 - 75% depending on the construct and the polarization conditions . retroviral mediated overexpression of genes is a powerful way to analyze function in helper t cells , as their development and function is often determined by the expression level of key regulators . however , cautious interpretation of the results is required because expression levels significantly above those of the endogenous gene can introduce many artifacts . therefore , this technique should be combined with others to verify the relevance of function . for example , overexpression should be complemented by reduced expression using sirnas or gene knockouts if available . with mirnas , we complemented overexpression experiments with those of blocking by using viruses that overexpressed artificial mirna targeting sites that acted as competitive inhibitors for a mirna . retroviral transduced cells can also be utilized in biochemical assays involving rna and protein analysis . however , a major limitation of these experiments is efficiency of transduction resulting in a mixed population of transduced and untransduced cells . finally , in vitro differentiation assays should be combined with in vivo experiments , and one way this can be achieved is by adoptively transferring the transduced t cells into mice and following their differentiation and their effect on the immune response . one of the key limitations to this system is the size of the rna genome that can be packaged into the retroviral capsid . in our experience , the maximum insert size for mig retroviral system that gives good virus production is 3 - 3.5 kb . therefore , larger genes can not be analyzed with this system , as they give poor virus titers . however , most genes are smaller than this size so this system is useful for a wide variety of gene studies . with retroviral transduction , several alternatives within these protocols have been used . many researchers have utilized packaging cell lines that stably express the retroviral genes ( for example reference ) . however , we have obtained the highest titers using standard hek 293 t cells with co - transfection of the pcl - eco helper virus vector . isolation of nave helper t cells can also be achieved through cell sorting rather than the magnetic bead and cell separation column protocol , but this requires access to a cell sorter , and the costs for sort time are typically higher than the bead reagents . finally , there are variations on activation conditions used to differentiate helper t cells into the different subsets . for example , tcr stimulation of cells for too long before exposure to treg inducing conditions can inhibit their induction . this can be a problem because retroviral expression requires cell division induced by stimulation of cells . nevertheless , we have found efficient treg induction using this protocol with o / n activation prior to retroviral transduction . within these protocols , high titer retrovirus preparations need efficient transfection of the hek 293 t cells so high quality dna and precisely prepared 2x hbs are important . in addition , the cell density of the hek 293 t cells needs to be roughly 50% at point of transfection because good expression of the transfected dna requires that the cells are actively growing , and this will be inhibited if the cells are too sparse or dense . cells at the optimal density during transfection should reach confluence at some point during the virus collection steps , but they will continue producing high titer virus stocks all the way through to the last collection . efficient differentiation of the helper t cells requires good cell quality so ensure that isolated cells are to the purity illustrated in figure 1 . likewise , the quality of the cells is dependent on the mice from which they were isolated . older mice can have less nave cells , and other strains may differ in their differentiation . for example , balb / c mice are more prone to th2 responses than c57bl/6 mice so as stated above , c57bl/6 t cells can be difficult to induce a th2 response . in addition , any of the differentiation conditions may vary slightly from lab to lab , and the effect of gene overexpression may only become apparent in sub - optimal conditions so cytokine concentrations in the various polarization conditions may need to be titrated . finally , effects of the overexpressed gene or the polarization conditions on cell proliferation can influence the transduction efficiency so measuring effects of the gene of interest may require optimizing the timing and concentration of polarizing reagents .

helper t cell development and function must be tightly regulated to induce an appropriate immune response that eliminates specific pathogens yet prevents autoimmunity . many approaches involving different model organisms have been utilized to understand the mechanisms controlling helper t cell development and function . however , studies using mouse models have proven to be highly informative due to the availability of genetic , cellular , and biochemical systems . one genetic approach in mice used by many labs involves retroviral transduction of primary helper t cells . this is a powerful approach due to its relative ease , making it accessible to almost any laboratory with basic skills in molecular biology and immunology . therefore , multiple genes in wild type or mutant forms can readily be tested for function in helper t cells to understand their importance and mechanisms of action . we have optimized this approach and describe here the protocols for production of high titer retroviruses , isolation of primary murine helper t cells , and their transduction by retroviruses and differentiation toward the different helper subsets . finally , the use of this approach is described in uncovering mechanisms utilized by micrornas ( mirnas ) to regulate pathways controlling helper t cell development and function .

Introduction Protocol 1. Retroviral Production 2. Isolation of Primary Nave CD4 3. Retroviral Transduction of Activated CD4 Representative Results Discussion Disclosures

the immune response must be highly regulated to eliminate infections but prevent attacks on self - tissue that lead to autoimmunity . helper t cells play an essential role in regulating the immune response , and a great deal of effort has been undertaken to understand their development and function ( illustrated in several recent reviews ) . however , many questions remain , and many approaches have been utilized to study the mechanisms controlling helper t cell development and function . these have ranged from the use of in vitro cell culture systems to whole animals . however , they suffer from their limited ability to reproduce the actual conditions occurring in an immune response . nevertheless , the vast majority of helper t cells studies today still require the use of whole animal experiments involving primary t cells because of the inability of cell lines to duplicate the exact steps occurring in the whole animal . therefore , it is essential to utilize cost effective approaches that are highly informative . genetics is one powerful tool to study helper t cell development and function , yet traditional methods involving gene knockouts or transgenes are time consuming and expensive so they are often out of reach of small labs . however , retroviral transduction offers a powerful , rapid and , cost effective genetic approach to study the mechanisms of specific gene products . therefore , it is commonly used in papers studying helper t cell development and function . the vector was originally derived from the murine stem cell virus ( mscv ) vectors , which contain mutations in repressor binding sites in the ltrs making them resistant to silencing and thus , giving high expression in many cell types including helper t cells . production of high titer retrovirus requires a simple transient transfection protocol of human embryonic kidney ( hek ) 293 t cells with the mig vector and a helper virus vector that expresses the retroviral gag , pol , and env genes . here these protocols for retroviral production and transduction of primary murine t cells are described in addition to some of our results using this approach to study mirna regulation of gene expression controlling helper t cell differentiation . in helper t cells mirnas play multiple roles and are required for regulatory t cell ( treg ) development . we used retroviral transduction as a means to dissect the mechanisms of mirna regulation of treg differentiation . through such studies important subsequently , relevant genes regulated by these mirnas were identified in order to understand the molecular pathways regulated by mirnas in helper t cell differentiation . prior to proceeding obtain all required approvals for producing genetically modified organisms and the use of retroviruses in mammalian cells . growth of hek 293 t cells grow hek 293 t cells on 10 cm tissue culture dishes in hek 293 t medium ( dulbecco 's modified eagle 's medium ( dmem ) with 10% fetal bovine serum ( fbs ) , 100 units / ml penicillin 0.1 mg / ml streptomycin , and 2 mm l - glutamine ) . grow hek 293 t cells on 10 cm tissue culture dishes in hek 293 t medium ( dulbecco 's modified eagle 's medium ( dmem ) with 10% fetal bovine serum ( fbs ) , 100 units / ml penicillin 0.1 mg / ml streptomycin , and 2 mm l - glutamine ) . this is the virus stock.spin out any residual hek 293 t cells and cellular debris by centrifuging at 600 x g for 5 min . therefore , the exact timing of each batch of red cell lysis buffer will need to be optimized.add 12 - 13 ml of r10 medium . therefore , the exact timing of each batch of red cell lysis buffer will need to be optimized . isolation of nave cd4 t cells ( cd25 and cd62l ) using cell separation columns centrifuge cd4 t cells at 600 x g for 5 min at 4 c and resuspend in 150 - 200 l of r10 medium per 10 cells . each ms column retains 10 cells , and since cd25 cells typically represent about 10% of total cd4 cells , use 1 column per 10 cd4 t cells . resuspend cells in 500 l of cell separation column buffer per 10 cells , but still use 500 l if there are fewer cells.apply 500 l of cell / bead suspension to the column and collect the flow through . to obtain cd25 cd62l t cells , centrifuge the cd25 t cells isolated above in step 2.3.6 at 600 x g for 5 min at 4 and resuspend in 150 - 200 l of r10 medium per 10 cells . since cd62l t cells typically represent about 60 - 70% of cd25 cells , use 1 ls cell separation column per 10 cells . collect the cd62l t cells as described in step 2.3.7 for the cd25 cell collection.centrifuge cells at 600 x g for 5 min at 4 c. resuspend cells in 1 ml of r10 and count to determine yield . each ms column retains 10 cells , and since cd25 cells typically represent about 10% of total cd4 cells , use 1 column per 10 cd4 t cells . to obtain cd25 cd62l t cells , centrifuge the cd25 t cells isolated above in step 2.3.6 at 600 x g for 5 min at 4 and resuspend in 150 - 200 l of r10 medium per 10 cells . since cd62l t cells typically represent about 60 - 70% of cd25 cells , use 1 ls cell separation column per 10 cells . add cell / bead mixture to ls cell separation column as described in step 2.3.6 collect the cd62l t cells as described in step 2.3.7 for the cd25 cell collection.centrifuge cells at 600 x g for 5 min at 4 c. resuspend cells in 1 ml of r10 and count to determine yield . since cd62l t cells typically represent about 60 - 70% of cd25 cells , use 1 ls cell separation column per 10 cells . facs staining strategy for cells pre and post all stages of isolation , stain with cd4-fitc , cd8a - percp - cy5.5 ( helper and cytotoxic t cells ) , and mhcii - pe ( mhc class ii expressing cells).for cells pre and post cd25 isolation , stain with cd4-fitc and cd25-pe ( tregs versus other helper t cells).for cells pre and post cd62l isolation stain with cd4-fitc , cd62l - pe and cd44-apc ( nave versus memory and effector helper t cells ) . facs staining strategy for cells pre and post all stages of isolation , stain with cd4-fitc , cd8a - percp - cy5.5 ( helper and cytotoxic t cells ) , and mhcii - pe ( mhc class ii expressing cells).for cells pre and post cd25 isolation , stain with cd4-fitc and cd25-pe ( tregs versus other helper t cells).for cells pre and post cd62l isolation stain with cd4-fitc , cd62l - pe and cd44-apc ( nave versus memory and effector helper t cells ) . for cells pre and post all stages of isolation , stain with cd4-fitc , cd8a - percp - cy5.5 ( helper and cytotoxic t cells ) , and mhcii - pe ( mhc class ii expressing cells ) . for cells pre and post cd25 isolation , stain with cd4-fitc and cd25-pe ( tregs versus other helper t cells ) . for cells pre and post cd62l isolation stain with cd4-fitc , cd62l - pe and cd44-apc ( nave versus memory and effector helper t cells ) . therefore , t cells must be activated o / n . while isolating the nave t cells , coat a 24 well tissue culture plate with anti - cd3 and anti - cd28 antibodies diluted in dpbs . activate cells o / n ( 14 - 16 hr).the following day , centrifuge cells in the plate at 900 x g for 5 min at 30 c to attach cells to the bottom of the wells.collect and save the medium being careful not to displace the cells , and replace with 1 ml virus culture supernatant prepared from the virus production protocol . centrifuge cells in plate at 900 x g for 90 min at 30 c. while isolating the nave t cells , coat a 24 well tissue culture plate with anti - cd3 and anti - cd28 antibodies diluted in dpbs . the following day , centrifuge cells in the plate at 900 x g for 5 min at 30 c to attach cells to the bottom of the wells . collect and save the medium being careful not to displace the cells , and replace with 1 ml virus culture supernatant prepared from the virus production protocol . prior to proceeding obtain all required approvals for producing genetically modified organisms and the use of retroviruses in mammalian cells . growth of hek 293 t cells grow hek 293 t cells on 10 cm tissue culture dishes in hek 293 t medium ( dulbecco 's modified eagle 's medium ( dmem ) with 10% fetal bovine serum ( fbs ) , 100 units / ml penicillin 0.1 mg / ml streptomycin , and 2 mm l - glutamine ) . grow hek 293 t cells on 10 cm tissue culture dishes in hek 293 t medium ( dulbecco 's modified eagle 's medium ( dmem ) with 10% fetal bovine serum ( fbs ) , 100 units / ml penicillin 0.1 mg / ml streptomycin , and 2 mm l - glutamine ) . this is the virus stock.spin out any residual hek 293 t cells and cellular debris by centrifuging at 600 x g for 5 min . isolation of leukocyte cells euthanize mice by cervical dislocation , co2 asphyxiation , or other humane protocol appropriate to the animal handling rules of the institution.dissect freshly euthanized mice and remove spleen and desired lymph nodes . therefore , the exact timing of each batch of red cell lysis buffer will need to be optimized.add 12 - 13 ml of r10 medium . therefore , the exact timing of each batch of red cell lysis buffer will need to be optimized . isolation of cd4 t cells using magnetic beads kit to remove cd8 , cd11b , cd16/32 , cd45r , mhc class ii and ter-119 cells . isolation of nave cd4 t cells ( cd25 and cd62l ) using cell separation columns centrifuge cd4 t cells at 600 x g for 5 min at 4 c and resuspend in 150 - 200 l of r10 medium per 10 cells . each ms column retains 10 cells , and since cd25 cells typically represent about 10% of total cd4 cells , use 1 column per 10 cd4 t cells . resuspend cells in 500 l of cell separation column buffer per 10 cells , but still use 500 l if there are fewer cells.apply 500 l of cell / bead suspension to the column and collect the flow through . to obtain cd25 cd62l t cells , centrifuge the cd25 t cells isolated above in step 2.3.6 at 600 x g for 5 min at 4 and resuspend in 150 - 200 l of r10 medium per 10 cells . collect the cd62l t cells as described in step 2.3.7 for the cd25 cell collection.centrifuge cells at 600 x g for 5 min at 4 c. resuspend cells in 1 ml of r10 and count to determine yield . each ms column retains 10 cells , and since cd25 cells typically represent about 10% of total cd4 cells , use 1 column per 10 cd4 t cells . to obtain cd25 cd62l t cells , centrifuge the cd25 t cells isolated above in step 2.3.6 at 600 x g for 5 min at 4 and resuspend in 150 - 200 l of r10 medium per 10 cells . since cd62l t cells typically represent about 60 - 70% of cd25 cells , use 1 ls cell separation column per 10 cells . add cell / bead mixture to ls cell separation column as described in step 2.3.6 collect the cd62l t cells as described in step 2.3.7 for the cd25 cell collection.centrifuge cells at 600 x g for 5 min at 4 c. resuspend cells in 1 ml of r10 and count to determine yield . since cd62l t cells typically represent about 60 - 70% of cd25 cells , use 1 ls cell separation column per 10 cells . collect the cd62l t cells as described in step 2.3.7 for the cd25 cell collection . facs staining strategy for cells pre and post all stages of isolation , stain with cd4-fitc , cd8a - percp - cy5.5 ( helper and cytotoxic t cells ) , and mhcii - pe ( mhc class ii expressing cells).for cells pre and post cd25 isolation , stain with cd4-fitc and cd25-pe ( tregs versus other helper t cells).for cells pre and post cd62l isolation stain with cd4-fitc , cd62l - pe and cd44-apc ( nave versus memory and effector helper t cells ) . facs staining strategy for cells pre and post all stages of isolation , stain with cd4-fitc , cd8a - percp - cy5.5 ( helper and cytotoxic t cells ) , and mhcii - pe ( mhc class ii expressing cells).for cells pre and post cd25 isolation , stain with cd4-fitc and cd25-pe ( tregs versus other helper t cells).for cells pre and post cd62l isolation stain with cd4-fitc , cd62l - pe and cd44-apc ( nave versus memory and effector helper t cells ) . for cells pre and post all stages of isolation , stain with cd4-fitc , cd8a - percp - cy5.5 ( helper and cytotoxic t cells ) , and mhcii - pe ( mhc class ii expressing cells ) . for cells pre and post cd25 isolation , stain with cd4-fitc and cd25-pe ( tregs versus other helper t cells ) . for cells pre and post cd62l isolation stain with cd4-fitc , cd62l - pe and cd44-apc ( nave versus memory and effector helper t cells ) . therefore , t cells must be activated o / n . while isolating the nave t cells , coat a 24 well tissue culture plate with anti - cd3 and anti - cd28 antibodies diluted in dpbs . activate cells o / n ( 14 - 16 hr).the following day , centrifuge cells in the plate at 900 x g for 5 min at 30 c to attach cells to the bottom of the wells.collect and save the medium being careful not to displace the cells , and replace with 1 ml virus culture supernatant prepared from the virus production protocol . centrifuge cells in plate at 900 x g for 90 min at 30 c. while isolating the nave t cells , coat a 24 well tissue culture plate with anti - cd3 and anti - cd28 antibodies diluted in dpbs . the following day , centrifuge cells in the plate at 900 x g for 5 min at 30 c to attach cells to the bottom of the wells . collect and save the medium being careful not to displace the cells , and replace with 1 ml virus culture supernatant prepared from the virus production protocol . the success of this experimental system requires highly pure populations of t cells and high titer retrovirus preparations . figure 1 shows the typical purity of pre- and post - selected populations at each stage of the nave helper t cell isolation protocol . also , the number of gfp t cells can vary depending on the polarization conditions . furthermore , the mean expression level of gfp and the inserted gene can vary depending on the number of virus copies integrated , the effect of the integration site on transcription , and post - transcriptional regulatory mechanisms affecting the viral transcript . finally , figure 4 shows some typical results we have observed with helper t cell differentiation when the mirnas mir-15b/16 are overexpressed . these results show some of the variability that can occur within an individual experiment so true effects must be substantiated by statistical analysis of multiple repeat experiments using different preparations of helper t cells . these illustrate the enrichment of helper t cells and the loss of cytotoxic t cells and mhc class ii expressing cells . after cd62l selection virtually all memory cells ( cd44 ) are removed leaving a highly enriched population of nave helper t cells that contains 10 - 15% effector cells ( cd62l ) . gfp expression is shown in retroviral - transduced helper t cells after differentiation in th0 , th1 , th2 , th9 , th17 , and treg polarization conditions for three days . retroviral mediated overexpression of genes is a powerful way to analyze function in helper t cells , as their development and function is often determined by the expression level of key regulators . finally , in vitro differentiation assays should be combined with in vivo experiments , and one way this can be achieved is by adoptively transferring the transduced t cells into mice and following their differentiation and their effect on the immune response . however , we have obtained the highest titers using standard hek 293 t cells with co - transfection of the pcl - eco helper virus vector . isolation of nave helper t cells can also be achieved through cell sorting rather than the magnetic bead and cell separation column protocol , but this requires access to a cell sorter , and the costs for sort time are typically higher than the bead reagents . finally , there are variations on activation conditions used to differentiate helper t cells into the different subsets . nevertheless , we have found efficient treg induction using this protocol with o / n activation prior to retroviral transduction . within these protocols , high titer retrovirus preparations need efficient transfection of the hek 293 t cells so high quality dna and precisely prepared 2x hbs are important . in addition , the cell density of the hek 293 t cells needs to be roughly 50% at point of transfection because good expression of the transfected dna requires that the cells are actively growing , and this will be inhibited if the cells are too sparse or dense . cells at the optimal density during transfection should reach confluence at some point during the virus collection steps , but they will continue producing high titer virus stocks all the way through to the last collection . efficient differentiation of the helper t cells requires good cell quality so ensure that isolated cells are to the purity illustrated in figure 1 . older mice can have less nave cells , and other strains may differ in their differentiation . for example , balb / c mice are more prone to th2 responses than c57bl/6 mice so as stated above , c57bl/6 t cells can be difficult to induce a th2 response . in addition , any of the differentiation conditions may vary slightly from lab to lab , and the effect of gene overexpression may only become apparent in sub - optimal conditions so cytokine concentrations in the various polarization conditions may need to be titrated .

despite greater public and professional awareness of sexual and gender diversity and more positive work climates in recent years , lesbian , gay , bisexual , transgender , and queer ( lgbtq ) people continue to encounter invisibility , overt and more subtle discrimination and violence , and other barriers to relevant , respectful care . according to the registered nurses association of ontario ( rnao ) , sexual diversity relates to sexual orientation ( lesbian , gay , bisexual , and heterosexual ) and gender diversity refers to gender identity ( e.g. , transgender ) which refers to one 's sense of being male and/or female or neither . this last decade has brought greater attention to transgender health , the t in lgbtq , in health care and research has shown that ethnicity , race , geography , age , education , and other social categories across genders influence how particular trans - identified individuals experience and understand their health . typically , transgender is used in the broadest sense to refer to people who transgress gender norms in some way . in this paper , [ t]rans refers to transgender , transsexual , gender nonconforming , and gender questioning ( people ) and transmen to those who identify with a masculine trans identity . within trans communities , these include transmen , transwomen , and genderqueer , and trans identities may shift over a lifetime . gender expression and/or transition processes can involve dress , electrolysis , and/or sex reassignment surgery . encounters with violence , transphobia , and discrimination related to sexuality and gender norms across home , community , work , and educational settings , and challenges to finding meaningful social support , shape trans people 's disclosure with respect to trans issues and identities , their health status , and access to care [ 57 ] . considerable hurdles also exist to locating sensitive and knowledgeable health providers , trans - positive services and obtaining hormone therapy or surgery if so desired , especially outside of large urban cities [ 6 , 8 ] . all of these factors contribute to trans people 's vulnerability to health inequities . as social determinants of health , employment , and working conditions for trans people both shape and are shaped by multiple factors such as support networks , social environments , income and social status , shelter , and personal health practices [ 6 , 7 , 9 , 10 ] . decisions to transition in the workplace are informed by many factors that include underemployment , available support systems , and weighing the costs of job - loss or unpredictable discrimination that mark many environments for trans people [ 8 , 9 , 11 ] . certainly income level and access to education lgbtq workplace issues have recently become high profile in north american media in relation to same - sex benefits , legal protections , and organizational policy and diversity education initiatives , and guides to support transition in the workplace are becoming increasingly visible [ 10 , 12 , 13 ] . yet , workplace support for gender - variant individuals and those who self - identify on the trans spectrum remains highly politicized , even within lgbtq communities . in canada , inclusive policy change related to diversity is part of broader social policy and health professional advocacy for equity ; nursing in particular has taken leadership in a canadian context of the professions . however , the policy context in which discrimination occurs for trans people is still fraught . in ontario , canada , findings from the trans pulse project indicate that there is little known about trans people 's work , although it is well documented that underemployment and barriers to education exist . many are forced to leave education before completing high school due to experiences of harassment , bullying , and violence , yet there is some evidence that despite this , many are able to achieve some level of higher education [ 8 , 10 ] . narratives published in print , or online , offer insight into the dynamics of work and health and the complexities of diverse trans people 's lived experiences of gender expression and transitioning in a canadian context [ 1416 ] . in 2008 , the canadian professional association for transgender health ( cpath ) also emerged to offer resources and professional support for those involved in trans health provision , creating a community for trans - identified providers and allies to be visible in a professional context . however , few trans - identified members of the regulated health professions ( e.g. , nurses ) are visible in the health sector and there is limited research that focuses on their work , and even less on their career trajectories , especially in the professional context ( this includes both alternative and regulated health providers ) ( e.g. , ) . yet , gender and its intersections with race , sexuality , and other social categories inform a body of research on professionals and the nature of their work ; dominant notions of masculinities and femininities as well as gender conformity have also been noted as relevant to lgbtq health professionals ' career / work dynamics ( e.g. , [ 1820 ] ) . as a result , research that fosters an understanding of factors that influence the career , work , and health of diverse trans providers is needed to illustrate the complexities of their lives with the goal of creating supportive policies and practices . in a nursing context , recent attention to the need to broaden understandings of culturally competent care in order to meet the diverse and holistic needs of patients and clients has pointed to the importance of addressing sexual diversity , and to some extent , transgender health [ 2124 ] . yet , when this is addressed , most health studies , including nursing research , focus on the context in which trans people are assumed to be patients or clients . by default , the assumption is that all care providers are not trans - identified , but cisgendered ( a term used to describe people who are not transgendered ) and this has important consequences for the ways in which the health issues of diverse trans people are identified and articulated , and thus the practice strategies that are deemed relevant to supporting them . in fact , a search of the literature using cinahl , pubmed , social science abstracts , and psychinfo databases using the terms transgender and/or transsexual and work in titles and abstracts yielded a handful of articles that focus on transgender or transsexual people and their workplace experiences . typically across the health literature , transgender people and work get taken up in the context of sex work , sexual risk for hiv / aids and/or in relation to how providers a recent study suggests that such an approach erases the complexity of trans people 's lives [ 22 , 24 ] . a paucity of workplace - focused health literature exists that offers insight into trans people 's workplaces in relation to experiences of transitioning , and/or relevant policy dynamics ; in effect , their lives are equated with a patient context rather than a work context which has limitations to understanding health in a social determinants approach . this paper foregrounds the experiences of health providers who themselves are trans - identified and illustrates the links in their work , careers and health and wellbeing , and the complex ways that gender is implicated in their lives . in particular , this paper focuses on how transmen who are health providers make meaning through their careerlives , that is , meaningful life activities encompassing work and personal endeavors , with implications for their health and wellbeing . this paper explores intersections of gender , health and work or career for transmen health providers in relation to their everyday lived realities . in particular , this paper explores two research questions.how do transmen who are health providers make meaning through their careerlives?what are the implications for their health and wellbeing ? how do transmen who are health providers make meaning through their careerlives ? this study in the naturalistic and critical paradigms was appropriate for this exploratory study that examined sensitive dynamics of identity , work and career , and complex aspects of power . this life history approach attends to processes of narration , collaboration , and contextualization so that the researcher has an active role in situating participants ' narratives in the larger sociocultural , political , economic , and historical contexts . critical gender analysis of career histories , within - case and cross - case analysis , can also identify critical incidents and influences , such as identity , that shape diverse professionals ' careerlives . in this approach to policy research , the goal is to generate understandings of policy and related processes and practices through the eyes of policy actors themselves in the field . for trans professionals and health providers whose work lives are shaped by policy and whose work entails practices of implementing and creating policy , their lives as trans people are also inscribed by human rights and organizational policies that shape the ways in which trans people are variously included , marginalized , and/or legitimized in their work and larger social worlds [ 22 , 28 ] . in this study , careers are conceptualized as moving beyond the organizational context to encompass meaningful life activities across the lifespan . gender - based analysis in this paper , addresses both sex , biological differences between women and men , and gender , which takes into account the social dynamics or gender roles , identities , and norms which shape individuals ' holistic health . historically , sex has referred to biological characteristics that were used to determine discrete binary categories of female or male . however , trans people 's attention to and experiences of gender variance and the ways that their biological characteristics are at odds with their sense of being male , female , and/or expressing their gender , blur the discrete boundaries of sex and gender . for transgender people , for example , binary categories of male and female are inadequate to represent the fluid and socially constructed nature of sex and gender . to understand their health requires simultaneous attention to the ways that gender norms influence gender expression and identities [ 22 , 30 ] . rather than an exclusive focus on health risks , a critical gender - based approach foregrounds trans people 's agency , the ways that they negotiate their social worlds in particular times and places , and material and social dynamics characterizing their careerlives and the ways that gender intersects with work and other social determinants . it also encompasses how both material ( e.g. , physical embodiment ) and social dimensions ( e.g. , identities , norms ) are gendered . although gender - based analysis has typically been used to examine how gender matters in the lives of cisgendered women and men , johnson et al . explain that it could be applied to understand transgender / transsexual issues , and more recently it has been taken up in health research on trans people [ 21 , 22 ] . in this study , we incorporate this gender lens from the outset of the study , mindful of the ways that this methodology influences the way the study is conceptualized and questions asked , the data collection , analysis , and sharing of findings . as a type of feminist research , there is also an assumption that the research has political goals with an aim to improve the everyday lives of transgender people . ethics approval from york university research ethics board was received prior to commencing the study . at that point , a call for advisory group members for the study was sent out , and two trans - identified community members responded and provided feedback on the study materials and procedures . these two community members represented transwomen 's and transmen 's communities and had insight into health and social service delivery as providers in paid and/or volunteer roles ; they opted not to be interviewed , instead showing their support for this study in this advisory capacity . materials were revised with respect to the language used in the consent forms / recruitment material and the way that the researcher specified how she was situated with respect to the trans communities . the principal researcher , a health professional , conceptualized the study and was involved with recruitment , data collection , analysis and presentation of initial findings . the authors of this article , both of whom been involved with lgbtq advocacy and research , collaborated on analysis and developed this article . the call out for participants was for individuals who self - identified as trans health providers working in the health and social services sector initially in ontario ; and at a later date , recruitment was expanded to include those working in canada . thus the study aimed to recruit both health and social service providers who were self - identified as trans ( e.g. , transgender , transsexual , two - spirited , genderqueer ) and cisgendered providers who were involved in service delivery for trans communities and thus were key informants . this paper , however , reports on the trans - identified providers and specifically , trans providers who self - identified as transmen . this included rainbow health ontario and rainbow health network online listserves which have broad reach to gender diverse communities and health providers throughout canada , as well as the researcher 's well - developed professional networks that included a focus on health equity - related research . the interviews focused on career histories , everyday activities , experiences of health and work , as well as factors that shaped career and workplace decisions and practice dynamics . interview questions were created for the study based on the literature and the principal researcher 's engagement with issues that trans health communities had previously identified as relevant . interview questions focused on the everyday lived experiences and challenges shaping many trans people 's worklives : gender presentation , transitioning , health concerns , discrimination , violence , underemployment , or unemployment , and social support . for example , the researcher asked about a typical day in their life as a trans - identified health provider , pivotal incidents , or key influences that played a part in their health , as well as workplace dynamics , policies , or issues that would contribute to trans providers ' health and wellbeing and their vision for strategies that could improve practice environments for trans providers . the advisory group affirmed the focus of these questions in relation to the purpose and scope of the project . face - to - face or telephone semi - structured interviews were completed from april 2008 to april 2010 with four participants who self - identified as transmen . probes were used to encourage participants to provide thick description and elaboration on the context of points raised . this was especially relevant given the small pool of out trans - identified people in professional positions whose identification could have enormous personal and professional implications , including career risks . conventional content analysis was chosen as an inductive approach , a method that is appropriate for examining qualitative data for a study that is exploratory and whose aim is to describe a phenomenon which is not well understood . analysis of descriptive data emerging from the interviews was guided by the gender - based analytical approach with its focus , for example , on social dynamics , gender norms , and experiences of gender variance . both researchers independently coded the transcripts for themes and subthemes that were guided by an understanding of gender dynamics ( e.g. , the notion that both material and social factors are relevant to worklives ) . however , the researchers were also attuned to unexpected themes that emerged , such as some participants ' experiences of congruence between their work interest and their social identities as members of trans communities . transcripts were read as a whole for immersion in the data and then line - by - line coding was carried out in which key phrases were identified as initial themes and notations made with researchers ' reflections on emerging codes , allowing the categories and names for categories to flow from the data ( , page 1279 ) . where possible , verbatim phrases were used as codes . for example gender identity as career " based on one participant saying , being able to transcend the limits of um physicality my gender identity is my career in a sense or vice versa you know . this code later became part of the theme fitting in " . under the theme of critical influences , we identified a subcategory : feminist work that influenced me most in terms of identifying as queer and coming out as queer . researchers identified themes , codes , and subcategories which were later clustered in a coding scheme . the two researchers shared these initial themes , which were then validated using consensus process and were informed by the existing literature . when there was disagreement on emerging themes , discussion ensued with return to the transcripts until the researchers reached consensus . thus the way gender - based analysis was used in this study considers the ways that gender normativity provides a reference point for understanding the experience of people who do not fit into the binary categories of male and female . as well , it affirms the unique experiences and identities of diverse trans people who share the common experience of knowing themselves to be a gender that is not congruent with their birth sex paper , analysis focuses on the career histories of four health providers who self - identified as transmen . these participants , who ranged in age from their 20s to their 50s , had varied gender histories and experiences from those who had transitioned recently , to those who had done so over 10 years previously . the narratives represented experiences of students in professional and alternative health programs and providers with decades of work experience , some also with international experience . professional work and/or education over their lifetimes included social work , medicine , nursing , midwifery , naturopathy , massage therapy , as well as other care work such as teaching . because of the extremely small pool of openly trans - identified health providers and specifically those with professional credentials in a canadian context , narrative excerpts and findings are reported in this paper in ways that will not identify participants ; information such as age , profession , and place of work are omitted . tm identifies the participant as a transman , and a page number from the transcript is cited . in this paper , we report on themes that represent how participants articulated gender identities in the context of work , their careerlives and larger lives , so that the focus is on meaning - making through gender / transition and doing gender in their work and other meaningful life activities . themes relate to their engagement with their trans identities and careerlives with implications for fostering or challenging their health and wellbeing . all participants described themselves in ways that identified themselves as transmen . over their lifetimes however , they reported quite different identities , which , in part , shaped and were shaped by their particular engagement with gender identities , transition processes , and other ways in which gender factored into their social worlds . for instance , deeply entrenched homophobia , heterosexism , and biphobia contribute to gender norms and invisibility , lack of awareness , and transphobia as factors that hinder diverse trans people 's ability to locate respectful , responsive , and meaningful health care in their lives [ 7 , 22 ] . these narratives point to the ways that such dynamics can also influence their narratives of becoming aware of a masculine trans identity as relevant to their wellbeing . all four participants shared that their engagement with a trans identity occurred as adults , with three having also incorporated a same - sex identity for most , or part of their teenaged years . for instance , while coming out and exploring his bisexual and queer identities in his late teens , one of the participants noted that hemet a very different gendered world . at first i did n't really want to do it , but it was very helpful in the end , because i did n't feel that pressure to conform to that gender world anymore , so i could come out and go out to something more myself in a different way . ( tm , page 18 ) . met a very different gendered world . at first i did n't really want to do it , but it was very helpful in the end , because i did n't feel that pressure to conform to that gender world anymore , so i could come out and go out to something more myself in a different way . ( tm , page 18 ) . another described the initial trigger not from becoming attracted to someone , which i think is not the usual way just the access to those kinds of books , or the encouragement to read books that were exploring gender identities and sexuality this awareness emerged in more gradual and complex ways:who am i really ? and what choices can i make to be more at ease with myself ? i do n't want to be a woman like this anymore , because it 's not working for me people are rejecting me as i am and i 'm being rejected by my partner and i ca n't be a mother in the way i want to because of my career . and i started to think , well maybe i was just made atypically , you know . who am i really ? and what choices can i make to be more at ease with myself ? i do n't want to be a woman like this anymore , because it 's not working for me people are rejecting me as i am and i 'm being rejected by my partner and i ca n't be a mother in the way i want to because of my career . and i started to think , well maybe i was just made atypically , you know . ( tm , page 26 ) . in each case , taking on a trans identity was incredibly affirming for these participants on a personal level , even as they encountered unpredictable challenges for disclosure and gender expression , which prompted both feelings of elation when things were working well and also at times , deeply wrenching emotional upheaval . moreover , their narratives illustrated how their education into a profession and/or workplace context was linked to these dynamics . as one noted , the journey of becoming queer - identified led me into the health sector was wanting to take care of my own health ( tm , page 4 ) , while another suggested that trans people definitely have a better chance of making it in health care or social services . having a trans- [ identified ] worker would make sense for them , but you know that is not common ( tm , page 14 ) . in these ways , transmen in this study proposed that their careerlives and career choices represented a holistic , or spiritual component rooted in their gendered identities and their experiences of experiencing marginalization in social institutions . financial factors surfaced for some participants who acquired debts from education or weighed transition - related costs on top of living and/or education expenses . working in contract positions or in alternative health contexts , which were not publicly funded , prompted some to take on heavy workloads and juggle several jobs both in health or research and those that were not health - related . such pressures exacerbated participants ' everyday stresses . at times , the connection between work and identity was complicated by the material consequences of transitioning . pressures to conform to rigid gendered expectations for roles and appearance carried over to their presentation in professional contexts and the unease that they experienced . as one participant shared , part of his transition included a gradual change in dress and appearance while working and this visibility in relation to his gender expression was an important step towards enhancing his psychological wellbeing:so , i kind of on the one hand rejected traditional womanhood i suppose and i started to dress in a way that made me feel comfortable for the first time . i cut my hair and i started to feel much better ( tm , page 27 ) . womanhood i suppose and i started to dress in a way that made me feel comfortable for the first time . another participant pointed out some of the challenges of hormonally transitioning while working , indicating that he often avoided interactions with his peers because his voice was not yet low enough for him to feel confident that he would n't be singled out , noting , i did n't want them to notice me , basically , and i definitely did n't want them to think poorly of me another related that while commuting to work during the transition period he experienced negative incidents riding the bus : like the bus driver being rude , you know , or interacting with someone who 's being transphobic . further , he added that while , wearing pants to work was a blessing when transitioning from female to male generally , but having to ride a bike to work to save money presented issues when binding his breasts . several participants also shared stories of the struggle to get registered as a working professional or to apply to a professional program . in each case , this professional context was a turning point for the provider . working and transitioning , whether as a credentialed professional or professional - in - training , meant that these transmen had to worry about how to change their documentation and professional registration , in addition to dealing with the multiple decisions related to , and demands of , disclosing ( or not ) and living and working as a transman in various community and work contexts . as a result of this , application to professional schools and professional bodies was fraught . as one participant shared , the struggle to get registered was like a warning of how hard it was going to be another participant justified the timing of changing his name at the beginning of his professional career having suddenly realized that , you start meeting [ colleagues ] you 'll be working with people who you 'll want the support of for the rest of your career . i should be ready to go as the person that i want to be for the rest of my life another explained the complicated nature of having to determine how and when to disclose professional education and experiences obtained pretransition:before , i was , you know , passing when i was first transitioning . what i struggled with was when to come out . because if you put it on your cover letter you might not get called , but if you do n't address it fairly on when you show up for the interview than they sort of look at you and they call your references . and especially in social health care your reputation is , like , being able to verify your character is a very important part of people being able to hire you , right ? before , i was , you know , passing when i was first transitioning . what i struggled with was when to come out . because if you put it on your cover letter you might not get called , but if you do n't address it fairly on when you show up for the interview than they sort of look at you and they call your references . and especially in social health care your reputation is , like , being able to verify your character is a very important part of people being able to hire you , right ? ( tm , page 8) . in response to the question , why did n't you use your legal name ? by an administrator , one participant reported that he disclosed being trans , saying , well i 've been like transitioning , not physically , but i 'm in the process of questioning my gender identity and i feel more comfortable with [ chosen name ] ( tm , page 28 ) . although such processes were eventually resolved for this participant , disclosure of trans identity had significant negative impacts for other participants . two participants reported that disclosure engendered larger questions about their credibility , mental health , maturity , and fit for the profession itself . one of the participants described that hehad a meeting with support [ staff who ] asked me if this was the right time for me to be in [ professional ] school until i had sorted out my gender and like until i could pass as male , did i really think that this was the right time for me to be here ? had a meeting with support [ staff who ] asked me if this was the right time for me to be in [ professional ] school until i had sorted out my gender and like until i could pass as male , did i really think that this was the right time for me to be here ? at times , the struggle to disclose or provide professional documentation was avoided by changing their legal name only and deferring gender transition until later . participating transmen also reported having to move jobs and/or change careers to be able to work in their chosen genders . one participant further illustrated that even in a professional school environment that offered the potential of strong group support , disclosure and transition were not straightforward:i was coming out in my personal life in my second to last semester and so then in my last semester i was doing my practicum and i was n't quite confident to share that with the whole school . it seems like that was too much outing to deal with at once and i knew that they would have to process it because we were social workers . i was coming out in my personal life in my second to last semester and so then in my last semester i was doing my practicum and i was n't quite confident to share that with the whole school . it seems like that was too much outing to deal with at once and i knew that they would have to process it because we were social workers . those affiliated with mainstream education programs or work settings such as hospitals were thus likely to be the sole trans - identified provider in their workspaces . professional isolation in particular , emerged in several career histories , with experiences of physical and mental distress as a result . this was a pervasive dynamic for at least one participant in several workplaces , exacerbated by the nature of the precarious work and related working conditions , and by the need to move to different job locations in order to find work and support the family : it was just an incredible experience just feeling utterly alone through the whole year . the experience of transitioning at work or in educational contexts meant that at times trans people 's health suffered with the potential for adverse outcomes . one spoke of a long period of recovery after several spells of serious illness which made it impossible for him to work : with no sick benefits [ or ] money to tide me over . i was coming up against the health care system and not being able to get started with transition and then being all stressed out at work and obviously in my personal life as well going with lots of stuff at the beginning of transition . i was coming up against the health care system and not being able to get started with transition and then being all stressed out at work and obviously in my personal life as well going with lots of stuff at the beginning of transition . participants spoke highly of allies they encountered in professional and educational contexts , who often , but not always , were members of lgbtq communities . this played out in various ways , where individual mentors and champions advocated on their behalf , provided important emotional support , challenged systems to become more responsive to their needs and provided role modeling in clinical placements where they could engage with trans health . participants in particular identified that being trans and working in health care meant that education and coming out were constant worries or struggles , and that employers who supported them through medical transition were highly valued . , some of the participants could also tap into lgbtq professional networks in their work and educational settings , which relived some of the isolation and marginalization that they felt:it really kept it all together for me in school because it can be overwhelming and it 's isolating to feel like you 're the only person who is like you . you know , there 's no other trans people that i 've encountered working . so , going to [ a lgbtq - positive agency ] really just re - energized me for the rest of the year . it really kept it all together for me in school because it can be overwhelming and it 's isolating to feel like you 're the only person who is like you . you know , there 's no other trans people that i 've encountered working . so , going to [ a lgbtq - positive agency ] really just re - energized me for the rest of the year . yet , their career histories showed that such support and resources were not always consistent , even within the same organization , and this created significant anxiety either throughout their experience , or at particular points . this prompted one participant to take action using a formal complaint process , despite having a supportive manager , remarking that his colleagues were making inappropriate remarks towards me . i 'm getting upset and that 's not fair ( tm , page 5 ) . transitioning at school , or living as a transman outside of work , also posed challenges in intensive programs where students were immersed in environments with professional colleagues and fellow students . as one participant shared , there 's just no space for me , you know , and the locker rooms even us students some of them are both genders but some of them are not ( tm , page 28 ) . working in professions which were historically known for being female - dominated , or which had strong male roots , especially in institutional settings , prompted concerns more often than working in alternative health or small agencies that focused on the care of highly marginalized populations . i was afraid i would have someone who would refuse to call me he or like be ignorant or discriminatory , and , i just lucked out . these are the things that make people believe in , like , a higher power the more structured and hierarchical an organization is , the more you need to be just like a little cookie cutout so everybody is comfortable . it 's one less thing to worry about in the high stress situations , i guess ( tm , page 7 ) . lack of fit within the profession surfaced as a theme for participants both before gender transition began , and afterward . at times lack of fit was also used by employers to discriminate against participants after they began to transition , and this influenced job opportunities and job security . there 's a point beyond which you ca n't reach and it 's kind of invisible and you just kind of grind to a halt and there 's no real explanation you 're nice , dependable but there 's something that just does n't quite gel . ( tm , page 6 ) . the glass ceiling' . there 's a point beyond which you ca n't reach and it 's kind of invisible and you just kind of grind to a halt and there 's no real explanation you 're nice , dependable but there 's something that just does n't quite gel . ( tm , page 6 ) . he recalled being stunned by employers ' feedback . they said , we just do n't feel you 're a good fit . ' and i asked them to elaborate but they absolutely could n't ( tm , page 23 ) . all of the narratives revealed instances of explicit work - related discrimination or challenges of fitting in that were insidious . one participant , who perceived discomfort from peers when he began a new job , questioned how much education his current coworkers had about working with trans people on a team : another participant received a clear message from several higher - ups that gender transition in the professional training context was a weakness . their attitudes conveyed that people need to give you a hard time about it until you 're tough enough , you know , you 're thick - skinned enough that it wo n't bother you ( tm , page 34 ) . on the other hand , fit was also associated with a positive change after transition consistent with processes that engendered participants ' resilience and growth . for some , fit meant that how they did their job changed after transition because they identified a new niche for their practice , or a different way of doing gender that added a new element or a new community . one participant explained that he felt that he does masculinity differently than other men and that this is advantageous in his professional work with men who often have complex health concerns and who seek him out to discuss tough issues that they would not discuss with straight male or female providers . another spoke of being wowed by the possibility of being able to weave together his trans and professional identities in ways that are valued in a health care context such as participating openly in lgbtq - positive education , although he recognized that there is still much to be done before this is possible in all facets of his work as a provider : so that was the beginning of a new phase . i 'm beginning to integrate my identity with my health care provision ( tm , page 33 ) . for another participant , career choices involving alternative health care and health services research paralleled his emergent identity that for him were congruent , because both challenged the prevailing biomedical and binary discourses in health in ways that not only fostered the creation of trans - positive workplaces and professional practice , but also had profoundly positive consequences on a personal level . his narrative shows links between meaning - making in a career context and the process of coming to identify as trans:my gender identity is my career in a sense or vice versa you know . i 've , i feel that it is also significant that i have come to both of them at a similar time , like i feel like my life has really changed course as i 've um shifted as how i identify as both my sexual orientation and my gender identity . ( tm , pages 7 - 8 ) my gender identity is my career in a sense or vice versa you know . i 've , i feel that it is also significant that i have come to both of them at a similar time , like i feel like my life has really changed course as i 've um shifted as how i identify as both my sexual orientation and my gender identity . ( tm , pages 7 - 8 ) certainly , the opportunity to find supportive communities both within and beyond their workplaces where participants could validate their holistic lives and identities was enormously valuable for wellbeing . this was represented by one participant who shared , i 'm not going to go stealth . i do n't want to erase my past as a female and i think that being trans gives me a unique position in the world and . another spoke of connecting to queer community [ through ] freelance writing and through the gender expression performance yet for another , his decision to disclose as a transman , which was sparked by professional concerns , prompted unexpected ripples of family support . well i 'm thrilled everyone is just okay with it ( tm , page 14 ) . finding community or re - reconnecting unexpectedly with colleagues or family surfaced as significant effects of the processes of expressing trans identities for all participants in some context , although support varied considerably among the narratives . this life history study , with its attention to analysis within and across narratives , points to critical events and influences that shape trans health providers ' engagement with their identities as transmen and as health providers . gender and sexuality are determinants of health that are relevant to diverse trans people , whose everyday lives reflect a continuum of femininities and masculinities in a context of cisnormativity . cisnormativity , which is embedded in all social institutions and our everyday worlds , refers to the assumption that all people are cisgendered . in these narratives , gender and work emerged as the most salient social determinants in trans people 's career and worklives . the findings of this study show that trans people have unique , and gender - related , critical events and influences that shape continuities and discontinuities in their careerlives . examples of continuity are reflected in their decision to remain involved in health provision despite experiencing immense challenges brought on by experiences of discrimination , harassment , and ill health . despite this , the narratives of the participants also reveal that at times their career trajectories were curtailed or limited by these challenges , and for some eventually necessitated an unanticipated shift in the scope of work or a change in health profession . in fact , these narratives show how personal and professional identities , in conjunction with practices of gender expression , are intimately linked to professional socialization processes . as a result , this study adds to the necessity of expanding organizational and educational environments by bringing forth trans providers ' narratives and experiences . as one participant indicated , it is particularly crucial to bring [ ] forth trans voices that have this reality made known to a wider audience ( tm , page 3 ) with a focus on gender fluidity and as opposed to sexuality , especially in a context of health care where transmen are often invisible . the focus on ongoing transitions without an endpoint that was demonstrated in this study challenges the more dominant before / after transition models of trans people 's lives with stages ( e.g. , ) and this is consistent with what we have foregrounded in this paper as a more fluid approach to gender and career / work transitions more broadly . the narratives revealed that both the professional and personal lives of trans providers reflect continuities and discontinuities that have particular consequences for their career and worklives . although continuities between the personal and professional may be relevant to a variety of individuals , findings of this study suggest that these continuities and discontinuities also have health - related effects that are both health - promoting and health - depleting in effect deleterious . in fact , the intersection of work , gender , and health are clearly depicted through these narratives , in terms of employment , working conditions , and professional culture , which all have implications for nursing . for instance , although it is crucially important for nurses to open spaces and contribute to the development of workplace policies that will foster transition processes for providers , as these findings suggest , ongoing workplace support of diverse colleagues encompasses a range of practices that move beyond the transition process . these include an awareness of the everyday challenges of living and working in one 's chosen gender , the significance of one 's chosen profession as it connects to one 's trans identity , and the importance of interpersonal , organizational , and professional supports to enhance broader community and professional environments for trans people across their lifespan . thus these findings bring to bear a complex understanding and novel way of exploring health for trans people as workers and in particular as health care workers . while we appreciate that there is an assumption that lgbtq health care providers experience similar workplace concerns in general , such as stigma , management of identity and social exclusion of minorities that factor into career trajectories and a range of workplace - related issues such as health , social support , and policy , we maintain that such literature is limited in its relation to our study 's focus on the career / work context . in fact , some lgbtq - focused literature on the workplace appears to address trans issues and focus on trans people ; however in effect it actually elaborates on sexual orientation rather than gender identity . for example , bell et al . , while offering important insights into workplace dynamics for sexually diverse groups , do not address the unique needs of trans people in comparison to lgb people , despite a title that espouses a focus on lgbtq workplace concerns . call for the development of transgender - specific versions of vocational measures to study trans workplace experiences given the ways that lgbq - focused measures are insufficient to capture the unique issues of trans people . as they note , many of the issues discussed in lgb vocational scholarship may be pertinent to transgender people , but the unique manifestations of experiences of workplace harassment and discrimination , workplace climate , and identity management are shaped specifically by gender identity and presentation ( , page 61 ) . thus , a key contribution of our paper is the juxtaposition of career , work , and health for trans - identified people who are health and social service providers , and in particular the gender - based analysis with its focus on transgender people is unique . this paper complements the main foci of health literature on trans people which assumes that they are clients , and the workplace literature which mainly addresses gender transition and related policies important as they are to trans people 's worklives ( e.g. , [ 10 , 12 , 17 , 36 ] ) . the findings also extend the dearth of trans - focused literature in the counselling and vocational literature ( e.g. , [ 11 , 33 , 3539 ] ) . this study contributes to a small , but increasing , body of nursing and health professional studies on gender diversity in clinical care , cultural competence , and gender nonconformity in the professions ( e.g. , [ 19 , 20 , 23 , 25 , 28 , 4044 ] ) . the findings build on research on transmen and trans people 's work experiences which address transition policies and practices , identities , disclosure , discrimination , and employment counseling ( e.g. , [ 17 , 37 , 45 , 46 ] ) , and on lgbtq - affirmative counselling and lgbtq - positive climates in higher education ( e.g. , [ 39 , 47 ] ) . the one nursing study that examines workplace experiences of lgbt - identified nurses in an american context offers important survey data on three respondents who are trans - identified . short narrative insights from these participants and same - sex - identified nurses as key informants indicate that trans nurses encounter challenging workplace dynamics with some positive support noted . our contribution , a gender - based analysis of qualitative data , in - depth interviews with transmen whose lifetime careers represent a variety of health care professions , including nursing , offers new and unique insights . given the significant gap a silence in the literature in relation to trans people who are health care providers and the gap in nursing publications in relation to trans issues in general , this study provides much needed information . without a doubt , making visible the career histories of these , often invisible , trans providers in ways that show resilience and complexity , provides not just stories of role modeling for other trans people and other marginalized people in the professions , but also acts as a counterpoint to the often grim evidence base that comprises trans health . certainly , professional education and activism that aim to alter structural dynamics are needed to foster the creation of equitable spaces for diverse trans people to thrive as health providers ( e.g. , [ 7 , 17 , 47 ] ) . these findings address the invisibility of trans people as health providers rather than as clients in ways that are strength - based and move beyond a focus on marginalization and mental health concerns . this qualitative research study provides thick description and illustrates the specific and multiple contexts of trans people 's lives that show how challenging employment settings , poverty , ill health , and marginalization may be part of trans health professionals ' life experiences . yet , these are also positive narratives of people who are successful in their lives and careers and who recognize their privilege as trans people who can work and thrive in particular professional contexts . certainly this small exploratory study of a selected group of trans providers who have had access to professional training and/or acquired professional credentials and who are working in the field reflects trans people with high social privilege and does not aim to represent all providers ' or transmen 's experiences , but instead aims for understanding . more research is needed to examine a range of trans people 's careerlives that accounts for a diversity of identities , practice settings , and ethnoracial differences , for instance . the findings show how work and career are mutually informing , and how work is meaning - making or community - finding all of the participants talk about finding or establishing a community at some point . these narratives also point to the negotiation , compromise , and resistance that figure into their everyday decision - making in the education and workplace environments where they are doing this gender work . as they work within structures that are shaped by gender normativity and which prescribe fit within professional practice , they also often push the structures to change through their visibility when they are able . these are not client or patient narratives of transition or of being trans , but rather these are insider narratives of the health profession that expand the spaces for talking about gender and transgender in health care contexts [ 48 , 49 ] . meaning - making through work and career , and finding community through gendered practices , resilience , and lack of linear trajectory are about process rather than an endpoint . such career experiences in a professional environment are characterized by both continuities and discontinuities , as noted earlier . the findings extend what is typically discussed about the physical and emotional upheaval that transition can bring . by foregrounding discontinuity we see multiple critical incidents and influences in trans people 's personal and professional lives that complicate what is often represented as a linear trajectory of professional work and career decision - making in general , and for trans people more specifically [ 33 , 46 ] . as transmen negotiate their work and identities , making meaning through their career paths , they are un / doing gender and doing transgender . this work builds on the work of west and zimmermen on doing gender , and connell 's extension of that in relation to the ways in which transgender people do transgender work by foregrounding the lived experiences of providers who are transmen who experience policy on the ground both as clients of health systems and as policy actors within health systems . the focus on spiritual values [ 51 , 52 ] in a professional context , which has often been in opposition to a medical model , is consistent with a holistic and hopeful framing of transgender that informs these participants ' expression and articulation of their trans identities and practices as providers . this spirituality component in its focus on meaning - making is a unique contribution to the literature on gendered identities for trans people . relating both to themes of continuity and fitting in , for some of the participants , gender identity and transitioning have catalyzed an expanded meaningful and positive connection to their professional identity / role . in spite of discontinuities that they experience in relation to identities , and at times experiences of rejection in work and/or professional environments , there is a congruency in the ways they create career paths in relation to their practices as health providers . they are working inside and outside the work context in political ways to challenge gender norms in the ways that they participate in professional , queer and/or trans community politics , connections and professional education ; connell would suggest that this is doing transgender work . there are thus implications for nursing research , education , and practice in terms of understanding how trans providers do transgender work and supporting them in that process . certainly , research and educational strategies which make visible the lives of trans people as care providers in a nursing , and larger provider contexts , are important steps ; trans - inclusive education and training for cultural competence to enhance individual nurses ' awareness , as well as organizational support , are warranted in order to create positive working environments that support providers who are trans - identified . such dynamics are intimately linked to nurses ' capacity to practice in inclusive ways as advocates for sexual and gender minority clients and communities . as indicated in life history research with other diverse nurses , these work and practice links have health implications that point to fostering providers ' wellbeing as well as their capacity to provide responsive care [ 19 , 42 ] . in order to create trans - positive spaces , findings of this paper support the development of a working paper on the process of formally integrating trans providers into the professional context . this paper thus takes up the call by eliason et al . for emancipatory nursing research which examines more fully the complexity of diverse lgbtq people 's lives .

well - documented health research points to trans people 's vulnerability to health inequities that are linked to deeply embedded structural and social determinants of health . gender and work , as social determinants of health for trans people , both shape and are shaped by multiple factors such as support networks , social environments , income and social status , shelter , and personal health practices . there is a gap in the nursing literature in regards to research on work and health for diverse trans people and a virtual silence on the particular issues of trans - identified health providers . this qualitative study used comparative life history methodology and purposeful sampling to examine links among work , career , and health for transmen who are health providers . semistructured interviews were completed with four canadian transmen involved in health care professional and/or practice contexts with diverse professions , age , work , and transitioning experiences . critical gender analysis showed that unique and gender - related critical events and influences shape continuities and discontinuities in their careerlives . this strength - based approach foregrounds how resilience and growth emerged through participants ' articulation with everyday gender dynamics . these findings have implications for nursing research , education , and practice that include an understanding of how trans providers do transgender work and supporting them in that process .

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

this last decade has brought greater attention to transgender health , the t in lgbtq , in health care and research has shown that ethnicity , race , geography , age , education , and other social categories across genders influence how particular trans - identified individuals experience and understand their health . encounters with violence , transphobia , and discrimination related to sexuality and gender norms across home , community , work , and educational settings , and challenges to finding meaningful social support , shape trans people 's disclosure with respect to trans issues and identities , their health status , and access to care [ 57 ] . all of these factors contribute to trans people 's vulnerability to health inequities . as social determinants of health , employment , and working conditions for trans people both shape and are shaped by multiple factors such as support networks , social environments , income and social status , shelter , and personal health practices [ 6 , 7 , 9 , 10 ] . decisions to transition in the workplace are informed by many factors that include underemployment , available support systems , and weighing the costs of job - loss or unpredictable discrimination that mark many environments for trans people [ 8 , 9 , 11 ] . in ontario , canada , findings from the trans pulse project indicate that there is little known about trans people 's work , although it is well documented that underemployment and barriers to education exist . narratives published in print , or online , offer insight into the dynamics of work and health and the complexities of diverse trans people 's lived experiences of gender expression and transitioning in a canadian context [ 1416 ] . in 2008 , the canadian professional association for transgender health ( cpath ) also emerged to offer resources and professional support for those involved in trans health provision , creating a community for trans - identified providers and allies to be visible in a professional context . , nurses ) are visible in the health sector and there is limited research that focuses on their work , and even less on their career trajectories , especially in the professional context ( this includes both alternative and regulated health providers ) ( e.g. yet , gender and its intersections with race , sexuality , and other social categories inform a body of research on professionals and the nature of their work ; dominant notions of masculinities and femininities as well as gender conformity have also been noted as relevant to lgbtq health professionals ' career / work dynamics ( e.g. as a result , research that fosters an understanding of factors that influence the career , work , and health of diverse trans providers is needed to illustrate the complexities of their lives with the goal of creating supportive policies and practices . yet , when this is addressed , most health studies , including nursing research , focus on the context in which trans people are assumed to be patients or clients . by default , the assumption is that all care providers are not trans - identified , but cisgendered ( a term used to describe people who are not transgendered ) and this has important consequences for the ways in which the health issues of diverse trans people are identified and articulated , and thus the practice strategies that are deemed relevant to supporting them . in fact , a search of the literature using cinahl , pubmed , social science abstracts , and psychinfo databases using the terms transgender and/or transsexual and work in titles and abstracts yielded a handful of articles that focus on transgender or transsexual people and their workplace experiences . typically across the health literature , transgender people and work get taken up in the context of sex work , sexual risk for hiv / aids and/or in relation to how providers a recent study suggests that such an approach erases the complexity of trans people 's lives [ 22 , 24 ] . a paucity of workplace - focused health literature exists that offers insight into trans people 's workplaces in relation to experiences of transitioning , and/or relevant policy dynamics ; in effect , their lives are equated with a patient context rather than a work context which has limitations to understanding health in a social determinants approach . this paper foregrounds the experiences of health providers who themselves are trans - identified and illustrates the links in their work , careers and health and wellbeing , and the complex ways that gender is implicated in their lives . in particular , this paper focuses on how transmen who are health providers make meaning through their careerlives , that is , meaningful life activities encompassing work and personal endeavors , with implications for their health and wellbeing . this paper explores intersections of gender , health and work or career for transmen health providers in relation to their everyday lived realities . in particular , this paper explores two research questions.how do transmen who are health providers make meaning through their careerlives?what are the implications for their health and wellbeing ? how do transmen who are health providers make meaning through their careerlives ? this study in the naturalistic and critical paradigms was appropriate for this exploratory study that examined sensitive dynamics of identity , work and career , and complex aspects of power . this life history approach attends to processes of narration , collaboration , and contextualization so that the researcher has an active role in situating participants ' narratives in the larger sociocultural , political , economic , and historical contexts . critical gender analysis of career histories , within - case and cross - case analysis , can also identify critical incidents and influences , such as identity , that shape diverse professionals ' careerlives . for trans professionals and health providers whose work lives are shaped by policy and whose work entails practices of implementing and creating policy , their lives as trans people are also inscribed by human rights and organizational policies that shape the ways in which trans people are variously included , marginalized , and/or legitimized in their work and larger social worlds [ 22 , 28 ] . gender - based analysis in this paper , addresses both sex , biological differences between women and men , and gender , which takes into account the social dynamics or gender roles , identities , and norms which shape individuals ' holistic health . however , trans people 's attention to and experiences of gender variance and the ways that their biological characteristics are at odds with their sense of being male , female , and/or expressing their gender , blur the discrete boundaries of sex and gender . rather than an exclusive focus on health risks , a critical gender - based approach foregrounds trans people 's agency , the ways that they negotiate their social worlds in particular times and places , and material and social dynamics characterizing their careerlives and the ways that gender intersects with work and other social determinants . although gender - based analysis has typically been used to examine how gender matters in the lives of cisgendered women and men , johnson et al . explain that it could be applied to understand transgender / transsexual issues , and more recently it has been taken up in health research on trans people [ 21 , 22 ] . at that point , a call for advisory group members for the study was sent out , and two trans - identified community members responded and provided feedback on the study materials and procedures . the call out for participants was for individuals who self - identified as trans health providers working in the health and social services sector initially in ontario ; and at a later date , recruitment was expanded to include those working in canada . this paper , however , reports on the trans - identified providers and specifically , trans providers who self - identified as transmen . this included rainbow health ontario and rainbow health network online listserves which have broad reach to gender diverse communities and health providers throughout canada , as well as the researcher 's well - developed professional networks that included a focus on health equity - related research . the interviews focused on career histories , everyday activities , experiences of health and work , as well as factors that shaped career and workplace decisions and practice dynamics . interview questions focused on the everyday lived experiences and challenges shaping many trans people 's worklives : gender presentation , transitioning , health concerns , discrimination , violence , underemployment , or unemployment , and social support . for example , the researcher asked about a typical day in their life as a trans - identified health provider , pivotal incidents , or key influences that played a part in their health , as well as workplace dynamics , policies , or issues that would contribute to trans providers ' health and wellbeing and their vision for strategies that could improve practice environments for trans providers . face - to - face or telephone semi - structured interviews were completed from april 2008 to april 2010 with four participants who self - identified as transmen . however , the researchers were also attuned to unexpected themes that emerged , such as some participants ' experiences of congruence between their work interest and their social identities as members of trans communities . as well , it affirms the unique experiences and identities of diverse trans people who share the common experience of knowing themselves to be a gender that is not congruent with their birth sex paper , analysis focuses on the career histories of four health providers who self - identified as transmen . professional work and/or education over their lifetimes included social work , medicine , nursing , midwifery , naturopathy , massage therapy , as well as other care work such as teaching . because of the extremely small pool of openly trans - identified health providers and specifically those with professional credentials in a canadian context , narrative excerpts and findings are reported in this paper in ways that will not identify participants ; information such as age , profession , and place of work are omitted . in this paper , we report on themes that represent how participants articulated gender identities in the context of work , their careerlives and larger lives , so that the focus is on meaning - making through gender / transition and doing gender in their work and other meaningful life activities . for instance , deeply entrenched homophobia , heterosexism , and biphobia contribute to gender norms and invisibility , lack of awareness , and transphobia as factors that hinder diverse trans people 's ability to locate respectful , responsive , and meaningful health care in their lives [ 7 , 22 ] . as one noted , the journey of becoming queer - identified led me into the health sector was wanting to take care of my own health ( tm , page 4 ) , while another suggested that trans people definitely have a better chance of making it in health care or social services . working and transitioning , whether as a credentialed professional or professional - in - training , meant that these transmen had to worry about how to change their documentation and professional registration , in addition to dealing with the multiple decisions related to , and demands of , disclosing ( or not ) and living and working as a transman in various community and work contexts . those affiliated with mainstream education programs or work settings such as hospitals were thus likely to be the sole trans - identified provider in their workspaces . participants in particular identified that being trans and working in health care meant that education and coming out were constant worries or struggles , and that employers who supported them through medical transition were highly valued . on the other hand , fit was also associated with a positive change after transition consistent with processes that engendered participants ' resilience and growth . another spoke of being wowed by the possibility of being able to weave together his trans and professional identities in ways that are valued in a health care context such as participating openly in lgbtq - positive education , although he recognized that there is still much to be done before this is possible in all facets of his work as a provider : so that was the beginning of a new phase . for another participant , career choices involving alternative health care and health services research paralleled his emergent identity that for him were congruent , because both challenged the prevailing biomedical and binary discourses in health in ways that not only fostered the creation of trans - positive workplaces and professional practice , but also had profoundly positive consequences on a personal level . this life history study , with its attention to analysis within and across narratives , points to critical events and influences that shape trans health providers ' engagement with their identities as transmen and as health providers . gender and sexuality are determinants of health that are relevant to diverse trans people , whose everyday lives reflect a continuum of femininities and masculinities in a context of cisnormativity . in these narratives , gender and work emerged as the most salient social determinants in trans people 's career and worklives . the findings of this study show that trans people have unique , and gender - related , critical events and influences that shape continuities and discontinuities in their careerlives . examples of continuity are reflected in their decision to remain involved in health provision despite experiencing immense challenges brought on by experiences of discrimination , harassment , and ill health . the narratives revealed that both the professional and personal lives of trans providers reflect continuities and discontinuities that have particular consequences for their career and worklives . although continuities between the personal and professional may be relevant to a variety of individuals , findings of this study suggest that these continuities and discontinuities also have health - related effects that are both health - promoting and health - depleting in effect deleterious . in fact , the intersection of work , gender , and health are clearly depicted through these narratives , in terms of employment , working conditions , and professional culture , which all have implications for nursing . these include an awareness of the everyday challenges of living and working in one 's chosen gender , the significance of one 's chosen profession as it connects to one 's trans identity , and the importance of interpersonal , organizational , and professional supports to enhance broader community and professional environments for trans people across their lifespan . thus these findings bring to bear a complex understanding and novel way of exploring health for trans people as workers and in particular as health care workers . while we appreciate that there is an assumption that lgbtq health care providers experience similar workplace concerns in general , such as stigma , management of identity and social exclusion of minorities that factor into career trajectories and a range of workplace - related issues such as health , social support , and policy , we maintain that such literature is limited in its relation to our study 's focus on the career / work context . , while offering important insights into workplace dynamics for sexually diverse groups , do not address the unique needs of trans people in comparison to lgb people , despite a title that espouses a focus on lgbtq workplace concerns . call for the development of transgender - specific versions of vocational measures to study trans workplace experiences given the ways that lgbq - focused measures are insufficient to capture the unique issues of trans people . as they note , many of the issues discussed in lgb vocational scholarship may be pertinent to transgender people , but the unique manifestations of experiences of workplace harassment and discrimination , workplace climate , and identity management are shaped specifically by gender identity and presentation ( , page 61 ) . thus , a key contribution of our paper is the juxtaposition of career , work , and health for trans - identified people who are health and social service providers , and in particular the gender - based analysis with its focus on transgender people is unique . this paper complements the main foci of health literature on trans people which assumes that they are clients , and the workplace literature which mainly addresses gender transition and related policies important as they are to trans people 's worklives ( e.g. the findings also extend the dearth of trans - focused literature in the counselling and vocational literature ( e.g. this study contributes to a small , but increasing , body of nursing and health professional studies on gender diversity in clinical care , cultural competence , and gender nonconformity in the professions ( e.g. the findings build on research on transmen and trans people 's work experiences which address transition policies and practices , identities , disclosure , discrimination , and employment counseling ( e.g. the one nursing study that examines workplace experiences of lgbt - identified nurses in an american context offers important survey data on three respondents who are trans - identified . our contribution , a gender - based analysis of qualitative data , in - depth interviews with transmen whose lifetime careers represent a variety of health care professions , including nursing , offers new and unique insights . given the significant gap a silence in the literature in relation to trans people who are health care providers and the gap in nursing publications in relation to trans issues in general , this study provides much needed information . without a doubt , making visible the career histories of these , often invisible , trans providers in ways that show resilience and complexity , provides not just stories of role modeling for other trans people and other marginalized people in the professions , but also acts as a counterpoint to the often grim evidence base that comprises trans health . certainly , professional education and activism that aim to alter structural dynamics are needed to foster the creation of equitable spaces for diverse trans people to thrive as health providers ( e.g. these findings address the invisibility of trans people as health providers rather than as clients in ways that are strength - based and move beyond a focus on marginalization and mental health concerns . this qualitative research study provides thick description and illustrates the specific and multiple contexts of trans people 's lives that show how challenging employment settings , poverty , ill health , and marginalization may be part of trans health professionals ' life experiences . yet , these are also positive narratives of people who are successful in their lives and careers and who recognize their privilege as trans people who can work and thrive in particular professional contexts . certainly this small exploratory study of a selected group of trans providers who have had access to professional training and/or acquired professional credentials and who are working in the field reflects trans people with high social privilege and does not aim to represent all providers ' or transmen 's experiences , but instead aims for understanding . more research is needed to examine a range of trans people 's careerlives that accounts for a diversity of identities , practice settings , and ethnoracial differences , for instance . as they work within structures that are shaped by gender normativity and which prescribe fit within professional practice , they also often push the structures to change through their visibility when they are able . meaning - making through work and career , and finding community through gendered practices , resilience , and lack of linear trajectory are about process rather than an endpoint . by foregrounding discontinuity we see multiple critical incidents and influences in trans people 's personal and professional lives that complicate what is often represented as a linear trajectory of professional work and career decision - making in general , and for trans people more specifically [ 33 , 46 ] . this work builds on the work of west and zimmermen on doing gender , and connell 's extension of that in relation to the ways in which transgender people do transgender work by foregrounding the lived experiences of providers who are transmen who experience policy on the ground both as clients of health systems and as policy actors within health systems . this spirituality component in its focus on meaning - making is a unique contribution to the literature on gendered identities for trans people . in spite of discontinuities that they experience in relation to identities , and at times experiences of rejection in work and/or professional environments , there is a congruency in the ways they create career paths in relation to their practices as health providers . there are thus implications for nursing research , education , and practice in terms of understanding how trans providers do transgender work and supporting them in that process . certainly , research and educational strategies which make visible the lives of trans people as care providers in a nursing , and larger provider contexts , are important steps ; trans - inclusive education and training for cultural competence to enhance individual nurses ' awareness , as well as organizational support , are warranted in order to create positive working environments that support providers who are trans - identified . as indicated in life history research with other diverse nurses , these work and practice links have health implications that point to fostering providers ' wellbeing as well as their capacity to provide responsive care [ 19 , 42 ] . in order to create trans - positive spaces , findings of this paper support the development of a working paper on the process of formally integrating trans providers into the professional context .

redox properties are essential functional characteristics of electron transfer proteins , and most electron transfer proteins are metalloproteins . however , while experimentally synthesized analogs of the iron sulfur proteins have played a critical role in understanding their redox properties , the synthesis of redox site analogs for the copper proteins has been difficult due to the flexible coordination of copper . so far , spectroscopically similar analogs have been synthesized , but they exhibit weak structural similarity to even the simplest copper protein redox sites . however , determining reduction potentials of redox - active metalloproteins by computational chemistry methods is challenging . for instance , quantum mechanical calculations of first - row transition metals found in these proteins must include electron correlation . since advances in density functional theory ( dft ) include functionals with effective electron correlation , dft is usually the method of choice because of computational efficiency . however , unlike conventional ab initio molecular orbital theory , dft suffers from the lack of a clear pathway for improvement fock ( hf ) exchange ) or hybrid meta ( i.e. , in addition , including the spin kinetic energy densities ) generalized gradient approximation ( gga ) density functionals appear more accurate for treating transition - metal systems than local density approximations and even some post - hf methods . moreover , efforts have been made to develop range - separated ( rs ) functionals in which the hf exchange is treated differently at different ranges for further improving upon global hybrids . however , benchmarking of density functionals and basis sets by comparisons with experimental measurements of structural and energetic properties of relevant compounds remain essential , with a goal of a balance of accuracy and computational efficiency . in particular , valence photoelectron spectroscopy ( pes ) of well - defined gaseous metal complexes has become important for testing the electronic structure and energetics , independent of environmental perturbations such as solvent , crystal field , or surrounding protein , since the calculation can be performed under the same conditions without any approximations for the environment . our previous benchmark studies for analogs of fe s proteins used gaseous pes experimental data , the ligand metal bond covalency from x - ray absorption spectroscopy ( xas ) , and structures from x - ray crystallography . good structures can be obtained using broken - symmetry ( bs ) dft with the b3lyp functional and a double- basis set with polarization functions such as 6 - 31g**. in addition , good single - point energies of these structures can be obtained when diffuse functions are added to the sulfurs , even comparable to coupled cluster ( cc ) methods . the calculated vertical ( vde ) and adiabatic ( ade ) detachment energies agree well with the gaseous pes data of [ 1fe ] , [ 2fe2s ] , and [ 4fe4s ] protein analogs , while the calculated percentage of ligand character of the fe 3d orbitals correlate well with ligand k - edge xas measurements . furthermore , calculations at this level also give reliable inner - sphere reduction free energies for reduction potential calculations of iron since spectroscopically and structurally similar analogs of copper proteins have not yet been synthesized , understanding the structural , electronic , and redox properties of simple copper complexes is especially important for benchmarking calculations of copper redox site analogs . furthermore , calculations with reliable methods may lead to better design strategies for synthesizing new analogs . in the current article , cu s and cu n bonding interactions are investigated in the simple cu complexes , [ cux2 ] ( x = sch3 , ncs ; n = 1 and 0 ) , as a prelude to investigations of the type i copper site found in the blue copper proteins . the type - i copper site consists of a distorted tetrahedral structure with four ligands : two histidines , a cysteine , and a weak axial fourth ligand , which is usually a methionine . since our long - term goal is to find functionals and basis sets that work well for the transition metals found in metalloprotein redox sites , which include mixed metal sites , the fe complexes , [ fex4 ] ( x = cl , sch3 ; n = 1 and 0 ) , which were used in our previous studies for redox sites of fe s proteins , we focus on contrasting the ability of the methods to handle the closed shell , low - spin cu complexes ( s = 1/2 ) with the open shell , high - spin fe complexes ( s = 5/2 and 2 ) rather than a statistical survey of many different transition - metal complexes . while a general criteria is good agreement of geometries with the x - ray crystallographic structures , our essential criteria is good agreement of energetics with pes electron detachment energies since the methods are being tested for redox calculations . several double- basis sets were evaluated against two triple- basis sets . also , several hybrid gga and meta - gga functionals and range - separated gga and meta - gga functionals were compared to cc methods . spin - unrestricted dft calculations with broken - symmetry ( bs ) molecular orbital ( mo ) wavefunctions were performed for geometries and energies . the energies were also calculated at the ccsd and ccsd(t ) levels , with the inner shells up to and including the 3s and 3p orbitals frozen in the correlation calculations . if the energy is calculated using a different method than the geometry optimization , the notation level2/basis2//level1/basis1 is used in which level1/basis1 calculations are for the geometry optimization and level2/basis2 calculations are used for single - point energies of the level1/basis1 geometry . the first three , dzvp2 , def2-svp , and 6 - 31 g * * , are double- basis sets with polarization functions . the next two , def2-svpd and 6 - 31++g * * , contain diffuse functions , while 6 - 31(++)lg * * has diffuse functions added only to the subscripted ligand atoms . finally , def2-tzvppd and aug - cc - pvtz are triple- basis sets with polarization and diffuse functions . the hybrid functionals tested with percentage hf exchange indicated in parentheses are the b3lyp gga ( 20% hf ) ; b3lyp * , a modified b3lyp gga ( 15% hf ) ; the b97 gga ( 19.43% hf ) ; the pbe1pbe gga ( 25% hf ) ; the m06 meta - gga ( 27% hf ) ; and the b(38hf)p86 gga ( 38% hf ) . in addition , one set of rs density functionals with percentage hf exchange at short - range ( sr ) and at long - range ( lr ) and range parameter indicated in parentheses tested are the bnl gga ( 0% sr , 100% lr , 0.33 a0 ) ; the cam - b3lyp gga ( 19% sr , 65% lr , 0.33 a0 ) ; and the lrc-pbeh gga ( 20% sr , 100% lr , 0.2 a0 ) . finally , another set of rs density functionals with highly parametrized and reoptimized short - range dft exchange ( referred to here as highly optimized rs ) are the b97 gga ( 0% sr , 100% lr , 0.4 a0 ) ; the b97x gga ( 15.77% sr , 100% lr , 0.3 a0 ) ; and the m11 meta - gga ( 42.8% sr , 100% lr , 0.25 a0 ) . ( note many different notations are used for rs functions ; for instance , % sr = , % lr = + , and = in refs ( 12 and 46 ) . ) the initial wavefunctions of the oxidized open - shell species were generated either from the wavefunctions of the reduced species or of its higher spin states at a reasonable initial geometry . since the hf and kohn sham ( ks ) electron configurations of the ground state of open - shell cu and fe complexes were often different , both were used as initial electron configurations for the dft and cc calculations to help establish that the solution was not an excited state . stability analysis of the second derivatives of the energy with respect to occupied orbital variation in the wavefunction was further utilized to confirm the ground state of the complexes in the hf and dft calculations . stationary points in the dft calculations that were uncertain due to a flat potential energy surface were examined further using an extra fine integration grid , with an energy convergence of 1 10 au . detachment energies of complexes were approximately calculated by the difference of total electronic energies between the reduced and oxidized species using the geometry of the reduced species ( vdes ) and by the negative energy of the highest occupied molecular orbital ( homo ) of the reduced species . the t1 diagnostic and the largest t2 amplitude values were determined from the ccsd(t)/def2-svp(d)l//m06/def2-svp calculations to evaluate multireference effects . all hybrid dft calculations were performed using the nwchem program package , the rs dft ( except lrc-pbeh ) , ccsd , and ccsd(t ) calculations utilized the gaussian09 program package , and the lrc-pbeh calculations utilized the q - chem program package . the molecular orbital visualizations were performed using the extensible computational chemistry environment ( ecce ) application software . the pes experiments were performed on a magnetic - bottle apparatus equipped with an electrospray ionization ( esi ) source . the only modification was the shortening of the electron flight tube of the magnetic - bottle pes analyzer from 4 to 2.5 m. moreover , the energy resolution of the pes spectra of both species is enhanced using our newly built cryogenically cooled ion trap operated at 20 k. briefly , a 1 mm solution of cu2o with nasch3 in a ch3cn / h2o mixed solvent ( 3:1 volume ratio ) was prepared to generate the [ cu(sch3)2 ] anion , and a 1 mm solution of cu(ch3coo)2 with kscn in a pure ch3cn solvent was prepared to generate the [ cu(ncs)2 ] anion . after being accumulated in the ion trap for 0.1 s , the desired anions were mass - selected via time - of - flight mass spectrometry and then decelerated before being intercepted by a probe laser beam . the laser wavelengths employed were 213 nm ( 5.821 ev ) from a dye laser and 266 nm ( 4.661 ev ) from a nd : yag laser . photoelectron time - of - flight spectra were collected and converted to kinetic energy spectra , calibrated by the known spectra of i and au . the electron kinetic energy resolution was about 3% , i.e. 30 mev for 1 ev electrons . the vde of each feature was measured from the maximum of each detachment band . in addition , pes vdes of the fe complexes were obtained from our previous studies . no crystal structure is available for [ cu(sch3)2 ] , so [ cu(s - t - butyl)2 ] ( csd i d : kobvaz ) was used . in addition , the [ cu(ncs)2 ] ( viccit ) , [ fecl4 ] ( ahepot ) , and [ fe(sch3)4 ] ( jurhin ) were used . the photoelectron spectra of [ cu(sch3)2 ] and [ cu(ncs)2 ] ( figure 1 ) were obtained at 20 k , which led to sharper and much better resolved peaks than at room temperature . the spectrum of [ cu(sch3)2 ] at 266 nm ( figure 2a ) reveals four prominent detachment bands , labeled as x , a , b , and c. the ground state x band is very broad , suggesting a large geometry change between the ground state of [ cu(sch3)2 ] and its neutral , and overlaps the a feature . in contrast , the b and c features are much sharper , suggesting they are from nonbonding cu 3d type orbitals . the spectrum of [ cu(ncs)2 ] at 157 nm ( figure 2b ) shows a detachment feature labeled as x , with a short vibrational progression with frequency 1780 50 cm . the detachment from the ground state ( x band ) is of relevance to redox reactions in proteins , and both the vde and ade from x can be determined . however , given the possible large geometric change upon oxidation of [ cu(sch3)2 ] , we focus on the vde for comparisons with the calculated results since crystal structures of the oxidized cu complexes are apparently not available . ball - stick renderings of [ cu(ncs)2 ] and [ cu(sch3)2 ] , m06/aug - cc - pvtz geometries . photoelectron spectra at 20 k of ( a ) [ cu(sch3)2 ] at 266 nm and ( b ) [ cu(ncs)2 ] at 213 nm . ks mo interaction diagrams between the cu and the ligands for [ cu(sch3)2 ] and [ cu(ncs)2 ] ( figure 3 ) were examined to consistency with the pes results . this figure is based on the m06/dzvp2 calculations but is representative of the b3lyp results as well . a full analysis of the calculated electronic structure and the pes spectrum will be presented elsewhere . schematic kohn sham molecular orbital interaction diagrams based on m06/dzvp2 calculations ( a ) between cu and sch3and the homos of [ cu(sch3)2 ] and ( b ) between cu and ncsand the homos of [ cu(ncs)2 ] . the two homos of [ cu(sch3)2 ] are formed by interactions between the cu dyz and dxz orbitals and the higher - lying s lone - pairs . the two cu s bonds are formed by interactions of the cu 4s , 4p , and dz2 orbitals and the lower - lying s lone - pairs with s the five occupied cu 3d orbitals lie below the symmetric s lone - pair orbitals and above the two cu s orbitals and an asymmetric s lone - pair orbital with s the oxidation of [ cu(sch3)2 ] involves the near - degenerate s lone - pair mos , which should induce a significant structural distortion ( consistent with the broad pes x band ) toward a more planar structure with strong spin - polarization . consequently , the singly occupied mo ( somo ) and the lowest unoccupied mo ( lumo ) upon oxidation increase the cu dxz character , while the other mos ( homo-1 and homo ) become pure lp(s ) without any cu 3d character . also , since the mo ( lumo ) that is oxidized has *cu s antibonding character ( consistent with s k - edge xas experiments of the redox site of the blue copper proteins ) , the cu the two degenerate homos of [ cu(ncs)2 ] are formed by interactions between the cu dyz and dxz orbitals and the higher - lying s lone - pair orbitals with *c s and n c character , even though the metal ligates to the n ; the asymmetric homos are stabilized by the electron back - donation into the high - lying unoccupied *n c orbitals , which increases the n c bonding character in the homos . the two cu n bonds are stabilized by interactions of the cu 4s , 4p , and dz2 orbitals and the lower - lying n lone - pairs [ lp(n ) ] with n c bonding character . the five occupied cu 3d orbitals lie below the four s lone - pair orbitals and above the four n the oxidation of [ cu(ncs)2 ] should differ from [ cu(sch3)2 ] because of stabilization of the homos by electron back - donation . for instance , electron detachment from the stabilized homo is more difficult so the vde should be larger than that in [ cu(sch3)2 ] , which is consistent with the pes data . in addition , the linear structure is unlikely to distort upon oxidation as does the [ cu(sch3)2 ] structure since the degenerate homos are stabilized by the back - donation . thus , although oxidation of both [ cu(ncs)2 ] and [ cu(sch3)2 ] involve the s lone - pair electron , the ground state x band in the pes spectra ( figure 2 ) of [ cu(ncs)2 ] has a very different shape from [ cu(sch3)2 ] . finally , the decrease in the *cu n antibonding , *c s antibonding , and n c bonding character upon oxidation should lead to a decrease in the cu n and c s bond lengths and an increase in the n c bond lengths . the basis sets were first evaluated for their performance in geometry optimization and energetics using the b3lyp and m06 hybrid functionals . first , the double- sets are examined for agreement with the triple- sets , and then agreement with experiment is assessed . the complete dft optimized geometries of [ cu(sch3)2 ] and [ cu(ncs)2 ] as well as crystal structures for the reduced species are given in the supporting information ( tables s1 and s2 ) . the calculations all show that [ cu(sch3)2 ] has a gauche conformation with c2 symmetry while [ cu(ncs)2 ] has a linear structure ( figure 1 ) , both in agreement with the x - ray structures . all of the basis sets show similar performance for most of the geometry . however , the s cu s angle of [ cu(sch3)2 ] is more than 20 larger in the 6 - 31(++)lg * * and 6 - 31++g * * ( not shown ) basis set compared to experiment , which causes larger deviations in the cu s c angle as well , so we do not recommend adding diffuse functions into the 6 - 31 g basis sets for geometry optimization . also , when the same basis set is used , the m06 functional gives slightly shorter bond lengths than the b3lyp functional , while the bond angles are quite similar . upon oxidation , similar changes are observed in the double- basis sets as in the triple- basis sets , although no experimental data are available for the neutral species . [ cu(sch3)2 ] is distorted from the gauche conformation of the reduced species into a planar trans conformation with c2h symmetry ( figure 1 ) , which is consistent with the broad pes x band ( figure 2a ) and may give rise to a large reorganization energy upon oxidation . the decrease in the cu s and s c bond lengths of [ cu(sch3)2 ] is also consistent with the mo analysis . in addition , [ cu(ncs)2 ] remains a linear structure and its cu n and c s bond lengths are slightly shorter while the n c bond lengths are longer relative to the reduced site , consistent with the mo analysis and the vibrational progression observed for the x feature . the metal ligand bond lengths are perhaps the most important geometrical feature for assessing the computational methods , so they are examined for the cu complexes and for fecl4 and [ fe(sch3)4 ] ( figure 4 ) . the double- basis sets are consistent with the triple- basis sets and give m l bond lengths for that are generally less than 0.05 longer than the crystal structures . however , the 6 - 31 g * * and 6 - 31(++)lg * * basis sets give significantly shorter bond lengths for cu(sch3)2 ] and [ cu(ncs)2 ] , which brings the calculated results further from experiment for [ cu(ncs)2 ] but closer for [ cu(sch3)2 ] ; however , the experimental m l bond length for the latter comes from [ cu(s - t - butyl)2 ] , which may be shorter than in [ cu(sch3)2 ] due to the better electron donating ability of the -s - t - butyl group than the -sch3 group that leads to stronger interactions with the cu . thus , of the small double- basis sets , the dzvp2 and def2-svp basis sets appear to give good m l bond lengths , with results similar to the triple- basis sets and the addition of diffuse functions to the latter ( i.e. , def2-svpd ) makes little difference . in addition , the m06 functional gives slightly shorter bond lengths than the b3lyp functional , so that the m l bond lengths for [ cu(ncs)2 ] ( triangle ) , [ cu(sch3)2 ] ( square ) , fecl4 ( circle ) , and [ fe(sch3)4 ] ( diamond ) , using the b3lyp ( open symbols ) and m06 ( solid symbols ) functionals as a function of basis set plotted on a scale of the logarithm of the number of basis functions in [ cu(ncs)2 ] . from left to right , the basis sets are dzvp2 , def2-svp , 6 - 31 g * * , 6 - 31(++)lg * * , def2-svpd , def2-tzvppd , and aug - cc - pvtz . the symbols are connected by dotted lines to guide the eye , and the results for the fe complexes are shifted upward by 0.1 to avoid overlaps . l bond lengths ( gray line with error indicated approximately by width of line ) are also shown . the complete dft vdes and ades of [ cu(sch3)2 ] and [ cu(ncs)2 ] are given in the supporting information ( table s3 ) . the vdes for the same cu and fe complexes are used to examine the redox energetics ( figure 5 ) . of the double- basis sets , the dzvp2 results are equivalent to the triple- basis set with the diffuse functions results , while the def2-svp and 6 - 31 g * * are much lower . of the double- basis sets with the diffuse functions , the 6 - 31(++)lg * * and 6 - 31++g * * ( not shown ) basis sets do not improve agreement as much for the cu complexes as for the fe complexes , while def2-svpd is in good agreement with the triple- basis set results , consistent with the better performance of the karlsruhe def2 basis sets with diffuse functions in the m06 calculations of barrier heights , ionization potentials , and electron affinities . in addition , because the differences in geometry are small between def2-svp and def2-svpd , the def2-svpd energy of the def2-svp geometry is almost identical to that of the def2-svpd geometry , which suggests geometry optimization with def2-svp and a single point energy calculation with def2-svpd for larger molecules . although the dzvp2 nondiffuse basis set is 2/3 the size of def2-svpd diffuse basis set for these redox sites , the results are remarkably similar , presumably because the dft optimized gaussian dzvp2 basis sets provide high quality valence orbitals in comparison with the energies and orbitals obtained by numerical solutions of the kohn sham equations . in particular , the smallest exponential parameters for each angular momentum are smaller than in def2-svp , which is more diffuse than standard nondiffuse basis sets , indicating dzvp2 is even more diffuse . however , while the b3lyp and m06 calculations show the increase in the vde of [ cu(ncs)2 ] over [ cu(sch3)2 ] observed experimentally , both underestimate the experimental values considerably by 400 mev although m06 is generally somewhat closer than b3lyp . calculated vde for [ cu(ncs)2 ] ( triangle ) , [ cu(sch3)2 ] ( square ) , fecl4 ( circle ) , and [ fe(sch3)4 ] ( diamond ) , using the b3lyp ( open symbols ) and m06 ( solid symbols ) functionals as a function of a basis set plotted on a scale of the logarithm of the number of basis functions in [ cu(ncs)2 ] . from left to right , the basis sets are dzvp2 , def2-svp , 6 - 31 g * * , 6 - 31(++)lg * * , def2-svpd , def2-tzvppd , and aug - cc - pvtz . the symbols are connected by dotted lines to guide the eye , and the results for the fe complexes are shifted upward by 3 ev to avoid overlaps . l bond lengths ( gray line with error indicated approximately by width of line ) are also shown . thus , dft calculations using def2-svpd agree well with those using the triple- basis sets with the diffuse functions for geometry and energetics for both the cu and fe complexes and appear to be a good balance of speed and accuracy for larger molecules . in addition , using either dzvp2 or else calculating single point energies using def2-svpd on def2-svp geometries , which are very similar to def2-svpd geometries , are options for much larger molecules or when computational speed is important . however , none of the dft results give good agreement for energetics compared to experiment for the cu complexes . given the poor energetics of b3lyp and m06 for the cu complexes , other functionals were tested . our strategy was first to examine trends and narrow down the functionals in bs - dft calculations with the dzvp2 double- basis set , since it compares well with larger basis sets with diffuse functions but its small size makes the calculations faster . l bonds ( figure 6 ) , so the discussion will focus on the vde . in addition , the criterion of equality of the negative of the homo energy ( ) with the scf vde proposed by baer and co - workers was examined , since they should be equal in exact ks theory . then , calculations of the functionals with the best performance with the dzvp2 basis set but now using the def2-svpd basis set are compared . the origins of differences with different functionals are discussed with the caveat that only two cu and two fe complexes are also examined . l bond lengths for [ cu(ncs)2 ] ( triangle ) , [ cu(sch3)2 ] ( square ) , fecl4 ( circle ) , and [ fe(sch3)4 ] ( diamond ) , using the dzvp2 basis set for different functionals in order of increasing ( short - range ) hf exchange . from left to right , the hybrid functionals ( open symbols ) are b3lyp * , b97 , b3lyp , pbe1pbe , m06 , and b(38hf)p86 ; the rs functionals ( solid symbols ) are bnl , cam - b3lyp , and lrc-pbeh ; and the highly optimized rs functionals ( gray symbols ) are b97 , b97x , and m11 . the symbols are connected by dotted lines to guide the eye , and the results for the fe complexes are shifted upward by 0.1 to avoid overlaps . l bond lengths ( gray line with error indicated approximately by width of line ) are also shown . the vde for these functionals generally increases with % hf exchange , despite having different exchange - correlation functionals ( figure 7 ) . thus , b(38hf)p86 , which was optimized against experimental cucl4 spin densities , agrees quite well for the cu complexes compared to experimental vde but is too large for the fe complexes , and generally none of the hybrid functionals can predict vde accurately for both the cu and fe complexes . in addition , is generally significantly lower than the scf vde in the hybrid functionals , although the agreement improves somewhat with the % hf exchange ( figure 7 ) . calculated vde from the difference in energy between the reduced form and a franck condon transition ( black ) and from the ( blue ) for [ cu(ncs)2 ] ( triangle ) , [ cu(sch3)2 ] ( square ) , fecl4 ( circle ) , and [ fe(sch3)4 ] ( diamond ) , using the dzvp2 basis set for hybrid functionals in order of increasing ( short - range ) hf exchange . from left to right , the functionals ( open symbols ) are b3lyp * , b97 , b3lyp , pbe1pbe , m06 , and b(38hf)p86 . the symbols are connected by dotted lines to guide the eye , and the results for the fe complexes are shifted upward by 3 ev to avoid overlaps . the experimental pes values ( gray line with error indicated approximately by width of line ) are also shown . the poor prediction of vde by the hybrid functionals may be due to ( among other things ) spin contamination , multireference effects , or errors in the exchange - correlation functional . spin contamination due to the bs approach can be ruled out since the expectation values of the total spin s ( calculated using m06/def2-svp ) of [ cul2 ] ( s = 1/2 ) , [ fel4 ] ( s = 5/2 ) , and [ fel4 ] ( s = 2 ) with the ligands studied here are equal to 0.76 , 8.77 , and 6.18 , respectively , which are almost the same as those of the pure states . in addition , since the homos in the cu and fe complexes are degenerate , multireference problems are possible . since empirically multireference character may be a problem in ccsd calculations when t1 diagnostic is greater than 0.02 or the largest t2 amplitude of the double excitations is greater than 0.2 , both conditions were checked ; the conditions for both diagnostics are satisfied for the reduced states but not always for the oxidized states ( table s4 ) . however , ccsd(t ) , which is often very effective in correcting for a single - reference treatment of weakly to moderately multireference problems , gives results that are in better agreement with experiment ( table s3 ) , indicating a single - reference treatment is possible . dft also appears less sensitive to multireference character than hf theory but , in a hybrid functional , is dependent on the amount of hf exchange included in the functional . in other words , while adding hf exchange tends to balance the self - interaction error in dft that leads to overdelocalization , it is expected to give worse performance on static correlation such as the overestimated spin polarization found in systems containing transition metals . since the vde generally becomes more positive with % hf exchange for both cu and fe complexes in the hybrid functionals so that no functional works well for both ( figure 7 ) ; another option is to look for a better , more flexible description of exchange in the density functional . of the less parametrized rs functionals ( bnl , cam - b3lyp , and lrc-pbeh ) , the bnl functional ( 0% sr hf ) appears to give a ground state with a different electron configuration from the b3lyp , m06 , and ccsd(t ) calculations for the vertical oxidation state of [ cu(sch3)2 ] and too low vde for both cu complexes , while both cam - b3lyp and lrc-pbeh with 20% sr hf exchange have reasonable ground states and vde for both cu complexes ( figure 8) , which supports that some hf exchange at short - range is necessary as others have concluded . however , all three functionals suffer from having what appears to be the incorrect electron configuration for the ground state for at least one of the fe complexes in the oxidized state : the ground states obtained by m06 , b3lyp , and ccsd(t ) are all symmetric with regard to distribution of the spins , while the ground states obtained by the rs functionals are generally asymmetric and are much like the electron configuration of the ground state obtained in a pure uhf calculation . consequently , the vdes are too high for the fe complexes . the of the less parametrized rs functionals ( bnl , cam - b3lyp , and lrc-pbeh ) are quite close to the vde ( figure s1 ) , although not so much for cam - b3lyp since it has only 65% lr hf exchange . of the highly optimized rs functionals ( b97 , b97x , and m11 ) , the incorrect electron configuration of the oxidized ground state is obtained for most of the cu and fe complexes , and the vdes are consistently too positive compared to experiment regardless of the amount of sr hf exchange ( i.e. , between 0 and 42.8% in figure 8) . since they also have slightly higher than vde ( figure s1 ) , the of the reduced state appears to have been increased too much . thus , even for the rs functionals , no functional using default parameters ( with the dzvp2 basis set ) works well for both cu and fe complexes , although cam - b3lyp and lrc-pbeh appear to work best since they at least give what appears to be the correct electron configuration of the ground state and reasonable vde for the cu complexes . calculated vde from the difference in energy between the reduced form and a franck condon transition for [ cu(ncs)2 ] ( triangle ) , [ cu(sch3)2 ] ( square ) , fecl4 ( circle ) , and [ fe(sch3)4 ] ( diamond ) , using the dzvp2 basis set for rs functionals in order of increasing ( short - range ) hf exchange . from left to right , the rs functionals ( solid symbols ) are bnl , cam - b3lyp , and lrc-pbeh , and the highly optimized rs functionals ( lighter colored symbols ) are b97 , b97x , and m11 . the symbols are connected by dotted lines to guide the eye , and the results for the fe complexes are shifted upward by 3 ev to avoid overlaps . the experimental pes values ( gray line with error indicated approximately by width of line ) are also shown . l bond length and vde ( tables 1 and 2 , respectively ) were also calculated using the def2-svpd basis set for b3lyp , m06 , cam - b3lyp , and lrc-pbeh using default parameters ; for comparison , appropriate values from experiment and ccsd and ccsd(t ) using the def2-tzvppd basis sets on the m06/def2-svp geometry are also given . highly optimized rs functionals were not investigated since the vdes are too high for both the cu and fe complexes . even with this larger basis set , although the rs separated functionals work well for the cu complexes , the vdes are too large for the fe complexes compared to experiment and give an incorrect ground state similar to the dzvp2 results upon oxidation . however , the lrc-pbeh appears superior to cam - b3lyp in the agreement of the vde with , as expected since the latter has only 65% lr hf exchange . cc / def2-tzvppd//m06/def2-svp except [ fe(sch3)4 ] are cc / def2-svpd//m06/dzvp2 . the ground state with incorrect electron configuration upon oxidation best dft values are in boldface ( see text ) . to investigate the source of the error in the rs functionals , the parameters altering the hf exchange were varied slightly using the def2-svpd basis set . rs functionals were not investigated because of the too high vde cited above and because the other parameters for these functionals were optimized for a specific amount of hf exchange . in addition , cam - b3lyp was not investigated because of the poor agreement of the vde with the , and the condition of 100% lr hf exchange is becoming widely accepted . for instance , baer and co - workers suggested that the range parameter is not necessarily universal to all molecules , while our results here indicate that some amount of short - range hf exchange is necessary . decreasing results in less hf exchange at intermediate ranges and as 0 , rs functionals became non - rs functionals , and rs hybrid functionals become non - rs hybrid functionals . thus , was reduced in lrc-pbeh from the default 0.2 a0 to 0.1 a0 , the correct ground state and much better agreement with experimental and ccsd(t ) vdes are obtained for the fe complexes . however , the default values give better vde for the cu complexes as well as better agreement of the with the vde for both cu and fe complexes . on the other hand , when the sr hf exchange was reduced to 10% and was kept at the default 0.2 a0 in lrc-pbeh , the vdes for the cu complexes are only slightly too low ( 0.1 to 0.2 ev ) while that for the fe complexes are slightly too high ( 0.07 ev ) with respect to the experimental values . in addition , the agreement of the with vde is somewhat better than when is reduced . together , this implies that the default value of is close to correct , while the altering amount of sr hf in lrc-pbeh for transition metals may be more important , with 10% representing a possible compromise for cu and fe complexes . furthermore , a good option may be to optimize the parameters for including range - separation into the dft exchange functional for each transition metal subject to certain additional criteria beyond the equality of with the scf vde such as those proposed by several workers . although no attempt is made here to determine the best criteria for optimization , the optimal value of 47.3% sr hf exchange found for cucl using the additional criteria of straight - line behavior of e(n ) , the energy as a function of a fractional electron number was examined for the cu complexes using the lrc-pbeh . however , the best agreement occurs when the sr hf exchange is closer to 20 than 47.3% and = 0.2 a0 for the cu complexes and 7.5% sr hf exchange and = 0.2 a0 are used for the fe complexes . in addition , spin - polarization effects may be a good criteria since for the open shell , high - spin fe complexes , spin polarization is generally important and a smaller amount of hf exchange is expected to improve performance , while for the closed shell , low - spin cu - complexes , spin polarization tends to be less important and the correction of self - interaction error by hf exchange may be more important . overall , the results here indicate that bs - dft with rs functionals and double- basis sets are able to model the electronic structure and geometry of two cu complexes , [ cu(ncs)2 ] and [ cu(sch3)2 ] , with metal ligand bonds found in the blue copper protein redox sites , as well as the two fe complexes used in previous studies for the fe s protein redox sites . based on comparisons with triple- basis sets with diffuse functions , the karlsruhe double- basis sets appear to be a good balance of size and accuracy for both geometry and energies as long as diffuse functions are used for energies . however , the vde can not be predicted reliably compared to experiment for both cu and fe complexes by hybrid density functionals . on the other hand , lrc-pbeh , a range - separated density functional , with 10% short - range and exact long - range hf exchange predicts vde better than the hybrid functionals for both metals , although even better values are obtained when differing amounts of short - range hf exchange are used based on the metal type . this suggests that parameters in rs functionals chosen to balance compensating errors ( i.e. , hf exchange vs self - interaction error in dft ) may differ between transition metals , for instance , because of the conflicting requirements of the low - spin , closed shell cu complexes versus the high - spin , open shell fe complexes . therefore , careful benchmarking of the dft functionals for redox energetics of smaller complexes against experimental and/or ccsd(t ) methods along with careful analysis of the ground states of both the oxidized and reduced forms still appears necessary for good vde of protein redox sites containing transition metals , especially when using rs functionals . moreover , the establishment of criteria for choosing different parameters for rs for transition metals is an important goal .

broken - symmetry density functional theory ( bs - dft ) calculations are assessed for redox energetics [ cu(sch3)2]1/0 , [ cu(ncs)2]1/0 , [ fecl4]1/0 , and [ fe(sch3)4]1/0 against vertical detachment energies ( vde ) from valence photoelectron spectroscopy ( pes ) , as a prelude to studies of metalloprotein analogs . the m06 and b3lyp hybrid functionals give vde that agree with the pes vde for the fe complexes , but both underestimate it by 400 mev for the cu complexes ; other hybrid functionals give vdes that are an increasing function of the amount of hartree fock ( hf ) exchange and so can not show good agreement for both cu and fe complexes . range - separated ( rs ) functionals appear to give a better distribution of hf exchange since the negative homo energy is approximately equal to the vdes but also give vdes dependent on the amount of hf exchange , sometimes leading to ground states with incorrect electron configurations ; the lrc-pbeh functional reduced to 10% hf exchange at short - range give somewhat better values for both , although still 150 mev too low for the cu complexes and 50 mev too high for the fe complexes . overall , the results indicate that while hf exchange compensates for self - interaction error in dft calculations of both cu and fe complexes , too much may lead to more sensitivity to nondynamical correlation in the spin - polarized fe complexes .

Introduction Methods Results and Discussion Conclusions

moreover , efforts have been made to develop range - separated ( rs ) functionals in which the hf exchange is treated differently at different ranges for further improving upon global hybrids . in particular , valence photoelectron spectroscopy ( pes ) of well - defined gaseous metal complexes has become important for testing the electronic structure and energetics , independent of environmental perturbations such as solvent , crystal field , or surrounding protein , since the calculation can be performed under the same conditions without any approximations for the environment . the calculated vertical ( vde ) and adiabatic ( ade ) detachment energies agree well with the gaseous pes data of [ 1fe ] , [ 2fe2s ] , and [ 4fe4s ] protein analogs , while the calculated percentage of ligand character of the fe 3d orbitals correlate well with ligand k - edge xas measurements . in the current article , cu s and cu n bonding interactions are investigated in the simple cu complexes , [ cux2 ] ( x = sch3 , ncs ; n = 1 and 0 ) , as a prelude to investigations of the type i copper site found in the blue copper proteins . since our long - term goal is to find functionals and basis sets that work well for the transition metals found in metalloprotein redox sites , which include mixed metal sites , the fe complexes , [ fex4 ] ( x = cl , sch3 ; n = 1 and 0 ) , which were used in our previous studies for redox sites of fe s proteins , we focus on contrasting the ability of the methods to handle the closed shell , low - spin cu complexes ( s = 1/2 ) with the open shell , high - spin fe complexes ( s = 5/2 and 2 ) rather than a statistical survey of many different transition - metal complexes . while a general criteria is good agreement of geometries with the x - ray crystallographic structures , our essential criteria is good agreement of energetics with pes electron detachment energies since the methods are being tested for redox calculations . spin - unrestricted dft calculations with broken - symmetry ( bs ) molecular orbital ( mo ) wavefunctions were performed for geometries and energies . the hybrid functionals tested with percentage hf exchange indicated in parentheses are the b3lyp gga ( 20% hf ) ; b3lyp * , a modified b3lyp gga ( 15% hf ) ; the b97 gga ( 19.43% hf ) ; the pbe1pbe gga ( 25% hf ) ; the m06 meta - gga ( 27% hf ) ; and the b(38hf)p86 gga ( 38% hf ) . in addition , one set of rs density functionals with percentage hf exchange at short - range ( sr ) and at long - range ( lr ) and range parameter indicated in parentheses tested are the bnl gga ( 0% sr , 100% lr , 0.33 a0 ) ; the cam - b3lyp gga ( 19% sr , 65% lr , 0.33 a0 ) ; and the lrc-pbeh gga ( 20% sr , 100% lr , 0.2 a0 ) . since the hf and kohn sham ( ks ) electron configurations of the ground state of open - shell cu and fe complexes were often different , both were used as initial electron configurations for the dft and cc calculations to help establish that the solution was not an excited state . all hybrid dft calculations were performed using the nwchem program package , the rs dft ( except lrc-pbeh ) , ccsd , and ccsd(t ) calculations utilized the gaussian09 program package , and the lrc-pbeh calculations utilized the q - chem program package . also , since the mo ( lumo ) that is oxidized has *cu s antibonding character ( consistent with s k - edge xas experiments of the redox site of the blue copper proteins ) , the cu the two degenerate homos of [ cu(ncs)2 ] are formed by interactions between the cu dyz and dxz orbitals and the higher - lying s lone - pair orbitals with *c s and n c character , even though the metal ligates to the n ; the asymmetric homos are stabilized by the electron back - donation into the high - lying unoccupied *n c orbitals , which increases the n c bonding character in the homos . thus , although oxidation of both [ cu(ncs)2 ] and [ cu(sch3)2 ] involve the s lone - pair electron , the ground state x band in the pes spectra ( figure 2 ) of [ cu(ncs)2 ] has a very different shape from [ cu(sch3)2 ] . finally , the decrease in the *cu n antibonding , *c s antibonding , and n c bonding character upon oxidation should lead to a decrease in the cu n and c s bond lengths and an increase in the n c bond lengths . however , the 6 - 31 g * * and 6 - 31(++)lg * * basis sets give significantly shorter bond lengths for cu(sch3)2 ] and [ cu(ncs)2 ] , which brings the calculated results further from experiment for [ cu(ncs)2 ] but closer for [ cu(sch3)2 ] ; however , the experimental m l bond length for the latter comes from [ cu(s - t - butyl)2 ] , which may be shorter than in [ cu(sch3)2 ] due to the better electron donating ability of the -s - t - butyl group than the -sch3 group that leads to stronger interactions with the cu . in addition , the m06 functional gives slightly shorter bond lengths than the b3lyp functional , so that the m l bond lengths for [ cu(ncs)2 ] ( triangle ) , [ cu(sch3)2 ] ( square ) , fecl4 ( circle ) , and [ fe(sch3)4 ] ( diamond ) , using the b3lyp ( open symbols ) and m06 ( solid symbols ) functionals as a function of basis set plotted on a scale of the logarithm of the number of basis functions in [ cu(ncs)2 ] . the vdes for the same cu and fe complexes are used to examine the redox energetics ( figure 5 ) . of the double- basis sets with the diffuse functions , the 6 - 31(++)lg * * and 6 - 31++g * * ( not shown ) basis sets do not improve agreement as much for the cu complexes as for the fe complexes , while def2-svpd is in good agreement with the triple- basis set results , consistent with the better performance of the karlsruhe def2 basis sets with diffuse functions in the m06 calculations of barrier heights , ionization potentials , and electron affinities . calculated vde for [ cu(ncs)2 ] ( triangle ) , [ cu(sch3)2 ] ( square ) , fecl4 ( circle ) , and [ fe(sch3)4 ] ( diamond ) , using the b3lyp ( open symbols ) and m06 ( solid symbols ) functionals as a function of a basis set plotted on a scale of the logarithm of the number of basis functions in [ cu(ncs)2 ] . the symbols are connected by dotted lines to guide the eye , and the results for the fe complexes are shifted upward by 3 ev to avoid overlaps . thus , dft calculations using def2-svpd agree well with those using the triple- basis sets with the diffuse functions for geometry and energetics for both the cu and fe complexes and appear to be a good balance of speed and accuracy for larger molecules . however , none of the dft results give good agreement for energetics compared to experiment for the cu complexes . in addition , the criterion of equality of the negative of the homo energy ( ) with the scf vde proposed by baer and co - workers was examined , since they should be equal in exact ks theory . l bond lengths for [ cu(ncs)2 ] ( triangle ) , [ cu(sch3)2 ] ( square ) , fecl4 ( circle ) , and [ fe(sch3)4 ] ( diamond ) , using the dzvp2 basis set for different functionals in order of increasing ( short - range ) hf exchange . the symbols are connected by dotted lines to guide the eye , and the results for the fe complexes are shifted upward by 0.1 to avoid overlaps . thus , b(38hf)p86 , which was optimized against experimental cucl4 spin densities , agrees quite well for the cu complexes compared to experimental vde but is too large for the fe complexes , and generally none of the hybrid functionals can predict vde accurately for both the cu and fe complexes . in addition , is generally significantly lower than the scf vde in the hybrid functionals , although the agreement improves somewhat with the % hf exchange ( figure 7 ) . calculated vde from the difference in energy between the reduced form and a franck condon transition ( black ) and from the ( blue ) for [ cu(ncs)2 ] ( triangle ) , [ cu(sch3)2 ] ( square ) , fecl4 ( circle ) , and [ fe(sch3)4 ] ( diamond ) , using the dzvp2 basis set for hybrid functionals in order of increasing ( short - range ) hf exchange . the symbols are connected by dotted lines to guide the eye , and the results for the fe complexes are shifted upward by 3 ev to avoid overlaps . spin contamination due to the bs approach can be ruled out since the expectation values of the total spin s ( calculated using m06/def2-svp ) of [ cul2 ] ( s = 1/2 ) , [ fel4 ] ( s = 5/2 ) , and [ fel4 ] ( s = 2 ) with the ligands studied here are equal to 0.76 , 8.77 , and 6.18 , respectively , which are almost the same as those of the pure states . in addition , since the homos in the cu and fe complexes are degenerate , multireference problems are possible . dft also appears less sensitive to multireference character than hf theory but , in a hybrid functional , is dependent on the amount of hf exchange included in the functional . in other words , while adding hf exchange tends to balance the self - interaction error in dft that leads to overdelocalization , it is expected to give worse performance on static correlation such as the overestimated spin polarization found in systems containing transition metals . since the vde generally becomes more positive with % hf exchange for both cu and fe complexes in the hybrid functionals so that no functional works well for both ( figure 7 ) ; another option is to look for a better , more flexible description of exchange in the density functional . of the less parametrized rs functionals ( bnl , cam - b3lyp , and lrc-pbeh ) , the bnl functional ( 0% sr hf ) appears to give a ground state with a different electron configuration from the b3lyp , m06 , and ccsd(t ) calculations for the vertical oxidation state of [ cu(sch3)2 ] and too low vde for both cu complexes , while both cam - b3lyp and lrc-pbeh with 20% sr hf exchange have reasonable ground states and vde for both cu complexes ( figure 8) , which supports that some hf exchange at short - range is necessary as others have concluded . however , all three functionals suffer from having what appears to be the incorrect electron configuration for the ground state for at least one of the fe complexes in the oxidized state : the ground states obtained by m06 , b3lyp , and ccsd(t ) are all symmetric with regard to distribution of the spins , while the ground states obtained by the rs functionals are generally asymmetric and are much like the electron configuration of the ground state obtained in a pure uhf calculation . consequently , the vdes are too high for the fe complexes . the of the less parametrized rs functionals ( bnl , cam - b3lyp , and lrc-pbeh ) are quite close to the vde ( figure s1 ) , although not so much for cam - b3lyp since it has only 65% lr hf exchange . of the highly optimized rs functionals ( b97 , b97x , and m11 ) , the incorrect electron configuration of the oxidized ground state is obtained for most of the cu and fe complexes , and the vdes are consistently too positive compared to experiment regardless of the amount of sr hf exchange ( i.e. thus , even for the rs functionals , no functional using default parameters ( with the dzvp2 basis set ) works well for both cu and fe complexes , although cam - b3lyp and lrc-pbeh appear to work best since they at least give what appears to be the correct electron configuration of the ground state and reasonable vde for the cu complexes . calculated vde from the difference in energy between the reduced form and a franck condon transition for [ cu(ncs)2 ] ( triangle ) , [ cu(sch3)2 ] ( square ) , fecl4 ( circle ) , and [ fe(sch3)4 ] ( diamond ) , using the dzvp2 basis set for rs functionals in order of increasing ( short - range ) hf exchange . the symbols are connected by dotted lines to guide the eye , and the results for the fe complexes are shifted upward by 3 ev to avoid overlaps . highly optimized rs functionals were not investigated since the vdes are too high for both the cu and fe complexes . even with this larger basis set , although the rs separated functionals work well for the cu complexes , the vdes are too large for the fe complexes compared to experiment and give an incorrect ground state similar to the dzvp2 results upon oxidation . however , the lrc-pbeh appears superior to cam - b3lyp in the agreement of the vde with , as expected since the latter has only 65% lr hf exchange . to investigate the source of the error in the rs functionals , the parameters altering the hf exchange were varied slightly using the def2-svpd basis set . rs functionals were not investigated because of the too high vde cited above and because the other parameters for these functionals were optimized for a specific amount of hf exchange . however , the default values give better vde for the cu complexes as well as better agreement of the with the vde for both cu and fe complexes . on the other hand , when the sr hf exchange was reduced to 10% and was kept at the default 0.2 a0 in lrc-pbeh , the vdes for the cu complexes are only slightly too low ( 0.1 to 0.2 ev ) while that for the fe complexes are slightly too high ( 0.07 ev ) with respect to the experimental values . although no attempt is made here to determine the best criteria for optimization , the optimal value of 47.3% sr hf exchange found for cucl using the additional criteria of straight - line behavior of e(n ) , the energy as a function of a fractional electron number was examined for the cu complexes using the lrc-pbeh . however , the best agreement occurs when the sr hf exchange is closer to 20 than 47.3% and = 0.2 a0 for the cu complexes and 7.5% sr hf exchange and = 0.2 a0 are used for the fe complexes . in addition , spin - polarization effects may be a good criteria since for the open shell , high - spin fe complexes , spin polarization is generally important and a smaller amount of hf exchange is expected to improve performance , while for the closed shell , low - spin cu - complexes , spin polarization tends to be less important and the correction of self - interaction error by hf exchange may be more important . overall , the results here indicate that bs - dft with rs functionals and double- basis sets are able to model the electronic structure and geometry of two cu complexes , [ cu(ncs)2 ] and [ cu(sch3)2 ] , with metal ligand bonds found in the blue copper protein redox sites , as well as the two fe complexes used in previous studies for the fe s protein redox sites . however , the vde can not be predicted reliably compared to experiment for both cu and fe complexes by hybrid density functionals . on the other hand , lrc-pbeh , a range - separated density functional , with 10% short - range and exact long - range hf exchange predicts vde better than the hybrid functionals for both metals , although even better values are obtained when differing amounts of short - range hf exchange are used based on the metal type . , hf exchange vs self - interaction error in dft ) may differ between transition metals , for instance , because of the conflicting requirements of the low - spin , closed shell cu complexes versus the high - spin , open shell fe complexes .

joint hypermobility syndrome ( jhs ) is a hereditary connective tissue disorder , characterized by musculoskeletal pain and an excessive range of motion in joints ( 2 ) . as there are no laboratory tests to indicate jhs ( 3 ) , it is usually subjectively assessed using the brighton criteria ( 4 ) , which include the beighton score ( 5 ) for joint hypermobility . it has been reported that symptomatic joint hypermobility affects around 5% of women and 0.6% of men ( 6 ) . the prevalence of jhs amongst those attending rheumatology and physiotherapy clinics has been estimated to be between 30% and 60% and is higher in non - caucasian populations ( 7,8 ) . however , the diverse and fluctuating symptoms associated with jhs may easily be attributed to other causes and the true prevalence of jhs may be much higher than previously estimated . it has been suggested that many patients presenting with painful non - inflammatory musculoskeletal problems may have unrecognized jhs ( 9 ) . although most individuals exhibiting joint hypermobility do not experience problems , a diagnosis of jhs may be given when symptoms such as arthralgia , proprioception difficulties , fatigue , soft tissue injury and joint instability are observed in the absence of genetic markers to indicate disorders such as osteogenesis imperfecta or marfan syndrome ( 10 ) . jhs , osteogenesis imperfecta , marfan syndrome and ehlers danlos syndrome share many symptoms and many experts now consider jhs to be indistinguishable from ehlers danlos syndrome , hypermobility type ( 11 ) . in this article primary - care practitioners are usually the patients first point of contact on entering the health - care system . they can do much to assist individuals to effectively self - manage their condition ( 12 ) and refer patients for appropriate secondary care such as physiotherapy , although the effectiveness of physiotherapy has yet to be established due to the lack of high - quality research in this area ( 13 ) . the aim of the current investigation is to examine patients lived experience of jhs , their views and experiences of jhs diagnosis and management . four focus groups were conducted between january and february 2013 in four locations in the uk . participants were recruited via physiotherapy services at two national health service ( nhs ) trusts , local members of the hypermobility syndromes association ( hmsa ) and patients who had previously expressed an interest in assisting with research activity at two university locations . eligible participants were aged 18 years or over , had previously received a diagnosis of jhs ( including ehlers danlos syndrome , hypermobility type ) , had attended physiotherapy within the preceding 12 months and were able to speak english . individuals with other known musculoskeletal pathology causing pain , particularly osteoarthritis and inflammatory musculoskeletal disease such as rheumatoid arthritis , were excluded . the purposive sampling strategy aimed for diversity with regard to age , gender , socio - economic situation and geographical location to capture maximum variation in views and experiences . ethical approval was obtained from the north east nhs research ethics committee ( 12/ne/0307 ) and all participants gave written consent . data reported in this article were collected from focus groups with individuals with jhs , aimed at developing a physiotherapy intervention for jhs management . topic guides used to facilitate discussions covered issues of living with jhs , day - to - day self - management and provision of support for symptom management . in line with an inductive approach , topic guides were revised in the light of emerging findings . the focus groups were conducted in non - clinical settings , facilitated by two researchers ( stp and jph ) , and open - ended questioning techniques were used to elicit participants own experiences and views . all focus groups were audio - recorded , fully transcribed , anonymized , checked for accuracy and imported into a qualitative software package ( nvivo 10 ) to aid data analysis . thematic analysis , using the constant comparison technique ( 14 ) , was used to scrutinize the data to identify and analyse patterns across the dataset and the data were scrutinized for negative cases . transcripts were examined on a line - by - line basis , with codes being assigned to segments of the data and an initial coding frame developed . rht and jph independently coded transcripts and any discrepancies were discussed to achieve a coding consensus and maximize rigour . the emergent themes were discussed by the multi - disciplinary research team to ensure credibility and confirmability . scrutiny of the data showed that data saturation had been reached at the end of the analysis , such that no new themes were arising from the data ( 15 ) . data reported in this article were collected from focus groups with individuals with jhs , aimed at developing a physiotherapy intervention for jhs management . topic guides used to facilitate discussions covered issues of living with jhs , day - to - day self - management and provision of support for symptom management . in line with an inductive approach , topic guides were revised in the light of emerging findings . the focus groups were conducted in non - clinical settings , facilitated by two researchers ( stp and jph ) , and open - ended questioning techniques were used to elicit participants own experiences and views . all focus groups were audio - recorded , fully transcribed , anonymized , checked for accuracy and imported into a qualitative software package ( nvivo 10 ) to aid data analysis . thematic analysis , using the constant comparison technique ( 14 ) , was used to scrutinize the data to identify and analyse patterns across the dataset and the data were scrutinized for negative cases . transcripts were examined on a line - by - line basis , with codes being assigned to segments of the data and an initial coding frame developed . rht and jph independently coded transcripts and any discrepancies were discussed to achieve a coding consensus and maximize rigour . the emergent themes were discussed by the multi - disciplinary research team to ensure credibility and confirmability . scrutiny of the data showed that data saturation had been reached at the end of the analysis , such that no new themes were arising from the data ( 15 ) . twenty - five individuals with jhs participated in the focus groups , aged 1966 years ( mean : 38.2 years ) , 22 were female and 23 were of the analysis led to the development of four key interrelated themes : the impact of jhs , jhs as a poorly understood condition , receiving a diagnosis and jhs management and self - care. verbatim extracts are provided to illustrate the findings . participants characteristics and demographic information relating to the participants of the four focus groups , carried out between january and february 2013 ( total n = 25 ) ses was measured as index of multiple deprivation ( imd ) quintile from home postcode . participants described in detail the impact of jhs , which included fatigue , pain and proprioception problems : day in day out you re managing your pain and it s a lot of pain , it s a dull ache and it makes you sleepy and it makes you tired and you re exhausted ( female g , age 30 , fg1 ) . it s on your mind the whole time because i m constantly thinking about where my hands and feet are ( female g , age 48 , fg2 ) . recurring joint dislocation and cycles of injury and recovery were common and participants frequently talked about the need to modify or restrict behaviours and activities . ... it s just difficult to know how much to push yourself because then you are worried about injuring and then you re setting yourself back , it s a vicious cycle really ( female b , age 27 , fg3 ) . however , participants acknowledged that the impact and consequences of these varied symptoms were different for each patient , with one participant noting , all of us are probably so different yet we re categorised as the same ( female d , age 21 , fg1 ) . thus , some participants found living with jhs symptoms to be very debilitating , ( male e , age 36 , fg3 ) , whilst others were determined to persevere with their chosen activities , in spite of their symptoms : i teach like rock - climbing , surfing , body boarding and all of that stuff , like , and i m not going to stop doing it because i m in pain like you ca nt live your whole life with pain dictating what you can and ca nt do ( female g , age 45 , fg3 ) . although jhs is characterized by pain , participants alluded to the complexity of their pain experience , for example , describing difficulties in distinguishing between chronic and acute pain , and in recognizing how or ifinjuries had occurred : how do we know whether we ve injured something ? because we ve got pain all the time ( female c , age 40 , fg1 ) . participants also described how prior experiences of repeated injuries led to heightened levels of anxiety and catastrophizing about future injuries : i feel like i m in a constant state of anxiety , waiting for the next injury and trying to pre - empt anything that s going to cause it ( female g , age 48 , fg2 ) . oh my god is this going to be like this for the next 60 years of my life? ( female b , age 27 , fg3 ) . participants perceived jhs to be a condition that was poorly understood by health professionals , and within society more widely . i think i was described as a biomechanical conundrum by one of the physiotherapists i saw [ ] and this is what i found repeated over and over again , that hypermobility should nt be causing pain , it s just the way you are ( female c , age 53 , fg2 ) . participants described feeling stigmatized and fraudulent : i m really struggling , but because of people s expectations and their perceptions and you do nt want to ruin that , you do nt want people to start thinking oh well , you know , [ unclear 1.02.47 ] , we do nt employ people with disabilities because this is what happens ( female c , age 40 , fg1 ) . ( female d , age 21 , fg1 ) . and felt they had been blamed for their symptoms : when i was at school i just had to sit at the side while they were doing all the games , they sort of almost , i felt they were blaming it on me the odd one out ( female b , age 34 , fg2 ) and tried to hide their experiences and appear normal : it s so exhausting mentally and physically to try and appear to be normal and do normal things throughout the day with everybody and pretend it s alright ( female g , age 48 , fg2 ) . participants felt that the unpredictable , diverse , evolving and fluctuating nature of their symptoms exacerbated others misunderstanding of the nature of jhs and contributed to a lack of social support : if you re inconsistent as well , they sort of go , she was alright with that last week , why is it this week she s saying that , you know , that s going to be difficult for her today ( female c , age 53 , fg2 ) . many participants reported lengthy diagnosis trajectories , and being treated for individual symptoms ( e.g. pain ) rather than jhs : we ve all been passed from pillar to post where people do nt recognise it or they just attribute a pain to something else , when a snap kind of diagnosis just comes out of the air and you know , you progress from there ( female g , age 45 , fg3 ) . often , obtaining a correct diagnosis was a coincidental occurrence : it was only because a locum happened to be in the day i went in because my gp was off sick , and he just started saying , well , to start bending everything but if had nt been for him i would nt have been put on the right track , because otherwise what other route do you really have if it s not through your gp ? ( female b , age 27 , fg3 ) . receiving a diagnosis was considered necessary in order to access appropriate care pathways , for example , referral for physiotherapy for jhs rather than for an acute single joint problem : i was originally seen by a physio who had nt diagnosed with the hypermobility and then went back to a musculo - skeletal specialist who then put me forward to specialist hypermobility physiotherapist and since then it s been amazing ; i feel like it s been worthwhile and it felt like the right thing to do and i ve been really enjoying it ( female b , age 27 , fg3 ) . in addition , a diagnosis helped to validate participants experiences and was psychologically helpful : getting a diagnosis on paper , this is what s wrong with me ? i mean that helped me hugely psychologically ( female a , age 60 , fg2 ) . a diagnosis in itself , however , still did not guarantee beneficial treatment : when i was first diagnosed , i was nt , i really felt i was nt given that much information about the condition [ ] and it just seemed to be all exercises they d given me at the time seemed to make me worse ( female b , age 27 , fg3 ) . similarly , a diagnosis did not necessarily lead to greater understanding and support from others in the participants social networks : there are people who do nt feel it s a genuine diagnosis , that it s something psychological , that you just need to be a bit braver ( female a , age 21 , fg2 ) . education for health professionals was a key issue for participants , to facilitate timely diagnosis of , and referral for , jhs . thus , education for health professionals was a prerequisite for diagnosis , and diagnosis was a prerequisite for participants to access education . participants recognized that individuals with jhs also need education , in order to find ways to self - manage their condition and to understand and engage with prescribed treatments . i suppose it s where someone who does nt really know about it , they ve got to learn about it first because you ca nt tell someone to do it [ ie engage with a particular treatment ] if they do nt understand it ( female d , age 21 , fg1 ) . participants also felt that they could provide valuable information about the nature of jhs to clinicians : i think actually it s the health professionals that need educating [ . ] there s lots of things i still need to know about hypermobility , but on the flip side i do think it s the health professionals that need to know more ( female g , age 42 , fg3 ) . participants described learning and understanding about jhs as a two - way process : so i think i get a huge amount of enlightenment from her [ the physiotherapist ] , and i lend her books and she lends me books about hypermobility and all that helps ( female d , age 54 , fg2 ) . participants accepted that treatment should aim to manage symptoms rather than provide a cure : it s all about them helping you to manage your pain , rather than cure it ( female , age 44 , fg1 ) . participants recognized the importance of self - care activities such as appropriate exercise , once a diagnosis had been received and a clear understanding of jhs had been developed . participants also recognised that activity pacing is really key to managing jhs symptoms ( female b , aged 32 ) : i have this balancing act , if i do too much it all hurts , do nt do enough , it all hurts , do it just right , i m okay ( male a , age 50 , fg1 ) . participants sought to deepen their understanding of jhs and access to health professionals who understood the nature of jhs in order to take a whole - body approach to self - care over a life course : the more you see the physio who knows what they re talking about the more you understand how the body works and the more you can apply your own sort of thinking to what you re doing in your exercises ( female e , age 44 , fg1 ) . it s got to be holistic , it really has to be ( female d , age 21 , fg1 ) . participants also felt that education about jhs should be provided at an early age , and therefore , early diagnosis was necessary : i m only 19 now but if you d have said that when i was 16 , i might not be in as much pain as i am now [ ] so if like if they d have told me more about how to treat it back then it probably would nt be as bad as it is now ( female f , age 19 , fg3 ) . participants described in detail the impact of jhs , which included fatigue , pain and proprioception problems : day in day out you re managing your pain and it s a lot of pain , it s a dull ache and it makes you sleepy and it makes you tired and you re exhausted ( female g , age 30 , fg1 ) . it s on your mind the whole time because i m constantly thinking about where my hands and feet are ( female g , age 48 , fg2 ) . recurring joint dislocation and cycles of injury and recovery were common and participants frequently talked about the need to modify or restrict behaviours and activities . ... it s just difficult to know how much to push yourself because then you are worried about injuring and then you re setting yourself back , it s a vicious cycle really ( female b , age 27 , fg3 ) . however , participants acknowledged that the impact and consequences of these varied symptoms were different for each patient , with one participant noting , all of us are probably so different yet we re categorised as the same ( female d , age 21 , fg1 ) . thus , some participants found living with jhs symptoms to be very debilitating , ( male e , age 36 , fg3 ) , whilst others were determined to persevere with their chosen activities , in spite of their symptoms : i teach like rock - climbing , surfing , body boarding and all of that stuff , like , and i m not going to stop doing it because i m in pain like you ca nt live your whole life with pain dictating what you can and ca nt do ( female g , age 45 , fg3 ) . although jhs is characterized by pain , participants alluded to the complexity of their pain experience , for example , describing difficulties in distinguishing between chronic and acute pain , and in recognizing how or ifinjuries had occurred : how do we know whether we ve injured something ? because we ve got pain all the time ( female c , age 40 , fg1 ) . participants also described how prior experiences of repeated injuries led to heightened levels of anxiety and catastrophizing about future injuries : i feel like i m in a constant state of anxiety , waiting for the next injury and trying to pre - empt anything that s going to cause it ( female g , age 48 , fg2 ) . oh my god is this going to be like this for the next 60 years of my life? ( female b , age 27 , fg3 ) . participants perceived jhs to be a condition that was poorly understood by health professionals , and within society more widely . i think i was described as a biomechanical conundrum by one of the physiotherapists i saw [ ] and this is what i found repeated over and over again , that hypermobility should nt be causing pain , it s just the way you are ( female c , age 53 , fg2 ) . i m really struggling , but because of people s expectations and their perceptions and you do nt want to ruin that , you do nt want people to start thinking oh well , you know , [ unclear 1.02.47 ] , we do nt employ people with disabilities because this is what happens ( female c , age 40 , fg1 ) . they think you re trying to cheat out of doing something ( female d , age 21 , fg1 ) . and felt they had been blamed for their symptoms : when i was at school i just had to sit at the side while they were doing all the games , they sort of almost , i felt they were blaming it on me the odd one out ( female b , age 34 , fg2 ) and tried to hide their experiences and appear normal : it s so exhausting mentally and physically to try and appear to be normal and do normal things throughout the day with everybody and pretend it s alright ( female g , age 48 , fg2 ) . participants felt that the unpredictable , diverse , evolving and fluctuating nature of their symptoms exacerbated others misunderstanding of the nature of jhs and contributed to a lack of social support : if you re inconsistent as well , they sort of go , she was alright with that last week , why is it this week she s saying that , you know , that s going to be difficult for her today ( female c , age 53 , fg2 ) . many participants reported lengthy diagnosis trajectories , and being treated for individual symptoms ( e.g. pain ) rather than jhs : we ve all been passed from pillar to post where people do nt recognise it or they just attribute a pain to something else , when a snap kind of diagnosis just comes out of the air and you know , you progress from there ( female g , age 45 , fg3 ) . often , obtaining a correct diagnosis was a coincidental occurrence : it was only because a locum happened to be in the day i went in because my gp was off sick , and he just started saying , well , to start bending everything but if had nt been for him i would nt have been put on the right track , because otherwise what other route do you really have if it s not through your gp ? ( female b , age 27 , fg3 ) . receiving a diagnosis was considered necessary in order to access appropriate care pathways , for example , referral for physiotherapy for jhs rather than for an acute single joint problem : i was originally seen by a physio who had nt diagnosed with the hypermobility and then went back to a musculo - skeletal specialist who then put me forward to specialist hypermobility physiotherapist and since then it s been amazing ; i feel like it s been worthwhile and it felt like the right thing to do and i ve been really enjoying it ( female b , age 27 , fg3 ) . in addition , a diagnosis helped to validate participants experiences and was psychologically helpful : getting a diagnosis on paper , this is what s wrong with me ? i mean that helped me hugely psychologically ( female a , age 60 , fg2 ) . a diagnosis in itself , however , still did not guarantee beneficial treatment : when i was first diagnosed , i was nt , i really felt i was nt given that much information about the condition [ ] and it just seemed to be all exercises they d given me at the time seemed to make me worse ( female b , age 27 , fg3 ) . similarly , a diagnosis did not necessarily lead to greater understanding and support from others in the participants social networks : there are people who do nt feel it s a genuine diagnosis , that it s something psychological , that you just need to be a bit braver ( female a , age 21 , fg2 ) . education for health professionals was a key issue for participants , to facilitate timely diagnosis of , and referral for , jhs . thus , education for health professionals was a prerequisite for diagnosis , and diagnosis was a prerequisite for participants to access education . participants recognized that individuals with jhs also need education , in order to find ways to self - manage their condition and to understand and engage with prescribed treatments . i suppose it s where someone who does nt really know about it , they ve got to learn about it first because you ca nt tell someone to do it [ ie engage with a particular treatment ] if they do nt understand it ( female d , age 21 , fg1 ) . participants also felt that they could provide valuable information about the nature of jhs to clinicians : i think actually it s the health professionals that need educating [ . ] there s lots of things i still need to know about hypermobility , but on the flip side i do think it s the health professionals that need to know more ( female g , age 42 , fg3 ) . participants described learning and understanding about jhs as a two - way process : so i think i get a huge amount of enlightenment from her [ the physiotherapist ] , and i lend her books and she lends me books about hypermobility and all that helps ( female d , age 54 , fg2 ) . participants accepted that treatment should aim to manage symptoms rather than provide a cure : it s all about them helping you to manage your pain , rather than cure it ( female , age 44 , fg1 ) . participants recognized the importance of self - care activities such as appropriate exercise , once a diagnosis had been received and a clear understanding of jhs had been developed . participants also recognised that activity pacing is really key to managing jhs symptoms ( female b , aged 32 ) : i have this balancing act , if i do too much it all hurts , do nt do enough , it all hurts , do it just right , i m okay ( male a , age 50 , fg1 ) . participants sought to deepen their understanding of jhs and access to health professionals who understood the nature of jhs in order to take a whole - body approach to self - care over a life course : the more you see the physio who knows what they re talking about the more you understand how the body works and the more you can apply your own sort of thinking to what you re doing in your exercises ( female e , age 44 , fg1 ) . it s got to be holistic , it really has to be ( female d , age 21 , fg1 ) . participants also felt that education about jhs should be provided at an early age , and therefore , early diagnosis was necessary : i m only 19 now but if you d have said that when i was 16 , i might not be in as much pain as i am now [ ] so if like if they d have told me more about how to treat it back then it probably would nt be as bad as it is now ( female f , age 19 , fg3 ) . education for health professionals was a key issue for participants , to facilitate timely diagnosis of , and referral for , jhs . thus , education for health professionals was a prerequisite for diagnosis , and diagnosis was a prerequisite for participants to access education . participants recognized that individuals with jhs also need education , in order to find ways to self - manage their condition and to understand and engage with prescribed treatments . i suppose it s where someone who does nt really know about it , they ve got to learn about it first because you ca nt tell someone to do it [ ie engage with a particular treatment ] if they do nt understand it ( female d , age 21 , fg1 ) . participants also felt that they could provide valuable information about the nature of jhs to clinicians : i think actually it s the health professionals that need educating [ . ] there s lots of things i still need to know about hypermobility , but on the flip side i do think it s the health professionals that need to know more ( female g , age 42 , fg3 ) . participants described learning and understanding about jhs as a two - way process : so i think i get a huge amount of enlightenment from her [ the physiotherapist ] , and i lend her books and she lends me books about hypermobility and all that helps ( female d , age 54 , fg2 ) . participants accepted that treatment should aim to manage symptoms rather than provide a cure : it s all about them helping you to manage your pain , rather than cure it ( female , age 44 , fg1 ) . participants recognized the importance of self - care activities such as appropriate exercise , once a diagnosis had been received and a clear understanding of jhs had been developed . participants also recognised that activity pacing is really key to managing jhs symptoms ( female b , aged 32 ) : i have this balancing act , if i do too much it all hurts , do nt do enough , it all hurts , do it just right , i m okay ( male a , age 50 , fg1 ) . participants sought to deepen their understanding of jhs and access to health professionals who understood the nature of jhs in order to take a whole - body approach to self - care over a life course : the more you see the physio who knows what they re talking about the more you understand how the body works and the more you can apply your own sort of thinking to what you re doing in your exercises ( female e , age 44 , fg1 ) . it s got to be holistic , it really has to be ( female d , age 21 , fg1 ) . participants also felt that education about jhs should be provided at an early age , and therefore , early diagnosis was necessary : i m only 19 now but if you d have said that when i was 16 , i might not be in as much pain as i am now [ ] so if like if they d have told me more about how to treat it back then it probably would nt be as bad as it is now ( female f , age 19 , fg3 ) . pain , fatigue and repeated episodes of injury were particularly challenging features of the wide - ranging symptoms of jhs . bury ( 16 ) describes how chronic illness brings about biographical disruption , and that the illness or disease requires the individual to make certain changes to their lives ( 17,18 ) . in our study , participants lives were disrupted repeatedly by fluctuating symptoms and cycles of injury and recovery . participants described the complex and individual nature of their pain experience and their responses to unpredictable symptoms . for some , previous episodes of pain or injury led to heightened levels of anxiety and catastrophizing about the future and their symptoms required them to modify or restrict their activities , while others refused to be dictated to by their symptoms . patients perceived jhs to be often poorly understood by health professionals and those in their wider social environment and reported feeling fraudulent and blamed for their symptoms . participants felt stigmatized , marked out as different ( 19) and alien to others in society and found it exhausting to try to appear normal. stigma can have wide - ranging negative biopsychosocial consequences including reduced participation in activities and an exacerbation of disability and disease , for example , through delayed diagnosis and treatment ( 20 ) . a lack of awareness of jhs amongst health professionals meant that obtaining a diagnosis of jhs was often difficult . participants had often been misdiagnosed or treated for symptoms ( e.g. pain ) rather than the condition itself and passed from pillar to post often until a serendipitous or coincidental diagnosis of jhs was made . previous studies have reported similar findings ( 9 ) and others have emphasized that being understood and believed by health professionals and significant others , along with social support , is instrumental in long - term pain management by facilitating ( or inhibiting ) pain acceptance ( 21,22 ) . participants highlighted the importance of a correct diagnosis in facilitating access to appropriate health care , support and education and helping to validate participants experiences . having lived with problematic symptoms of jhs sometimes for long periods of time , it is possible that the receipt of a diagnosis represented the beginning of a process during which they were able to understand and make sense of their symptoms , obtain appropriate treatment and subsequently find ways to self - manage the condition . participants experiences resonate with williams ( 23 ) who uses the term narrative re - construction to describe how individuals with chronic illness re - establish order and meaning in their lives . primary - care practitioners play an important role in helping patients to understand and self - manage long - term health conditions . a prerequisite of being able to provide support for patients is that primary - care practitioners are able to recognize and diagnose jhs and to refer patients to jhs - trained specialists . without a correct diagnosis , unsuitable treatments or information currently , primary - care practitioners and other health professionals such as physiotherapists do not routinely receive training related to jhs ( 9 ) and the validity of diagnostic criteria ( such as the beighton score ) has recently been questioned ( 25 ) . education and access to information was important for participants to allow them to make informed health - care choices . participants described the value of collaborating with health professionals and reciprocal learning between practitioners and patients in order to develop effective self - care strategies , and for holistic long - term management . recognizing that jhs would not be cured , participants felt that health professionals and those with jhs could potentially develop a deeper understanding of jhs management by learning from each other as a teachable dyad ( 26 , p. 682). to ensure validity , participants were recruited from four different geographical locations in the uk and had the experience of different health - care services . participants provided rich personal narratives of the day - to - day experiences of living with and managing jhs from patients perspectives and data analysis demonstrated general consensus and shared experiences . a limitation of this research is that participants were recruited through jhs support groups or via physiotherapy services for jhs and may therefore have been more active or aware of their condition than , for example , newly diagnosed individuals . the focus groups formed one phase of a study to develop a physiotherapy intervention to manage jhs . it was clear that the lived experience of those with jhs that emerged from the data analysis was an important story , which to date has received little attention . the issues raised in these focus groups highlight the need for more in - depth research in this area . future research could conduct interviews to provide a more detailed investigation of personal accounts of living with jhs , in particular those newly diagnosed with jhs . individuals with jhs experience diverse , fluctuating and often debilitating symptoms and diagnosis is often slow . without a correct diagnosis , following diagnosis , access to jhs - trained health professionals could help patients to effectively manage their condition over a life course and receive psychological support when needed . patients and practitioners may be able to learn from one another and so assist in developing a deeper understanding of a currently poorly understood condition . to ensure validity , participants were recruited from four different geographical locations in the uk and had the experience of different health - care services . participants provided rich personal narratives of the day - to - day experiences of living with and managing jhs from patients perspectives and data analysis demonstrated general consensus and shared experiences . a limitation of this research is that participants were recruited through jhs support groups or via physiotherapy services for jhs and may therefore have been more active or the focus groups formed one phase of a study to develop a physiotherapy intervention to manage jhs . it was clear that the lived experience of those with jhs that emerged from the data analysis was an important story , which to date has received little attention . the issues raised in these focus groups highlight the need for more in - depth research in this area . future research could conduct interviews to provide a more detailed investigation of personal accounts of living with jhs , in particular those newly diagnosed with jhs . individuals with jhs experience diverse , fluctuating and often debilitating symptoms and diagnosis is often slow . without a correct diagnosis , following diagnosis , access to jhs - trained health professionals could help patients to effectively manage their condition over a life course and receive psychological support when needed . patients and practitioners may be able to learn from one another and so assist in developing a deeper understanding of a currently poorly understood condition . funding : this project was funded by the national institute for health research health technology assessment programme ( project number 10/98/05 ) . the views and opinions expressed therein are those of the authors and do not necessarily reflect those of the health technology assessment programme , nihr , nhs or the department of health . ethical approval : this study received ethical approval from the north east nhs research ethics committee ( 12/ne/0307 ) .

background.musculoskeletal problems are common reasons for seeking primary health care . it has been suggested that many people with everyday non - inflammatory musculoskeletal problems may have undiagnosed joint hypermobility syndrome ( jhs ) , a complex multi - systemic condition . jhs is characterized by joint laxity , pain , fatigue and a wide range of other symptoms . physiotherapy is usually the preferred treatment option for jhs , although diagnosis can be difficult . the lived experience of those with jhs requires investigation.objective.the aim of the study was to examine patients lived experience of jhs , their views and experiences of jhs diagnosis and management.methods.focus groups in four locations in the uk were convened , involving 25 participants with a prior diagnosis of jhs . the focus groups were audio recorded , fully transcribed and analysed using the constant comparative method to inductively derive a thematic account of the data.results.pain , fatigue , proprioception difficulties and repeated cycles of injury were among the most challenging features of living with jhs . participants perceived a lack of awareness of jhs from health professionals and more widely in society and described how diagnosis and access to appropriate health - care services was often slow and convoluted . education for patients and health professionals was considered to be essential.conclusions.timely diagnosis , raising awareness and access to health professionals who understand jhs may be particularly instrumental in helping to ameliorate symptoms and help patients to self - manage their condition . physiotherapists and other health professionals should receive training to provide biopsychosocial support for people with this condition .

Introduction Methods Procedure Data analysis Results The impact of JHS JHSa poorly understood condition Receiving a diagnosis JHS management and self-care Education Identifying specific self-care activities Discussion and conclusions Strengths and limitations Implications for practice Declaration

joint hypermobility syndrome ( jhs ) is a hereditary connective tissue disorder , characterized by musculoskeletal pain and an excessive range of motion in joints ( 2 ) . as there are no laboratory tests to indicate jhs ( 3 ) , it is usually subjectively assessed using the brighton criteria ( 4 ) , which include the beighton score ( 5 ) for joint hypermobility . it has been reported that symptomatic joint hypermobility affects around 5% of women and 0.6% of men ( 6 ) . the prevalence of jhs amongst those attending rheumatology and physiotherapy clinics has been estimated to be between 30% and 60% and is higher in non - caucasian populations ( 7,8 ) . however , the diverse and fluctuating symptoms associated with jhs may easily be attributed to other causes and the true prevalence of jhs may be much higher than previously estimated . it has been suggested that many patients presenting with painful non - inflammatory musculoskeletal problems may have unrecognized jhs ( 9 ) . although most individuals exhibiting joint hypermobility do not experience problems , a diagnosis of jhs may be given when symptoms such as arthralgia , proprioception difficulties , fatigue , soft tissue injury and joint instability are observed in the absence of genetic markers to indicate disorders such as osteogenesis imperfecta or marfan syndrome ( 10 ) . jhs , osteogenesis imperfecta , marfan syndrome and ehlers danlos syndrome share many symptoms and many experts now consider jhs to be indistinguishable from ehlers danlos syndrome , hypermobility type ( 11 ) . in this article primary - care practitioners are usually the patients first point of contact on entering the health - care system . they can do much to assist individuals to effectively self - manage their condition ( 12 ) and refer patients for appropriate secondary care such as physiotherapy , although the effectiveness of physiotherapy has yet to be established due to the lack of high - quality research in this area ( 13 ) . the aim of the current investigation is to examine patients lived experience of jhs , their views and experiences of jhs diagnosis and management . four focus groups were conducted between january and february 2013 in four locations in the uk . eligible participants were aged 18 years or over , had previously received a diagnosis of jhs ( including ehlers danlos syndrome , hypermobility type ) , had attended physiotherapy within the preceding 12 months and were able to speak english . individuals with other known musculoskeletal pathology causing pain , particularly osteoarthritis and inflammatory musculoskeletal disease such as rheumatoid arthritis , were excluded . the purposive sampling strategy aimed for diversity with regard to age , gender , socio - economic situation and geographical location to capture maximum variation in views and experiences . data reported in this article were collected from focus groups with individuals with jhs , aimed at developing a physiotherapy intervention for jhs management . topic guides used to facilitate discussions covered issues of living with jhs , day - to - day self - management and provision of support for symptom management . the focus groups were conducted in non - clinical settings , facilitated by two researchers ( stp and jph ) , and open - ended questioning techniques were used to elicit participants own experiences and views . all focus groups were audio - recorded , fully transcribed , anonymized , checked for accuracy and imported into a qualitative software package ( nvivo 10 ) to aid data analysis . thematic analysis , using the constant comparison technique ( 14 ) , was used to scrutinize the data to identify and analyse patterns across the dataset and the data were scrutinized for negative cases . transcripts were examined on a line - by - line basis , with codes being assigned to segments of the data and an initial coding frame developed . data reported in this article were collected from focus groups with individuals with jhs , aimed at developing a physiotherapy intervention for jhs management . topic guides used to facilitate discussions covered issues of living with jhs , day - to - day self - management and provision of support for symptom management . the focus groups were conducted in non - clinical settings , facilitated by two researchers ( stp and jph ) , and open - ended questioning techniques were used to elicit participants own experiences and views . all focus groups were audio - recorded , fully transcribed , anonymized , checked for accuracy and imported into a qualitative software package ( nvivo 10 ) to aid data analysis . thematic analysis , using the constant comparison technique ( 14 ) , was used to scrutinize the data to identify and analyse patterns across the dataset and the data were scrutinized for negative cases . twenty - five individuals with jhs participated in the focus groups , aged 1966 years ( mean : 38.2 years ) , 22 were female and 23 were of the analysis led to the development of four key interrelated themes : the impact of jhs , jhs as a poorly understood condition , receiving a diagnosis and jhs management and self - care. participants characteristics and demographic information relating to the participants of the four focus groups , carried out between january and february 2013 ( total n = 25 ) ses was measured as index of multiple deprivation ( imd ) quintile from home postcode . participants described in detail the impact of jhs , which included fatigue , pain and proprioception problems : day in day out you re managing your pain and it s a lot of pain , it s a dull ache and it makes you sleepy and it makes you tired and you re exhausted ( female g , age 30 , fg1 ) . thus , some participants found living with jhs symptoms to be very debilitating , ( male e , age 36 , fg3 ) , whilst others were determined to persevere with their chosen activities , in spite of their symptoms : i teach like rock - climbing , surfing , body boarding and all of that stuff , like , and i m not going to stop doing it because i m in pain like you ca nt live your whole life with pain dictating what you can and ca nt do ( female g , age 45 , fg3 ) . although jhs is characterized by pain , participants alluded to the complexity of their pain experience , for example , describing difficulties in distinguishing between chronic and acute pain , and in recognizing how or ifinjuries had occurred : how do we know whether we ve injured something ? participants also described how prior experiences of repeated injuries led to heightened levels of anxiety and catastrophizing about future injuries : i feel like i m in a constant state of anxiety , waiting for the next injury and trying to pre - empt anything that s going to cause it ( female g , age 48 , fg2 ) . participants perceived jhs to be a condition that was poorly understood by health professionals , and within society more widely . i think i was described as a biomechanical conundrum by one of the physiotherapists i saw [ ] and this is what i found repeated over and over again , that hypermobility should nt be causing pain , it s just the way you are ( female c , age 53 , fg2 ) . participants felt that the unpredictable , diverse , evolving and fluctuating nature of their symptoms exacerbated others misunderstanding of the nature of jhs and contributed to a lack of social support : if you re inconsistent as well , they sort of go , she was alright with that last week , why is it this week she s saying that , you know , that s going to be difficult for her today ( female c , age 53 , fg2 ) . receiving a diagnosis was considered necessary in order to access appropriate care pathways , for example , referral for physiotherapy for jhs rather than for an acute single joint problem : i was originally seen by a physio who had nt diagnosed with the hypermobility and then went back to a musculo - skeletal specialist who then put me forward to specialist hypermobility physiotherapist and since then it s been amazing ; i feel like it s been worthwhile and it felt like the right thing to do and i ve been really enjoying it ( female b , age 27 , fg3 ) . similarly , a diagnosis did not necessarily lead to greater understanding and support from others in the participants social networks : there are people who do nt feel it s a genuine diagnosis , that it s something psychological , that you just need to be a bit braver ( female a , age 21 , fg2 ) . education for health professionals was a key issue for participants , to facilitate timely diagnosis of , and referral for , jhs . thus , education for health professionals was a prerequisite for diagnosis , and diagnosis was a prerequisite for participants to access education . participants recognized that individuals with jhs also need education , in order to find ways to self - manage their condition and to understand and engage with prescribed treatments . participants also felt that they could provide valuable information about the nature of jhs to clinicians : i think actually it s the health professionals that need educating [ . ] participants recognized the importance of self - care activities such as appropriate exercise , once a diagnosis had been received and a clear understanding of jhs had been developed . participants sought to deepen their understanding of jhs and access to health professionals who understood the nature of jhs in order to take a whole - body approach to self - care over a life course : the more you see the physio who knows what they re talking about the more you understand how the body works and the more you can apply your own sort of thinking to what you re doing in your exercises ( female e , age 44 , fg1 ) . participants described in detail the impact of jhs , which included fatigue , pain and proprioception problems : day in day out you re managing your pain and it s a lot of pain , it s a dull ache and it makes you sleepy and it makes you tired and you re exhausted ( female g , age 30 , fg1 ) . thus , some participants found living with jhs symptoms to be very debilitating , ( male e , age 36 , fg3 ) , whilst others were determined to persevere with their chosen activities , in spite of their symptoms : i teach like rock - climbing , surfing , body boarding and all of that stuff , like , and i m not going to stop doing it because i m in pain like you ca nt live your whole life with pain dictating what you can and ca nt do ( female g , age 45 , fg3 ) . although jhs is characterized by pain , participants alluded to the complexity of their pain experience , for example , describing difficulties in distinguishing between chronic and acute pain , and in recognizing how or ifinjuries had occurred : how do we know whether we ve injured something ? participants perceived jhs to be a condition that was poorly understood by health professionals , and within society more widely . i think i was described as a biomechanical conundrum by one of the physiotherapists i saw [ ] and this is what i found repeated over and over again , that hypermobility should nt be causing pain , it s just the way you are ( female c , age 53 , fg2 ) . participants felt that the unpredictable , diverse , evolving and fluctuating nature of their symptoms exacerbated others misunderstanding of the nature of jhs and contributed to a lack of social support : if you re inconsistent as well , they sort of go , she was alright with that last week , why is it this week she s saying that , you know , that s going to be difficult for her today ( female c , age 53 , fg2 ) . often , obtaining a correct diagnosis was a coincidental occurrence : it was only because a locum happened to be in the day i went in because my gp was off sick , and he just started saying , well , to start bending everything but if had nt been for him i would nt have been put on the right track , because otherwise what other route do you really have if it s not through your gp ? receiving a diagnosis was considered necessary in order to access appropriate care pathways , for example , referral for physiotherapy for jhs rather than for an acute single joint problem : i was originally seen by a physio who had nt diagnosed with the hypermobility and then went back to a musculo - skeletal specialist who then put me forward to specialist hypermobility physiotherapist and since then it s been amazing ; i feel like it s been worthwhile and it felt like the right thing to do and i ve been really enjoying it ( female b , age 27 , fg3 ) . similarly , a diagnosis did not necessarily lead to greater understanding and support from others in the participants social networks : there are people who do nt feel it s a genuine diagnosis , that it s something psychological , that you just need to be a bit braver ( female a , age 21 , fg2 ) . education for health professionals was a key issue for participants , to facilitate timely diagnosis of , and referral for , jhs . thus , education for health professionals was a prerequisite for diagnosis , and diagnosis was a prerequisite for participants to access education . participants recognized that individuals with jhs also need education , in order to find ways to self - manage their condition and to understand and engage with prescribed treatments . participants also felt that they could provide valuable information about the nature of jhs to clinicians : i think actually it s the health professionals that need educating [ . ] participants recognized the importance of self - care activities such as appropriate exercise , once a diagnosis had been received and a clear understanding of jhs had been developed . participants sought to deepen their understanding of jhs and access to health professionals who understood the nature of jhs in order to take a whole - body approach to self - care over a life course : the more you see the physio who knows what they re talking about the more you understand how the body works and the more you can apply your own sort of thinking to what you re doing in your exercises ( female e , age 44 , fg1 ) . education for health professionals was a key issue for participants , to facilitate timely diagnosis of , and referral for , jhs . thus , education for health professionals was a prerequisite for diagnosis , and diagnosis was a prerequisite for participants to access education . participants recognized that individuals with jhs also need education , in order to find ways to self - manage their condition and to understand and engage with prescribed treatments . participants also felt that they could provide valuable information about the nature of jhs to clinicians : i think actually it s the health professionals that need educating [ . ] participants recognized the importance of self - care activities such as appropriate exercise , once a diagnosis had been received and a clear understanding of jhs had been developed . participants sought to deepen their understanding of jhs and access to health professionals who understood the nature of jhs in order to take a whole - body approach to self - care over a life course : the more you see the physio who knows what they re talking about the more you understand how the body works and the more you can apply your own sort of thinking to what you re doing in your exercises ( female e , age 44 , fg1 ) . pain , fatigue and repeated episodes of injury were particularly challenging features of the wide - ranging symptoms of jhs . in our study , participants lives were disrupted repeatedly by fluctuating symptoms and cycles of injury and recovery . patients perceived jhs to be often poorly understood by health professionals and those in their wider social environment and reported feeling fraudulent and blamed for their symptoms . a lack of awareness of jhs amongst health professionals meant that obtaining a diagnosis of jhs was often difficult . previous studies have reported similar findings ( 9 ) and others have emphasized that being understood and believed by health professionals and significant others , along with social support , is instrumental in long - term pain management by facilitating ( or inhibiting ) pain acceptance ( 21,22 ) . participants highlighted the importance of a correct diagnosis in facilitating access to appropriate health care , support and education and helping to validate participants experiences . having lived with problematic symptoms of jhs sometimes for long periods of time , it is possible that the receipt of a diagnosis represented the beginning of a process during which they were able to understand and make sense of their symptoms , obtain appropriate treatment and subsequently find ways to self - manage the condition . primary - care practitioners play an important role in helping patients to understand and self - manage long - term health conditions . a prerequisite of being able to provide support for patients is that primary - care practitioners are able to recognize and diagnose jhs and to refer patients to jhs - trained specialists . without a correct diagnosis , unsuitable treatments or information currently , primary - care practitioners and other health professionals such as physiotherapists do not routinely receive training related to jhs ( 9 ) and the validity of diagnostic criteria ( such as the beighton score ) has recently been questioned ( 25 ) . education and access to information was important for participants to allow them to make informed health - care choices . participants described the value of collaborating with health professionals and reciprocal learning between practitioners and patients in order to develop effective self - care strategies , and for holistic long - term management . recognizing that jhs would not be cured , participants felt that health professionals and those with jhs could potentially develop a deeper understanding of jhs management by learning from each other as a teachable dyad ( 26 , p. 682). to ensure validity , participants were recruited from four different geographical locations in the uk and had the experience of different health - care services . participants provided rich personal narratives of the day - to - day experiences of living with and managing jhs from patients perspectives and data analysis demonstrated general consensus and shared experiences . the focus groups formed one phase of a study to develop a physiotherapy intervention to manage jhs . it was clear that the lived experience of those with jhs that emerged from the data analysis was an important story , which to date has received little attention . future research could conduct interviews to provide a more detailed investigation of personal accounts of living with jhs , in particular those newly diagnosed with jhs . individuals with jhs experience diverse , fluctuating and often debilitating symptoms and diagnosis is often slow . without a correct diagnosis , following diagnosis , access to jhs - trained health professionals could help patients to effectively manage their condition over a life course and receive psychological support when needed . patients and practitioners may be able to learn from one another and so assist in developing a deeper understanding of a currently poorly understood condition . to ensure validity , participants were recruited from four different geographical locations in the uk and had the experience of different health - care services . participants provided rich personal narratives of the day - to - day experiences of living with and managing jhs from patients perspectives and data analysis demonstrated general consensus and shared experiences . a limitation of this research is that participants were recruited through jhs support groups or via physiotherapy services for jhs and may therefore have been more active or the focus groups formed one phase of a study to develop a physiotherapy intervention to manage jhs . it was clear that the lived experience of those with jhs that emerged from the data analysis was an important story , which to date has received little attention . future research could conduct interviews to provide a more detailed investigation of personal accounts of living with jhs , in particular those newly diagnosed with jhs . individuals with jhs experience diverse , fluctuating and often debilitating symptoms and diagnosis is often slow . without a correct diagnosis , following diagnosis , access to jhs - trained health professionals could help patients to effectively manage their condition over a life course and receive psychological support when needed . the views and opinions expressed therein are those of the authors and do not necessarily reflect those of the health technology assessment programme , nihr , nhs or the department of health .

in 1989 , a local campaign against cancer and heart disease named clue ii was conducted in washington county , maryland . mobile trailers were stationed in a wide variety of locations in the county in an effort to give all segments of the community an opportunity to participate . at baseline , written informed consent was obtained , and a short questionnaire on demographics and medical history were administered to all participants . a total of 32,894 individuals , nearly one - third of the adult population of washington county at that time , took part in this study . to reduce the likelihood of a case subject not being identified through the cancer registries , the analysis cohort was limited to 25,076 clue ii participants who lived in washington county at baseline . we further excluded participants aged < 30 years ( n = 5,470 ) or with history of invasive cancer at baseline ( n = 1,360 ; individuals could be excluded for more than one reason ) . the final study sample consisted of 18,280 adults aged 30 years without a history of cancer at baseline . individuals were classified as having diabetes if they reported taking antidiabetes medications in the last 48 h on the baseline questionnaire . cancer cases in the clue ii cohort have been identified through linkage of the cohort participants with the washington county cancer registry and , since 1992 , with the maryland cancer registry . for all - cause mortality , we identified deaths among cohort members via searches of the national death index , maryland death certificates , reviews of obituaries of the local newspaper , and reports by next of kin . other information collected at baseline included demographic characteristics ( e.g. , race , marital status , and education ) ; self - reported height , weight , and weight at age 21 ; brief medical history ( e.g. , treated hypertension and treated high cholesterol ) ; use of medications in the past 48 h ; and a modified block food frequency questionnaire . clinical variables , including resting blood pressure and plasma total cholesterol , were measured for each participant . a follow - up questionnaire in 1996 ascertained whether participants had a first - degree family history of cancer . participants were grouped by diabetes status at baseline , and the test and t test were used to evaluate differences in proportions and means for the baseline characteristics between diabetic and nondiabetic participants . for cancer incidence , follow - up time for each participant was determined from the date of study entry to the date of cancer diagnosis , last date known to be free of a cancer diagnosis , death , or the end of follow - up ( 31 december 2006 ) , whichever came first . for cancer mortality , person - years of follow - up were calculated from the date of study entry to either the date of cancer death , the date of death from other causes , or the end of follow - up , whichever came first . we subsequently excluded cancer incidence and cancer death in the first 2 years of follow - up to minimize the potential that participants may have had subclinical cancer prior to baseline . to determine cancer - specific death in participants with cancer , we calculated follow - up time from the date of cancer diagnosis to either the date of death from that cancer , the date of death from other causes , or the end of follow - up , whichever came first . in a similar manner , for death from all causes , in participants with cancer , person - years of follow - up were calculated from the date of cancer diagnosis to either the date of death from any cause or the end of follow - up , whichever came first . the associations between treated diabetes and the cumulative risk of cancer incidence , cancer mortality , and survival after cancer diagnosis were first evaluated using the kaplan - meier method with log - rank tests to determine the significance of differences in cumulative risk using age as the time scale . cox proportional hazards models were subsequently used to calculate hazard ratios ( hrs ) and 95% cis for cancer incidence and cancer mortality , and in those diagnosed with cancer , death from cancer and death from all causes comparing baseline - treated diabetes to no diabetes after adjusting for age ( continuous ) , age squared ( continuous ) , sex , bmi ( < 25 , 2529 , 3035 , and > 35 kg / m ) , smoking ( never , former 20 cigarettes / day , former > 20 cigarettes / day , current 20 cigarettes / day , and current > 20 cigarettes / day ) , education ( < 12 vs. 12 years ) , hypertension treatment ( yes / no ) , and high cholesterol treatment ( yes / no ) . we confirmed the proportionality assumption by examining schoenfeld residuals and used the efron method to handle ties . additional analyses were performed to determine the association between diabetes and risk of advanced stage of major cancers , including colorectal ( stage 3/4 ) , breast ( stage 3/4 ) , and prostate cancer ( stage 3/4 or gleason score 7 ) . for participants with valid responses ( 10 ) to a dietary questionnaire ( n = 13,690 ; 75% of study population ) , we additionally adjusted for alcohol , fruit , vegetable , and red meat intakes and use of a multivitamin . to reduce possible detection bias from regular medical encounters in participants with treated diabetes , for the analysis of cancer incidence , a subgroup analysis was further conducted in adults who received treatment for hypertension and/or high cholesterol at baseline . to further reduce possible confounding as a result of the difference in the age distribution between diabetic and nondiabetic groups on mortality , we conducted a separate subgroup analysis limited to participants aged 60 years at baseline . afs ( 11 ) of cancer incidence , cancer case fatality , and all - cause mortality due to diabetes among individuals with preexisting , treated diabetes were estimated based on adjusted hrs to give crude , relative measures of the percentages of events that are expected to be the result of diabetes , should there be a causal relationship { af = [ ( hr 1)/hr ] 100}. tests of significance were two - tailed , with an -level of 0.05 . we performed analyses using sas version 9.2 ( cary , nc ) and stata / se version 10.0 ( college station , tx ) . individuals were classified as having diabetes if they reported taking antidiabetes medications in the last 48 h on the baseline questionnaire . cancer cases in the clue ii cohort have been identified through linkage of the cohort participants with the washington county cancer registry and , since 1992 , with the maryland cancer registry . for all - cause mortality , we identified deaths among cohort members via searches of the national death index , maryland death certificates , reviews of obituaries of the local newspaper , and reports by next of kin . other information collected at baseline included demographic characteristics ( e.g. , race , marital status , and education ) ; self - reported height , weight , and weight at age 21 ; brief medical history ( e.g. , treated hypertension and treated high cholesterol ) ; use of medications in the past 48 h ; and a modified block food frequency questionnaire . clinical variables , including resting blood pressure and plasma total cholesterol , were measured for each participant . a follow - up questionnaire in 1996 ascertained whether participants had a first - degree family history of cancer . participants were grouped by diabetes status at baseline , and the test and t test were used to evaluate differences in proportions and means for the baseline characteristics between diabetic and nondiabetic participants . for cancer incidence , follow - up time for each participant was determined from the date of study entry to the date of cancer diagnosis , last date known to be free of a cancer diagnosis , death , or the end of follow - up ( 31 december 2006 ) , whichever came first . for cancer mortality , person - years of follow - up were calculated from the date of study entry to either the date of cancer death , the date of death from other causes , or the end of follow - up , whichever came first . we subsequently excluded cancer incidence and cancer death in the first 2 years of follow - up to minimize the potential that participants may have had subclinical cancer prior to baseline . to determine cancer - specific death in participants with cancer , we calculated follow - up time from the date of cancer diagnosis to either the date of death from that cancer , the date of death from other causes , or the end of follow - up , whichever came first . in a similar manner , for death from all causes , in participants with cancer , person - years of follow - up were calculated from the date of cancer diagnosis to either the date of death from any cause or the end of follow - up , whichever came first . the associations between treated diabetes and the cumulative risk of cancer incidence , cancer mortality , and survival after cancer diagnosis were first evaluated using the kaplan - meier method with log - rank tests to determine the significance of differences in cumulative risk using age as the time scale . cox proportional hazards models were subsequently used to calculate hazard ratios ( hrs ) and 95% cis for cancer incidence and cancer mortality , and in those diagnosed with cancer , death from cancer and death from all causes comparing baseline - treated diabetes to no diabetes after adjusting for age ( continuous ) , age squared ( continuous ) , sex , bmi ( < 25 , 2529 , 3035 , and > 35 kg / m ) , smoking ( never , former 20 cigarettes / day , former > 20 cigarettes / day , current 20 cigarettes / day , and current > 20 cigarettes / day ) , education ( < 12 vs. 12 years ) , hypertension treatment ( yes / no ) , and high cholesterol treatment ( yes / no ) . we confirmed the proportionality assumption by examining schoenfeld residuals and used the efron method to handle ties . additional analyses were performed to determine the association between diabetes and risk of advanced stage of major cancers , including colorectal ( stage 3/4 ) , breast ( stage 3/4 ) , and prostate cancer ( stage 3/4 or gleason score 7 ) . for participants with valid responses ( 10 ) to a dietary questionnaire ( n = 13,690 ; 75% of study population ) , we additionally adjusted for alcohol , fruit , vegetable , and red meat intakes and use of a multivitamin . to reduce possible detection bias from regular medical encounters in participants with treated diabetes , for the analysis of cancer incidence , a subgroup analysis was further conducted in adults who received treatment for hypertension and/or high cholesterol at baseline . to further reduce possible confounding as a result of the difference in the age distribution between diabetic and nondiabetic groups on mortality , we conducted a separate subgroup analysis limited to participants aged 60 years at baseline . afs ( 11 ) of cancer incidence , cancer case fatality , and all - cause mortality due to diabetes among individuals with preexisting , treated diabetes were estimated based on adjusted hrs to give crude , relative measures of the percentages of events that are expected to be the result of diabetes , should there be a causal relationship { af = [ ( hr 1)/hr ] 100}. tests of significance were two - tailed , with an -level of 0.05 . we performed analyses using sas version 9.2 ( cary , nc ) and stata / se version 10.0 ( college station , tx ) . baseline characteristics are shown in table 1 by diabetes status . of 18,280 participants , 599 ( 3.3% ) had been treated for diabetes , including 354 taking oral medication , 197 receiving insulin , and 48 taking unspecified diabetes medications . in age - adjusted comparisons , compared with their nondiabetic counterparts , adults with diabetes were less educated , were more likely to be smokers , reported higher intake of red meat and fruit but lower alcohol consumption , were less likely to take multivitamins , had higher bmi at age 21 and at baseline , had higher systolic blood pressure , were more likely to have been treated for hypertension or high cholesterol , and were more likely to use aspirin ( all p < 0.05 ) . women with diabetes were less likely to have used oral contraceptives or hormone replacement therapy . age - adjusted baseline characteristics by treated diabetes status in 18,280 adults , clue ii , washington county , maryland , 19892006 during 17 years of follow - up , 116 diabetic and 2,365 nondiabetic adults developed cancer , corresponding to age - adjusted incidence rates of 13.25 and 10.58 per 1,000 person - years , respectively . the cumulative cancer incidence for adults with diabetes was elevated constantly after age 60 ( fig . compared with their nondiabetic counterparts , adults with diabetes were slightly more likely to develop cancer overall ( multivariable - adjusted hr 1.22 [ 95% ci 0.981.53 ] ) , especially cancers of the pancreas and digestive system and cancers related to smoking ( table 2 ) . diabetes was not statistically significantly associated with other cancers : the hrs for colorectal , lung , kidney , breast , and prostate were 1.41 ( 0.812.44 ) , 1.26 ( 0.732.18 ) , 2.06 ( 0.795.37 ) , 1.07 ( 0.611.87 ) , and 1.08 ( 0.661.77 ) , respectively . a : cumulative cancer incidence in 18,280 adults by treated diabetes status , log - rank p = 0.012 . b : cumulative cancer mortality in 18,280 adults by treated diabetes status , log - rank p = 0.015 . c : age - adjusted overall survival after cancer diagnosis in 2,481 participants diagnosed with cancer by treated diabetes status , log - rank p < 0.0001 . d : afs and ses of cancer incidence , cancer case fatality , and total mortality due to diabetes in participants with preexisting diabetes . association between treated diabetes and cancer incidence and cancer mortality in 18,280 adults , clue ii , washington county , maryland , 19892006 no statistically significant associations were observed between diabetes and risks of advanced stage of colorectal or prostate cancer . in the subset of participants with valid responses for the food frequency questionnaire , the results were similar to the overall cohort . in another subset analysis including participants receiving treatments for hypertension and/or high cholesterol , the fully adjusted hr of overall cancer incidence in the treated diabetic group was somewhat stronger than but not different from the estimate in the total study population ( hr 1.37 [ 95% ci 1.041.80 ] ) . a total of 51 diabetic and 856 nondiabetic participants died of cancer during follow - up , corresponding to age - adjusted cancer mortality rates of 4.17 and 3.12 per 1,000 person - years , respectively . similar to cancer incidence , diabetes was associated with an increased risk of cancer mortality after age 60 ( fig . the multivariable - adjusted hr of all - cancer mortality was 1.36 ( 95% ci 1.021.81 ) comparing adults with and without diabetes ( table 2 ) . diabetes - related cancer mortality risk was significantly increased for cancers of the digestive system , especially pancreatic cancer and colorectal cancer ( table 2 ) . there were no significant associations observed in other site - specific analyses ( data not shown ) . during follow - up , the mean age of cancer diagnosis was 71.7 8.8 and 67.9 11.4 years in adults with and without treated diabetes , respectively ( p < 0.0001 ) . cancer patients with diabetes experienced a rapid decline in survival within the first 3 years after cancer diagnosis ( fig . overall , 69% of participants with cancer died of cancer and 13% died of circulatory disease . cancer case fatality was higher in individuals with diabetes compared with those without diabetes ( table 3 ) . among participants with colorectal cancer , individuals with diabetes had a higher risk of death from colorectal cancer than those without diabetes . diabetes also conferred a higher risk of death from all causes in adults who developed a wide array of cancers , including cancers of the colorectum , breast , and prostate . pooling across cancers , diabetes was associated with a nearly 60% excess risk of all - cause mortality . in the subset of participants with valid responses for the food frequency questionnaire , the results were similar to the overall cohort after further adjustment for alcohol consumption ( data not shown ) . association between treated diabetes and cancer fatality and all - cause mortality in 2,481 adults diagnosed with cancer , clue ii , washington county , maryland , 19892006 to compare the impact of diabetes across the full range of cancer outcomes , we calculated a series of afs ( fig . the af can be interpreted as the percentage of events in people with preexisting diabetes attributable to diabetes itself , if the relationships are causal . in the clue ii cohort , the fraction of events attributable to diabetes rose in a graded fashion from incident cancer , to cancer case fatality , to all - cause mortality in cancer patients . the largest af for incident cancer risk was for pancreatic cancer ( > 60% ) . the largest afs for all - cause mortality in cancer patients were for colon , prostate , and breast all > 50% . colon cancer showed a moderate - to - strong influence of diabetes across its full natural history , from incidence to cancer case fatality and all - cause mortality . baseline characteristics are shown in table 1 by diabetes status . of 18,280 participants , 599 ( 3.3% ) had been treated for diabetes , including 354 taking oral medication , 197 receiving insulin , and 48 taking unspecified diabetes medications . in age - adjusted comparisons , compared with their nondiabetic counterparts , adults with diabetes were less educated , were more likely to be smokers , reported higher intake of red meat and fruit but lower alcohol consumption , were less likely to take multivitamins , had higher bmi at age 21 and at baseline , had higher systolic blood pressure , were more likely to have been treated for hypertension or high cholesterol , and were more likely to use aspirin ( all p < 0.05 ) . women with diabetes were less likely to have used oral contraceptives or hormone replacement therapy . age - adjusted baseline characteristics by treated diabetes status in 18,280 adults , clue ii , washington county , maryland , 19892006 during 17 years of follow - up , 116 diabetic and 2,365 nondiabetic adults developed cancer , corresponding to age - adjusted incidence rates of 13.25 and 10.58 per 1,000 person - years , respectively . the cumulative cancer incidence for adults with diabetes was elevated constantly after age 60 ( fig . compared with their nondiabetic counterparts , adults with diabetes were slightly more likely to develop cancer overall ( multivariable - adjusted hr 1.22 [ 95% ci 0.981.53 ] ) , especially cancers of the pancreas and digestive system and cancers related to smoking ( table 2 ) . diabetes was not statistically significantly associated with other cancers : the hrs for colorectal , lung , kidney , breast , and prostate were 1.41 ( 0.812.44 ) , 1.26 ( 0.732.18 ) , 2.06 ( 0.795.37 ) , 1.07 ( 0.611.87 ) , and 1.08 ( 0.661.77 ) , respectively . a : cumulative cancer incidence in 18,280 adults by treated diabetes status , log - rank p = 0.012 . b : cumulative cancer mortality in 18,280 adults by treated diabetes status , log - rank p = 0.015 . c : age - adjusted overall survival after cancer diagnosis in 2,481 participants diagnosed with cancer by treated diabetes status , log - rank p < 0.0001 . d : afs and ses of cancer incidence , cancer case fatality , and total mortality due to diabetes in participants with preexisting diabetes . association between treated diabetes and cancer incidence and cancer mortality in 18,280 adults , clue ii , washington county , maryland , 19892006 no statistically significant associations were observed between diabetes and risks of advanced stage of colorectal or prostate cancer . there were no advanced breast cancer cases in participants with diabetes . in the subset of participants with valid responses for the food frequency questionnaire , including participants receiving treatments for hypertension and/or high cholesterol , the fully adjusted hr of overall cancer incidence in the treated diabetic group was somewhat stronger than but not different from the estimate in the total study population ( hr 1.37 [ 95% ci 1.041.80 ] ) . a total of 51 diabetic and 856 nondiabetic participants died of cancer during follow - up , corresponding to age - adjusted cancer mortality rates of 4.17 and 3.12 per 1,000 person - years , respectively . similar to cancer incidence , diabetes was associated with an increased risk of cancer mortality after age 60 ( fig . the multivariable - adjusted hr of all - cancer mortality was 1.36 ( 95% ci 1.021.81 ) comparing adults with and without diabetes ( table 2 ) . diabetes - related cancer mortality risk was significantly increased for cancers of the digestive system , especially pancreatic cancer and colorectal cancer ( table 2 ) . there were no significant associations observed in other site - specific analyses ( data not shown ) . the mean age of cancer diagnosis was 71.7 8.8 and 67.9 11.4 years in adults with and without treated diabetes , respectively ( p < 0.0001 ) . cancer patients with diabetes experienced a rapid decline in survival within the first 3 years after cancer diagnosis ( fig . overall , 69% of participants with cancer died of cancer and 13% died of circulatory disease . cancer case fatality was higher in individuals with diabetes compared with those without diabetes ( table 3 ) . among participants with colorectal cancer , individuals with diabetes had a higher risk of death from colorectal cancer than those without diabetes . diabetes also conferred a higher risk of death from all causes in adults who developed a wide array of cancers , including cancers of the colorectum , breast , and prostate . pooling across cancers , diabetes was associated with a nearly 60% excess risk of all - cause mortality . in the subset of participants with valid responses for the food frequency questionnaire , the results were similar to the overall cohort after further adjustment for alcohol consumption ( data not shown ) . association between treated diabetes and cancer fatality and all - cause mortality in 2,481 adults diagnosed with cancer , clue ii , washington county , maryland , 19892006 to compare the impact of diabetes across the full range of cancer outcomes , we calculated a series of afs ( fig . the af can be interpreted as the percentage of events in people with preexisting diabetes attributable to diabetes itself , if the relationships are causal . in the clue ii cohort , the fraction of events attributable to diabetes rose in a graded fashion from incident cancer , to cancer case fatality , to all - cause mortality in cancer patients . the largest af for incident cancer risk was for pancreatic cancer ( > 60% ) . the largest afs for all - cause mortality in cancer patients were for colon , prostate , and breast all > 50% . colon cancer showed a moderate - to - strong influence of diabetes across its full natural history , from incidence to cancer case fatality and all - cause mortality . first , adults with treated diabetes were more likely to develop cancer than their nondiabetic counterparts , especially pancreatic cancer . second , diabetic adults were more likely to die of cancer than their nondiabetic counterparts . third , diabetes was associated with greater cancer - specific case fatality for adults with cancer , particularly with colorectal cancer . fourth , in patients with cancer , individuals with diabetes had higher all - cause mortality than those without diabetes . fifth , in individuals with diabetes , the afs due to diabetes were larger for cancer case fatality and total mortality than for cancer incidence , with the exception of pancreatic cancer . thus , diabetes appears to exert a greater influence downstream on the risk of mortality in people with cancer than on upstream risk of incident cancer . our study is consistent with prior literature in several ways , including our overall relationship of diabetes with incident cancer ( 7 ) and our estimates for diabetes - related risk of incident pancreatic cancer ( 5 ) , risk of mortality owing to cancer of the pancreas and colon ( 7,8 ) , and risk of all - cause mortality in cancer patients ( 9 ) . a unique strength of our study , however , is its quantification of the influence of diabetes on cancer outcomes across the full range of its clinical history , from risk of incident cancer to risk of cancer death , case fatality , and all - cause mortality in cancer patients . previous studies did not adopt this more global perspective , instead focusing more specifically on individual relationships with particular cancers , separating the impact on incidence from the impact on mortality . type 2 diabetes ( which accounts for > 90% of prevalent cases of diabetes in the u.s . ) these factors include obesity ( 12 ) and physical inactivity ( 13 ) , which increase the risk of insulin resistance and compensatory hyperinsulinemia . an important role of hyperinsulinemia in colorectal and pancreatic carcinogenesis is supported by in vitro studies , animal models , and several epidemiologic studies ( 14 ) . another mechanism may be that hyperglycemia itself promotes tumor growth ( 15 ) , but results are not consistent among studies ( 16 ) . we found preexisting diabetes to be an independent risk factor for death from cancer in adults with any cancer and for colorectal cancer death among adults with colorectal cancer . possible explanations for diabetes - related case fatality include tumor proliferation due to hyperinsulinemia and hyperglycemia ( 17 ) , less aggressive cancer treatment resulting from the presence of diabetes - related comorbidities ( 18 ) , poorer response to cancer treatment in adults with diabetes ( 19 ) , and suboptimal cancer screening practices related to diabetes status ( 20 ) . diabetes is a strong independent predictor of all - cause mortality in patients with a variety of cancer types . it is possible that cancer s adverse effects on thrombosis and oxygenation , as well as the cardiovascular risks imposed by cancer surgery , will create adverse biological interactions ( 21 ) . besides , the urgent need to treat cancer might distract from optimal care for diabetes and related conditions ( 22 ) . for these reasons , some experts favor all - cause mortality as an end point in cancer outcomes research that is not biased by attribution of cause of death and captures the full effect of possible interactions ( 23 ) . a main strength of our study is the availability of data on cancer incidence and mortality across multiple cancer types in the same population that allowed us to isolate the effects of diabetes on cancer incidence from its effects on cancer case fatality and death from all causes . other strengths of our study include a community - based cohort , comprehensive ascertainments of cancer and mortality , data on potential confounders , and 17 years of follow - up that offered the opportunity to study long - term associations . first , the diagnosis of diabetes and fasting glucose data were not available in the study , and diabetes status was not updated during follow - up . we therefore relied exclusively on self - reported use of diabetes medications at baseline to identify cases of diabetes . thus , the diabetic individuals in our study may be later in the natural history of their diabetes than in other studies . the consequent misclassification of adults with undiagnosed and untreated diabetes as nondiabetic likely biased our results toward the null . second , as a result of the lack of fasting blood assessment , we were not able to isolate the effects on cancer from hyperglycemia , diabetes , or antidiabetes treatment ( e.g. , insulin ) . third , it is possible that detection bias occurred if adults with diabetes had more frequent contact with their physicians and therefore were more likely to be detected with cancer ; although we attempted to rule out this bias by conducting a subgroup analysis among participants who presumably accessed care because they had been treated for hypertension and/or high cholesterol . fourth , we lacked optimal data to characterize adiposity : bmi was calculated based on self - reported weight and height ( which tend to underestimate true bmi ) , and we lacked data on waist circumference or percent body fat . fifth , we had a limited number of cases to investigate less common cancer sites , such as endometrial , lymphoma , esophageal , and liver cancers . sixth , given the significant difference between diabetic and nondiabetic individuals at baseline , even the multiple adjustments for covariates leave the possibility of residual confounding . finally , our study suggests that for many common cancers like colon , breast , and prostate , diabetes exerts a stronger adverse influence downstream , after cancer occurs , than upstream , in relation to incident cancer risk . whether improvements in diabetes management might reduce the risk of mortality in cancer patients with preexisting cancer deserves further attention .

objectiveto quantify the association of treated diabetes with cancer incidence and cancer mortality as well as cancer case fatality and all - cause mortality in adults who subsequently develop cancer and to calculate attributable fractions due to diabetes on various cancer outcomes.research design and methodsprospective data on 599 diabetic and 17,681 nondiabetic adults from the clue ii ( give us a clue to cancer and heart disease ) cohort in washington county , maryland , were analyzed . diabetes was defined by self - reported use of diabetes medications at baseline . cancer incidence was ascertained using county and state cancer registries . mortality data were obtained from death certificates.resultsfrom 1989 to 2006 , 116 diabetic and 2,365 nondiabetic adults developed cancer , corresponding to age - adjusted incidence of 13.25 and 10.58 per 1,000 person - years , respectively . adjusting for age , sex , education , bmi , smoking , hypertension treatment , and high cholesterol treatment using cox proportional hazards regression , diabetes was associated with a higher risk of incident cancer ( hazard ratio 1.22 [ 95% ci 0.981.53 ] ) and cancer mortality ( 1.36 [ 1.021.81 ] ) . in individuals who developed cancer , adults with diabetes had a higher risk of cancer case fatality ( 1.34 [ 1.0021.79 ] ) and all - cause mortality ( 1.61 [ 1.292.01 ] ) . for colorectal , breast , and prostate cancers , the attributable fractions resulting from diabetes were larger for cancer fatality and mortality than cancer incidence.conclusionsin this prospective cohort , diabetes appears to exert a greater influence downstream on the risk of mortality in people with cancer than on upstream risk of incident cancer .

RESEARCH DESIGN AND METHODS Diabetes status Cancer incidence and mortality assessment Other baseline variables Data analysis RESULTS Baseline characteristics Cancer incidence Cancer mortality Cancer case fatality and all-cause mortality after a cancer diagnosis AFs resulting from diabetes CONCLUSIONS

in 1989 , a local campaign against cancer and heart disease named clue ii was conducted in washington county , maryland . to reduce the likelihood of a case subject not being identified through the cancer registries , the analysis cohort was limited to 25,076 clue ii participants who lived in washington county at baseline . cancer cases in the clue ii cohort have been identified through linkage of the cohort participants with the washington county cancer registry and , since 1992 , with the maryland cancer registry . for all - cause mortality , we identified deaths among cohort members via searches of the national death index , maryland death certificates , reviews of obituaries of the local newspaper , and reports by next of kin . , race , marital status , and education ) ; self - reported height , weight , and weight at age 21 ; brief medical history ( e.g. for cancer incidence , follow - up time for each participant was determined from the date of study entry to the date of cancer diagnosis , last date known to be free of a cancer diagnosis , death , or the end of follow - up ( 31 december 2006 ) , whichever came first . for cancer mortality , person - years of follow - up were calculated from the date of study entry to either the date of cancer death , the date of death from other causes , or the end of follow - up , whichever came first . to determine cancer - specific death in participants with cancer , we calculated follow - up time from the date of cancer diagnosis to either the date of death from that cancer , the date of death from other causes , or the end of follow - up , whichever came first . in a similar manner , for death from all causes , in participants with cancer , person - years of follow - up were calculated from the date of cancer diagnosis to either the date of death from any cause or the end of follow - up , whichever came first . the associations between treated diabetes and the cumulative risk of cancer incidence , cancer mortality , and survival after cancer diagnosis were first evaluated using the kaplan - meier method with log - rank tests to determine the significance of differences in cumulative risk using age as the time scale . cox proportional hazards models were subsequently used to calculate hazard ratios ( hrs ) and 95% cis for cancer incidence and cancer mortality , and in those diagnosed with cancer , death from cancer and death from all causes comparing baseline - treated diabetes to no diabetes after adjusting for age ( continuous ) , age squared ( continuous ) , sex , bmi ( < 25 , 2529 , 3035 , and > 35 kg / m ) , smoking ( never , former 20 cigarettes / day , former > 20 cigarettes / day , current 20 cigarettes / day , and current > 20 cigarettes / day ) , education ( < 12 vs. 12 years ) , hypertension treatment ( yes / no ) , and high cholesterol treatment ( yes / no ) . additional analyses were performed to determine the association between diabetes and risk of advanced stage of major cancers , including colorectal ( stage 3/4 ) , breast ( stage 3/4 ) , and prostate cancer ( stage 3/4 or gleason score 7 ) . to reduce possible detection bias from regular medical encounters in participants with treated diabetes , for the analysis of cancer incidence , a subgroup analysis was further conducted in adults who received treatment for hypertension and/or high cholesterol at baseline . afs ( 11 ) of cancer incidence , cancer case fatality , and all - cause mortality due to diabetes among individuals with preexisting , treated diabetes were estimated based on adjusted hrs to give crude , relative measures of the percentages of events that are expected to be the result of diabetes , should there be a causal relationship { af = [ ( hr 1)/hr ] 100}. cancer cases in the clue ii cohort have been identified through linkage of the cohort participants with the washington county cancer registry and , since 1992 , with the maryland cancer registry . for all - cause mortality , we identified deaths among cohort members via searches of the national death index , maryland death certificates , reviews of obituaries of the local newspaper , and reports by next of kin . for cancer incidence , follow - up time for each participant was determined from the date of study entry to the date of cancer diagnosis , last date known to be free of a cancer diagnosis , death , or the end of follow - up ( 31 december 2006 ) , whichever came first . for cancer mortality , person - years of follow - up were calculated from the date of study entry to either the date of cancer death , the date of death from other causes , or the end of follow - up , whichever came first . to determine cancer - specific death in participants with cancer , we calculated follow - up time from the date of cancer diagnosis to either the date of death from that cancer , the date of death from other causes , or the end of follow - up , whichever came first . in a similar manner , for death from all causes , in participants with cancer , person - years of follow - up were calculated from the date of cancer diagnosis to either the date of death from any cause or the end of follow - up , whichever came first . the associations between treated diabetes and the cumulative risk of cancer incidence , cancer mortality , and survival after cancer diagnosis were first evaluated using the kaplan - meier method with log - rank tests to determine the significance of differences in cumulative risk using age as the time scale . cox proportional hazards models were subsequently used to calculate hazard ratios ( hrs ) and 95% cis for cancer incidence and cancer mortality , and in those diagnosed with cancer , death from cancer and death from all causes comparing baseline - treated diabetes to no diabetes after adjusting for age ( continuous ) , age squared ( continuous ) , sex , bmi ( < 25 , 2529 , 3035 , and > 35 kg / m ) , smoking ( never , former 20 cigarettes / day , former > 20 cigarettes / day , current 20 cigarettes / day , and current > 20 cigarettes / day ) , education ( < 12 vs. 12 years ) , hypertension treatment ( yes / no ) , and high cholesterol treatment ( yes / no ) . additional analyses were performed to determine the association between diabetes and risk of advanced stage of major cancers , including colorectal ( stage 3/4 ) , breast ( stage 3/4 ) , and prostate cancer ( stage 3/4 or gleason score 7 ) . to reduce possible detection bias from regular medical encounters in participants with treated diabetes , for the analysis of cancer incidence , a subgroup analysis was further conducted in adults who received treatment for hypertension and/or high cholesterol at baseline . afs ( 11 ) of cancer incidence , cancer case fatality , and all - cause mortality due to diabetes among individuals with preexisting , treated diabetes were estimated based on adjusted hrs to give crude , relative measures of the percentages of events that are expected to be the result of diabetes , should there be a causal relationship { af = [ ( hr 1)/hr ] 100}. in age - adjusted comparisons , compared with their nondiabetic counterparts , adults with diabetes were less educated , were more likely to be smokers , reported higher intake of red meat and fruit but lower alcohol consumption , were less likely to take multivitamins , had higher bmi at age 21 and at baseline , had higher systolic blood pressure , were more likely to have been treated for hypertension or high cholesterol , and were more likely to use aspirin ( all p < 0.05 ) . age - adjusted baseline characteristics by treated diabetes status in 18,280 adults , clue ii , washington county , maryland , 19892006 during 17 years of follow - up , 116 diabetic and 2,365 nondiabetic adults developed cancer , corresponding to age - adjusted incidence rates of 13.25 and 10.58 per 1,000 person - years , respectively . the cumulative cancer incidence for adults with diabetes was elevated constantly after age 60 ( fig . compared with their nondiabetic counterparts , adults with diabetes were slightly more likely to develop cancer overall ( multivariable - adjusted hr 1.22 [ 95% ci 0.981.53 ] ) , especially cancers of the pancreas and digestive system and cancers related to smoking ( table 2 ) . diabetes was not statistically significantly associated with other cancers : the hrs for colorectal , lung , kidney , breast , and prostate were 1.41 ( 0.812.44 ) , 1.26 ( 0.732.18 ) , 2.06 ( 0.795.37 ) , 1.07 ( 0.611.87 ) , and 1.08 ( 0.661.77 ) , respectively . c : age - adjusted overall survival after cancer diagnosis in 2,481 participants diagnosed with cancer by treated diabetes status , log - rank p < 0.0001 . d : afs and ses of cancer incidence , cancer case fatality , and total mortality due to diabetes in participants with preexisting diabetes . association between treated diabetes and cancer incidence and cancer mortality in 18,280 adults , clue ii , washington county , maryland , 19892006 no statistically significant associations were observed between diabetes and risks of advanced stage of colorectal or prostate cancer . in another subset analysis including participants receiving treatments for hypertension and/or high cholesterol , the fully adjusted hr of overall cancer incidence in the treated diabetic group was somewhat stronger than but not different from the estimate in the total study population ( hr 1.37 [ 95% ci 1.041.80 ] ) . a total of 51 diabetic and 856 nondiabetic participants died of cancer during follow - up , corresponding to age - adjusted cancer mortality rates of 4.17 and 3.12 per 1,000 person - years , respectively . similar to cancer incidence , diabetes was associated with an increased risk of cancer mortality after age 60 ( fig . the multivariable - adjusted hr of all - cancer mortality was 1.36 ( 95% ci 1.021.81 ) comparing adults with and without diabetes ( table 2 ) . during follow - up , the mean age of cancer diagnosis was 71.7 8.8 and 67.9 11.4 years in adults with and without treated diabetes , respectively ( p < 0.0001 ) . cancer case fatality was higher in individuals with diabetes compared with those without diabetes ( table 3 ) . among participants with colorectal cancer , individuals with diabetes had a higher risk of death from colorectal cancer than those without diabetes . diabetes also conferred a higher risk of death from all causes in adults who developed a wide array of cancers , including cancers of the colorectum , breast , and prostate . pooling across cancers , diabetes was associated with a nearly 60% excess risk of all - cause mortality . association between treated diabetes and cancer fatality and all - cause mortality in 2,481 adults diagnosed with cancer , clue ii , washington county , maryland , 19892006 to compare the impact of diabetes across the full range of cancer outcomes , we calculated a series of afs ( fig . in the clue ii cohort , the fraction of events attributable to diabetes rose in a graded fashion from incident cancer , to cancer case fatality , to all - cause mortality in cancer patients . the largest afs for all - cause mortality in cancer patients were for colon , prostate , and breast all > 50% . colon cancer showed a moderate - to - strong influence of diabetes across its full natural history , from incidence to cancer case fatality and all - cause mortality . in age - adjusted comparisons , compared with their nondiabetic counterparts , adults with diabetes were less educated , were more likely to be smokers , reported higher intake of red meat and fruit but lower alcohol consumption , were less likely to take multivitamins , had higher bmi at age 21 and at baseline , had higher systolic blood pressure , were more likely to have been treated for hypertension or high cholesterol , and were more likely to use aspirin ( all p < 0.05 ) . age - adjusted baseline characteristics by treated diabetes status in 18,280 adults , clue ii , washington county , maryland , 19892006 during 17 years of follow - up , 116 diabetic and 2,365 nondiabetic adults developed cancer , corresponding to age - adjusted incidence rates of 13.25 and 10.58 per 1,000 person - years , respectively . the cumulative cancer incidence for adults with diabetes was elevated constantly after age 60 ( fig . compared with their nondiabetic counterparts , adults with diabetes were slightly more likely to develop cancer overall ( multivariable - adjusted hr 1.22 [ 95% ci 0.981.53 ] ) , especially cancers of the pancreas and digestive system and cancers related to smoking ( table 2 ) . diabetes was not statistically significantly associated with other cancers : the hrs for colorectal , lung , kidney , breast , and prostate were 1.41 ( 0.812.44 ) , 1.26 ( 0.732.18 ) , 2.06 ( 0.795.37 ) , 1.07 ( 0.611.87 ) , and 1.08 ( 0.661.77 ) , respectively . c : age - adjusted overall survival after cancer diagnosis in 2,481 participants diagnosed with cancer by treated diabetes status , log - rank p < 0.0001 . d : afs and ses of cancer incidence , cancer case fatality , and total mortality due to diabetes in participants with preexisting diabetes . association between treated diabetes and cancer incidence and cancer mortality in 18,280 adults , clue ii , washington county , maryland , 19892006 no statistically significant associations were observed between diabetes and risks of advanced stage of colorectal or prostate cancer . in the subset of participants with valid responses for the food frequency questionnaire , including participants receiving treatments for hypertension and/or high cholesterol , the fully adjusted hr of overall cancer incidence in the treated diabetic group was somewhat stronger than but not different from the estimate in the total study population ( hr 1.37 [ 95% ci 1.041.80 ] ) . a total of 51 diabetic and 856 nondiabetic participants died of cancer during follow - up , corresponding to age - adjusted cancer mortality rates of 4.17 and 3.12 per 1,000 person - years , respectively . similar to cancer incidence , diabetes was associated with an increased risk of cancer mortality after age 60 ( fig . the multivariable - adjusted hr of all - cancer mortality was 1.36 ( 95% ci 1.021.81 ) comparing adults with and without diabetes ( table 2 ) . the mean age of cancer diagnosis was 71.7 8.8 and 67.9 11.4 years in adults with and without treated diabetes , respectively ( p < 0.0001 ) . overall , 69% of participants with cancer died of cancer and 13% died of circulatory disease . cancer case fatality was higher in individuals with diabetes compared with those without diabetes ( table 3 ) . among participants with colorectal cancer , individuals with diabetes had a higher risk of death from colorectal cancer than those without diabetes . diabetes also conferred a higher risk of death from all causes in adults who developed a wide array of cancers , including cancers of the colorectum , breast , and prostate . pooling across cancers , diabetes was associated with a nearly 60% excess risk of all - cause mortality . association between treated diabetes and cancer fatality and all - cause mortality in 2,481 adults diagnosed with cancer , clue ii , washington county , maryland , 19892006 to compare the impact of diabetes across the full range of cancer outcomes , we calculated a series of afs ( fig . the af can be interpreted as the percentage of events in people with preexisting diabetes attributable to diabetes itself , if the relationships are causal . in the clue ii cohort , the fraction of events attributable to diabetes rose in a graded fashion from incident cancer , to cancer case fatality , to all - cause mortality in cancer patients . the largest afs for all - cause mortality in cancer patients were for colon , prostate , and breast all > 50% . colon cancer showed a moderate - to - strong influence of diabetes across its full natural history , from incidence to cancer case fatality and all - cause mortality . first , adults with treated diabetes were more likely to develop cancer than their nondiabetic counterparts , especially pancreatic cancer . third , diabetes was associated with greater cancer - specific case fatality for adults with cancer , particularly with colorectal cancer . fourth , in patients with cancer , individuals with diabetes had higher all - cause mortality than those without diabetes . fifth , in individuals with diabetes , the afs due to diabetes were larger for cancer case fatality and total mortality than for cancer incidence , with the exception of pancreatic cancer . thus , diabetes appears to exert a greater influence downstream on the risk of mortality in people with cancer than on upstream risk of incident cancer . our study is consistent with prior literature in several ways , including our overall relationship of diabetes with incident cancer ( 7 ) and our estimates for diabetes - related risk of incident pancreatic cancer ( 5 ) , risk of mortality owing to cancer of the pancreas and colon ( 7,8 ) , and risk of all - cause mortality in cancer patients ( 9 ) . a unique strength of our study , however , is its quantification of the influence of diabetes on cancer outcomes across the full range of its clinical history , from risk of incident cancer to risk of cancer death , case fatality , and all - cause mortality in cancer patients . we found preexisting diabetes to be an independent risk factor for death from cancer in adults with any cancer and for colorectal cancer death among adults with colorectal cancer . possible explanations for diabetes - related case fatality include tumor proliferation due to hyperinsulinemia and hyperglycemia ( 17 ) , less aggressive cancer treatment resulting from the presence of diabetes - related comorbidities ( 18 ) , poorer response to cancer treatment in adults with diabetes ( 19 ) , and suboptimal cancer screening practices related to diabetes status ( 20 ) . diabetes is a strong independent predictor of all - cause mortality in patients with a variety of cancer types . for these reasons , some experts favor all - cause mortality as an end point in cancer outcomes research that is not biased by attribution of cause of death and captures the full effect of possible interactions ( 23 ) . a main strength of our study is the availability of data on cancer incidence and mortality across multiple cancer types in the same population that allowed us to isolate the effects of diabetes on cancer incidence from its effects on cancer case fatality and death from all causes . other strengths of our study include a community - based cohort , comprehensive ascertainments of cancer and mortality , data on potential confounders , and 17 years of follow - up that offered the opportunity to study long - term associations . first , the diagnosis of diabetes and fasting glucose data were not available in the study , and diabetes status was not updated during follow - up . we therefore relied exclusively on self - reported use of diabetes medications at baseline to identify cases of diabetes . third , it is possible that detection bias occurred if adults with diabetes had more frequent contact with their physicians and therefore were more likely to be detected with cancer ; although we attempted to rule out this bias by conducting a subgroup analysis among participants who presumably accessed care because they had been treated for hypertension and/or high cholesterol . fourth , we lacked optimal data to characterize adiposity : bmi was calculated based on self - reported weight and height ( which tend to underestimate true bmi ) , and we lacked data on waist circumference or percent body fat . finally , our study suggests that for many common cancers like colon , breast , and prostate , diabetes exerts a stronger adverse influence downstream , after cancer occurs , than upstream , in relation to incident cancer risk . whether improvements in diabetes management might reduce the risk of mortality in cancer patients with preexisting cancer deserves further attention .

the experimental procedures and protocols of this study conformed to the national institutes of health guide for the care and use of laboratory animals and were approved by the institutional animal care and use committee of the university of mississippi medical center . male sprague - dawley ( harlan , indianapolis , in ) rats ( 325350 g , n = 20 ) were anesthetized with 50 mg / kg sodium pentobarbital ( nembutal ) , and atropine sulfate ( 0.1 mg / kg ) was administered to prevent excess airway secretions . briefly , using aseptic techniques , a laparotomy was performed and a sterile nonocclusive polyvinyl catheter was inserted into the abdominal aorta , distal to the kidneys . through a left femoral vein incision , a sterile catheter was placed in the vena cava . both catheters were exteriorized through a subcutaneously implanted stainless steel button . immediately after arterial and venous catheter implantation , a stainless steel cannula ( 26 gauge , 10 mm long ) was implanted into the right lateral cerebral ventricle using the coordinates previously described ( 22 ) . the guide cannula was anchored into place with three stainless steel machine screws , a metal cap , and dental acrylic , and a stylet was inserted to seal the cannula until use . during stereotaxic manipulation , after 7 days of recovery from surgery , accuracy of the cannula placement was tested by measuring the dipsogenic response ( immediate drinking of at least 5 ml of water in 10 min ) to an intracerebroventricular injection of 100 ng angiotensin ii . after the experiment , the animals were killed and the brains removed and sectioned to confirm placement of the cannula . after recovery from anesthesia , the rats were housed in individual metabolic cages for determination of daily water and food consumption . the arterial and venous catheters were connected to a dual - channel infusion swivel ( instech ) . the arterial catheter was connected to a pressure transducer ( maxxim ) for continuous 24-h measurement of mean arterial pressure ( map ) and heart rate using computerized techniques as previously described ( 7,8 ) . the venous catheter was connected to a syringe pump for continuous infusion of saline ( 0.45% , 40 ml / day ) . total sodium intake was maintained constant at 3.1 meq / day via the continuous saline infusion combined with sodium - deficient rat chow ( 0.006 mmol sodium / g food , teklad ) . intravenous solutions were infused through a sterile filter ( 0.22 m , millipore ) , and the saline infusion was started immediately after placement of the rats into the metabolic cages . map , heart rate , urine volume , food , and water intake were measured 24 h / day and average values were recorded daily . after 45 days of stable control measurements , insulin - deficient diabetes was induced by a single intravenous injection of streptozotocin ( stz , 50 mg / kg [ sigma aldrich ] , dissolved in 0.5 ml of 0.05 m citrate buffer , ph 4.5 ) . five days after stz injection , leptin ( 0.62 g / h , 0.5 l / h ) was infused intracerebroventricularly for 10 days via osmotic minipump ( model 2002 , durect ) implanted subcutaneously in the scapular region as previously described ( 20 ) . we have shown that this rate of leptin infusion fully restores euglycemia in stz - diabetic rats ( 11 ) . five days after stz injection , the mc3/4r agonist , mtii ( 10 ng / h , 0.5 l / h , polypeptide laboratories ) , was infused intracerebroventricularly for 10 days via osmotic minipump as described above ( n = 5 ) . on the 10th day of mtii infusion at 10 ng / h , the rats were lightly anesthetized with isofluorane and the osmotic minipump was replaced by another pump to deliver the agonist at a higher concentration ( 50 ng / h ) . in a separate experiment , diabetic rats ( n = 5 ) were treated with mtii at the dose of 10 ng / h and were pair - fed the same amount of food consumed by diabetic rats during chronic leptin intracerebroventricular infusion . five days after stz injection , the mc3/4r antagonist shu-9119 ( 1 nmol / h , 0.5 l / h ; polypeptide laboratories ) was infused intracerebroventricularly for 17 days via osmotic minipump as described above . after the first 7 days of intracerebroventricular shu-9119 infusion , an additional minipump was implanted 12 cm apart from the other minipump and connected to the intracerebroventricular cannula via a y connector to deliver leptin intracerebroventricularly for 10 days at 0.62 g / h . the rate of shu-9119 infusion was based on our previous study showing that this dose effectively blocks the mc3/4r and the chronic dietary and cardiovascular effects of leptin in normal sprague - dawley rats ( 9 ) . blood glucose concentration was measured each morning between 9:00 and 10:00 a.m. for determination of blood glucose levels using glucose strips ( reli on ) . the data are expressed as mean se and analyzed by using two - factor anova with repeated measures . dunnett 's test was used for comparisons of experimental and control values within each group , when appropriate . the experimental procedures and protocols of this study conformed to the national institutes of health guide for the care and use of laboratory animals and were approved by the institutional animal care and use committee of the university of mississippi medical center . male sprague - dawley ( harlan , indianapolis , in ) rats ( 325350 g , n = 20 ) were anesthetized with 50 mg / kg sodium pentobarbital ( nembutal ) , and atropine sulfate ( 0.1 mg / kg ) was administered to prevent excess airway secretions . briefly , using aseptic techniques , a laparotomy was performed and a sterile nonocclusive polyvinyl catheter was inserted into the abdominal aorta , distal to the kidneys . through a left femoral vein incision , a sterile catheter was placed in the vena cava . immediately after arterial and venous catheter implantation , a stainless steel cannula ( 26 gauge , 10 mm long ) was implanted into the right lateral cerebral ventricle using the coordinates previously described ( 22 ) . the guide cannula was anchored into place with three stainless steel machine screws , a metal cap , and dental acrylic , and a stylet was inserted to seal the cannula until use . during stereotaxic manipulation , after 7 days of recovery from surgery , accuracy of the cannula placement was tested by measuring the dipsogenic response ( immediate drinking of at least 5 ml of water in 10 min ) to an intracerebroventricular injection of 100 ng angiotensin ii . after the experiment , the animals were killed and the brains removed and sectioned to confirm placement of the cannula . after recovery from anesthesia , the rats were housed in individual metabolic cages for determination of daily water and food consumption . the arterial and venous catheters were connected to a dual - channel infusion swivel ( instech ) . the arterial catheter was connected to a pressure transducer ( maxxim ) for continuous 24-h measurement of mean arterial pressure ( map ) and heart rate using computerized techniques as previously described ( 7,8 ) . the venous catheter was connected to a syringe pump for continuous infusion of saline ( 0.45% , 40 ml / day ) . total sodium intake was maintained constant at 3.1 meq / day via the continuous saline infusion combined with sodium - deficient rat chow ( 0.006 mmol sodium / g food , teklad ) . intravenous solutions were infused through a sterile filter ( 0.22 m , millipore ) , and the saline infusion was started immediately after placement of the rats into the metabolic cages . map , heart rate , urine volume , food , and water intake were measured 24 h / day and average values were recorded daily . after 45 days of stable control measurements , insulin - deficient diabetes was induced by a single intravenous injection of streptozotocin ( stz , 50 mg / kg [ sigma aldrich ] , dissolved in 0.5 ml of 0.05 m citrate buffer , ph 4.5 ) . five days after stz injection , leptin ( 0.62 g / h , 0.5 l / h ) was infused intracerebroventricularly for 10 days via osmotic minipump ( model 2002 , durect ) implanted subcutaneously in the scapular region as previously described ( 20 ) . we have shown that this rate of leptin infusion fully restores euglycemia in stz - diabetic rats ( 11 ) . five days after stz injection , the mc3/4r agonist , mtii ( 10 ng / h , 0.5 l / h , polypeptide laboratories ) , was infused intracerebroventricularly for 10 days via osmotic minipump as described above ( n = 5 ) . on the 10th day of mtii infusion at 10 ng / h , the rats were lightly anesthetized with isofluorane and the osmotic minipump was replaced by another pump to deliver the agonist at a higher concentration ( 50 ng / h ) . in a separate experiment , diabetic rats ( n = 5 ) were treated with mtii at the dose of 10 ng / h and were pair - fed the same amount of food consumed by diabetic rats during chronic leptin intracerebroventricular infusion . five days after stz injection , the mc3/4r antagonist shu-9119 ( 1 nmol / h , 0.5 l / h ; polypeptide laboratories ) was infused intracerebroventricularly for 17 days via osmotic minipump as described above . after the first 7 days of intracerebroventricular shu-9119 infusion , an additional minipump was implanted 12 cm apart from the other minipump and connected to the intracerebroventricular cannula via a y connector to deliver leptin intracerebroventricularly for 10 days at 0.62 g / h . the rate of shu-9119 infusion was based on our previous study showing that this dose effectively blocks the mc3/4r and the chronic dietary and cardiovascular effects of leptin in normal sprague - dawley rats ( 9 ) . blood glucose concentration was measured each morning between 9:00 and 10:00 a.m. for determination of blood glucose levels using glucose strips ( reli on ) . the data are expressed as mean se and analyzed by using two - factor anova with repeated measures . dunnett 's test was used for comparisons of experimental and control values within each group , when appropriate . induction of insulin - deficient diabetes with stz was associated with rapid development of hyperglycemia ( 433 28 mg/100 ml on day 5 post - stz injection , fig . 1b , table 1 ) , hyperphagia ( from 22 1 g / day in the control period to 45 2 g / day on day 5 after stz injection , fig . 1a ) , and increased water intake and urine volume ( table 1 ) . food intake ( a ) and blood glucose ( b ) responses to chronic intracerebroventricular infusion of leptin ( , n = 5 ) or the mc3/4r agonist mtii in ad libitum fed ( , n = 5 ) and pair - fed ( , n = 5 ) stz - diabetic rats . blood glucose , water intake , and urine output in stz - diabetic rats treated with leptin alone ( 0.62 g / h i.c.v . ) or during mc3/4r antagonism with shu-9119 ( 1 nmol / h i.c.v . ) and in diabetic rats fed ad libitum or pair - fed that were infused with the mc3/4r agonist mtii ( 10 ng / h i.c.v . ) values expressed are for day 5 of control period , 5 days after injection of stz , and during the experimental periods as indicated in the table . note : total daily fluid intake equals the water plus a fixed continuous intravenous saline infusion ( 40 ml / day , 0.45% saline ) throughout the study . * p < 0.05 compared with control ; p < 0.05 compared with stz day 5 . induction of stz - diabetes also caused marked bradycardia with heart rate rapidly falling by as much as 77 bpm 5 days after stz injection ( fig . map responses after the induction of diabetes were variable among the groups but remained unchanged on average during the first 5 days after induction of diabetes ( fig . we previously showed that it takes 1015 days after the stz injection for a significant reduction in map ( 11 ) . heart rate ( a ) and map ( b ) responses to chronic intracerebroventricular infusion of leptin ( , n = 5 ) or the mc3/4r agonist mtii in ad libitum fed ( , n = 5 ) and pair - fed ( , n = 5 ) stz - diabetic rats . baseline heart rate and map values for leptin , mtii ad libitum fed , and mtii pair - fed groups were 378 5 bpm and 100 3 mmhg , 366 11 bpm and 90 1 mmhg , and 398 8 bpm and 101 4 mmhg , respectively . chronic intracerebroventricular leptin infusion for 10 days in diabetic rats decreased food intake from 45 2 g / day to an average of 16 1 g / day during the last 5 days of leptin infusion ( fig . 1a ) and reduced blood glucose levels all the way back to pre - diabetic control values ( 118 19 mg/100 ml , fig . the normalization of blood glucose levels by intracerebroventricular leptin infusion was accompanied by a marked reduction in urine volume and water intake to values similar to pre - diabetic levels ( table 1 ) . in addition , chronic leptin treatment reversed the bradycardia and raised heart rate by 4050 bpm above the pre - diabetic control values ( fig . although leptin tended to raise map , the increase was not significant compared with the day before leptin infusion was started ( fig . chronic intracerebroventricular infusion of the mc3/4r agonist , mtii , at a dose of 10 ng / h for 10 days in ad libitum fed diabetic rats caused only a transient reduction in food intake lasting for 34 days , after which food intake returned to values observed before the mtii infusion was started ( fig . during the course of the 10-day treatment period , food intake continued to rise to a level that was more than double the initial control level . a similar pattern was observed for the effects of mtii on blood glucose levels , except for the less pronounced initial reduction in glucose levels ( fig . chronic mc3/4r activation also resulted in a transient reduction in water intake and urine output , in parallel with the transient changes in blood glucose levels ( table 1 ) . chronic mc3/4r activation in ad libitum fed diabetic rats also attenuated the bradycardia associated with induction of diabetes and raised heart rate by 50 bpm during the initial 5 days of infusion ( fig . however , this effect waned on days 67 of mtii infusion and heart rate gradually fell . we also observed a 10-mmhg initial elevation in map during the first 56 days of mtii infusion that was followed by a return of map to the same values observed on the day before mtii treatment was initiated ( fig . a fivefold increase in the dose of mtii from 1050 ng / h had no additional effect to prevent the hyperphagia , hyperglycemia , and bradycardia associated with stz - induced diabetes ( fig . these results indicate that chronic mc3/4r activation alone does not recapitulate the long - term effects of leptin on food intake , glucose homeostasis , and cardiovascular function in this model of diabetes . inclusion of a group of pair - fed diabetic rats treated with mtii to match the amount of food consumed by the leptin - treated group showed no additional effects of the pair - feeding on the long - term actions of mc3/4r activation on appetite , glucose homeostasis , or cardiovascular function in stz - diabetic rats ( fig . these results suggest that changes in food intake can not explain the lack of sustained long - term metabolic and cardiovascular actions during mc3/4r activation . although chronic mtii intracerebroventricular infusion for 17 days did not mimic the results observed in the leptin - treated group , a functional mc3/4r is required for leptin to exert its metabolic and cardiovascular effects in diabetic rats . chronic blockade of mc3/4r , using shu-9119 , completely abolished the effects of leptin to reduce food intake and to restore euglycemia in stz - diabetic rats . food intake remained elevated at 57 2 g / day and blood glucose averaged 436 14 mg/100 ml on day 10 of leptin plus shu-9119 intracerebroventricular infusion ( fig . 3a and b ) . the inability of leptin to reduce blood glucose levels and food consumption during mc3/4r antagonism was also reflected in the maintenance of markedly elevated water intake and urine output during intracerebroventricular leptin infusion , in contrast to the effects of leptin to completely restore these variables to pre - diabetic levels in rats with intact mc3/4r function ( table 1 ) . food intake ( a ) and blood glucose ( b ) responses to chronic intracerebroventricular infusion of mc3/4r antagonist ( shu-9119 ) and leptin during shu-9119 infusion in stz - diabetic rats ( n = 5 ) . in addition to preventing the antidiabetic and anorexic actions of leptin , shu-9119 treatment also abolished the rise in heart rate during intracerebroventricular leptin infusion ; heart rate continued to fall during leptin treatment ( 137 11 bpm on the last day of intracerebroventricular leptin infusion , fig . mc3/4r antagonism also blocked leptin 's ability to prevent the fall in map normally observed when stz - induced diabetes is maintained for periods longer than 2 weeks ( 92 3 vs. 80 3 mmhg for control and the last day of intracerebroventricular leptin infusion , respectively ; fig . 4b ) . heart rate ( a ) and map ( b ) responses to chronic intracerebroventricular infusion of mc3/4r antagonist ( shu-9119 ) and leptin during shu-9119 infusion in stz - diabetic rats ( n = 5 ) . induction of insulin - deficient diabetes with stz was associated with rapid development of hyperglycemia ( 433 28 mg/100 ml on day 5 post - stz injection , fig . 1b , table 1 ) , hyperphagia ( from 22 1 g / day in the control period to 45 2 g / day on day 5 after stz injection , fig . 1a ) , and increased water intake and urine volume ( table 1 ) . food intake ( a ) and blood glucose ( b ) responses to chronic intracerebroventricular infusion of leptin ( , n = 5 ) or the mc3/4r agonist mtii in ad libitum fed ( , n = 5 ) and pair - fed ( , n = 5 ) stz - diabetic rats . blood glucose , water intake , and urine output in stz - diabetic rats treated with leptin alone ( 0.62 g / h i.c.v . ) or during mc3/4r antagonism with shu-9119 ( 1 nmol / h i.c.v . ) and in diabetic rats fed ad libitum or pair - fed that were infused with the mc3/4r agonist mtii ( 10 ng / h i.c.v . ) values expressed are for day 5 of control period , 5 days after injection of stz , and during the experimental periods as indicated in the table . note : total daily fluid intake equals the water plus a fixed continuous intravenous saline infusion ( 40 ml / day , 0.45% saline ) throughout the study . * p < 0.05 compared with control ; p < 0.05 compared with stz day 5 . induction of stz - diabetes also caused marked bradycardia with heart rate rapidly falling by as much as 77 bpm 5 days after stz injection ( fig . map responses after the induction of diabetes were variable among the groups but remained unchanged on average during the first 5 days after induction of diabetes ( fig . we previously showed that it takes 1015 days after the stz injection for a significant reduction in map ( 11 ) . heart rate ( a ) and map ( b ) responses to chronic intracerebroventricular infusion of leptin ( , n = 5 ) or the mc3/4r agonist mtii in ad libitum fed ( , n = 5 ) and pair - fed ( , n = 5 ) stz - diabetic rats . baseline heart rate and map values for leptin , mtii ad libitum fed , and mtii pair - fed groups were 378 5 bpm and 100 3 mmhg , 366 11 bpm and 90 1 mmhg , and 398 8 bpm and 101 4 mmhg , respectively . chronic intracerebroventricular leptin infusion for 10 days in diabetic rats decreased food intake from 45 2 g / day to an average of 16 1 g / day during the last 5 days of leptin infusion ( fig . 1a ) and reduced blood glucose levels all the way back to pre - diabetic control values ( 118 19 mg/100 ml , fig . the normalization of blood glucose levels by intracerebroventricular leptin infusion was accompanied by a marked reduction in urine volume and water intake to values similar to pre - diabetic levels ( table 1 ) . in addition , chronic leptin treatment reversed the bradycardia and raised heart rate by 4050 bpm above the pre - diabetic control values ( fig . although leptin tended to raise map , the increase was not significant compared with the day before leptin infusion was started ( fig . chronic intracerebroventricular infusion of the mc3/4r agonist , mtii , at a dose of 10 ng / h for 10 days in ad libitum fed diabetic rats caused only a transient reduction in food intake lasting for 34 days , after which food intake returned to values observed before the mtii infusion was started ( fig . during the course of the 10-day treatment period , food intake continued to rise to a level that was more than double the initial control level . a similar pattern was observed for the effects of mtii on blood glucose levels , except for the less pronounced initial reduction in glucose levels ( fig . chronic mc3/4r activation also resulted in a transient reduction in water intake and urine output , in parallel with the transient changes in blood glucose levels ( table 1 ) . chronic mc3/4r activation in ad libitum fed diabetic rats also attenuated the bradycardia associated with induction of diabetes and raised heart rate by 50 bpm during the initial 5 days of infusion ( fig . however , this effect waned on days 67 of mtii infusion and heart rate gradually fell . we also observed a 10-mmhg initial elevation in map during the first 56 days of mtii infusion that was followed by a return of map to the same values observed on the day before mtii treatment was initiated ( fig . a fivefold increase in the dose of mtii from 1050 ng / h had no additional effect to prevent the hyperphagia , hyperglycemia , and bradycardia associated with stz - induced diabetes ( fig . these results indicate that chronic mc3/4r activation alone does not recapitulate the long - term effects of leptin on food intake , glucose homeostasis , and cardiovascular function in this model of diabetes . inclusion of a group of pair - fed diabetic rats treated with mtii to match the amount of food consumed by the leptin - treated group showed no additional effects of the pair - feeding on the long - term actions of mc3/4r activation on appetite , glucose homeostasis , or cardiovascular function in stz - diabetic rats ( fig . these results suggest that changes in food intake can not explain the lack of sustained long - term metabolic and cardiovascular actions during mc3/4r activation . although chronic mtii intracerebroventricular infusion for 17 days did not mimic the results observed in the leptin - treated group , a functional mc3/4r is required for leptin to exert its metabolic and cardiovascular effects in diabetic rats . chronic blockade of mc3/4r , using shu-9119 , completely abolished the effects of leptin to reduce food intake and to restore euglycemia in stz - diabetic rats . food intake remained elevated at 57 2 g / day and blood glucose averaged 436 14 mg/100 ml on day 10 of leptin plus shu-9119 intracerebroventricular infusion ( fig . 3a and b ) . the inability of leptin to reduce blood glucose levels and food consumption during mc3/4r antagonism was also reflected in the maintenance of markedly elevated water intake and urine output during intracerebroventricular leptin infusion , in contrast to the effects of leptin to completely restore these variables to pre - diabetic levels in rats with intact mc3/4r function ( table 1 ) . food intake ( a ) and blood glucose ( b ) responses to chronic intracerebroventricular infusion of mc3/4r antagonist ( shu-9119 ) and leptin during shu-9119 infusion in stz - diabetic rats ( n = 5 ) . in addition to preventing the antidiabetic and anorexic actions of leptin , shu-9119 treatment also abolished the rise in heart rate during intracerebroventricular leptin infusion ; heart rate continued to fall during leptin treatment ( 137 11 bpm on the last day of intracerebroventricular leptin infusion , fig . mc3/4r antagonism also blocked leptin 's ability to prevent the fall in map normally observed when stz - induced diabetes is maintained for periods longer than 2 weeks ( 92 3 vs. 80 3 mmhg for control and the last day of intracerebroventricular leptin infusion , respectively ; fig . heart rate ( a ) and map ( b ) responses to chronic intracerebroventricular infusion of mc3/4r antagonist ( shu-9119 ) and leptin during shu-9119 infusion in stz - diabetic rats ( n = 5 ) . first , we demonstrated that the chronic antidiabetic , appetite , and cardiovascular actions of leptin in insulin - deficient stz - diabetic rats require a functional cns melanocortin system and ultimately activation of mc3/4r ; blockade of these receptors completely prevented the chronic effects of leptin on food and water intake , blood glucose , heart rate , blood pressure , and urine volume . second , chronic mc3/4r activation alone transiently reduced appetite and blood glucose while raising heart rate in diabetic rats , but these effects gradually waned and did not mimic the responses observed during chronic hyperleptinemia . these results indicate that activation of the mc3/4r is required for leptin to exert its metabolic and cardiovascular actions , but is apparently not sufficient to mimic these actions of leptin that may involve interactions of multiple pathways . it is likely that other systems are triggered during prolonged mc3/4r stimulation to offset the reductions in food intake and blood glucose as well as the map and heart rate responses to mc3/4r activation . the identity of these compensatory systems is still uncertain , but may involve activation of orexigenic factors known to be suppressed by leptin ( i.e. , npy , melanin - concentrating hormone , agrp , and others ) and/or downregulation of anorexigenic factors that are stimulated by leptin ( i.e. , corticotrophin - releasing hormone , cocaine - amphetamine related peptide , and brain - derived neurotrophic factor , for example ) . it is possible that leptin - induced changes in one or more of these factors also contribute to the powerful antidiabetic effect of leptin and additional studies will be needed to answer these questions . the observation that mc3/4r antagonism abolished the antidiabetic and cardiovascular actions of leptin is consistent with our previous finding that an intact hypothalamic mc3/4r is necessary for leptin to reduce food intake and fasting insulin levels and to raise heart rate and blood pressure in normal nondiabetic rats ( 9 ) . however , the long - term responses to mc3/4r activation by mtii infusion in stz - diabetic rats differed from our previous observations in nondiabetic rats where , despite not causing sustained reductions in food intake , the elevations in heart rate and map were maintained through the entire period of mtii treatment ( 22,23 ) . increasing the dose of mtii fivefold did not alter the responses when compared with the lower dosage , indicating that the waning responses are not likely to be caused by an insufficient level of mc3/4r activation in diabetic rats . one possible explanation for these differences is that activation of compensatory mechanisms during chronic mc3/4r activation is even more pronounced in diabetes than under normal conditions . for instance , in stz - induced diabetes , npy / agrp neurons are markedly activated and may play a major role in promoting the hyperphagia in this model of diabetes ( 24,25 ) . another possible explanation for the inability of chronic mc3/4r activation to mimic the antidiabetic effects of leptin is that mc3/4r may have only a short - term anorectic action and that the initial fall in blood glucose is mainly caused by a reduction in food intake . it could be hypothesized that the hyperphagia in diabetes and increased intake of glucose and other nutrients ( fat and proteins ) that can be transformed into glucose overcomes the effects of mc3/4r to lower blood glucose . to test this hypothesis , we studied a group of diabetic rats in which food intake was prevented from increasing by pair - feeding them to match the amount of food consumed by the leptin - treated group . preventing the hyperphagia , however , did not improve the effectiveness of mtii to lower blood glucose levels , suggesting that the lack of sustained antidiabetic effect during chronic mc3/4r activation was not because of mtii inability to reduce appetite . these results also confirm our previous observation that the reduction of food intake in diabetic rats during intracerebroventricular leptin infusion does not play a major role in mediating the antidiabetic actions of leptin or in preventing the cardiovascular alterations associated with uncontrolled diabetes ( 11 ) . the precise mechanisms by which leptin exerts its powerful effect on peripheral glucose utilization even in insulin - deficient diabetic animals are still unclear , although our current study clearly indicates that a functional mc3/4r plays a crucial role . previous studies have implicated a role for the autonomic nervous system in mediating the acute effects of leptin on glucose regulation by showing that the increased insulin sensitivity observed during intracerebroventricular injection of single doses of leptin can be blocked by adrenergic receptor antagonism ( 26,27 ) , or that leptin 's ability to suppress liver glucose production can be prevented by denervation of the vagal fibers innervating the liver ( 28 ) . we recently showed that chronic blockade of the 1 , 1 , 2 , and 3 adrenergic receptors failed to attenuate leptin 's ability to restore euglycemia in stz - diabetic rats ( 11 ) . therefore , it is possible that leptin - induced sympathetic stimulation of nonadrenergic receptors may contribute to these effects of leptin and that the suppression of liver glucose output into the systemic circulation of diabetic rats may play an important role in mediating the chronic effects of leptin on glucose homeostasis , but additional studies are needed to test these possibilities . although our previous study indicates that adrenergic receptor activation does not mediate the chronic antidiabetic effects of leptin , 4050% of the rise in heart rate and prevention of the bradycardia in stz - diabetic rats during leptin infusion was dependent of adrenergic stimulation ( 11 ) . the mechanisms responsible for the remaining 5060% of leptin - induced rise in heart rate in diabetic rats are not well understood . however , the bradycardia caused by induction of stz - diabetes is associated with marked reductions in the intrinsic heart rate ( 29 ) , which is the heart rate in the absence of sympathetic and parasympathetic inputs to the heart . moreover , chronic intracerebroventricular leptin infusion completely restored intrinsic heart rate back to pre - diabetic values ( 29 ) . we have also shown that , similar to leptin , mc3/4r activation is associated with sustained sympathetic nervous system activation in nondiabetic rats ( 30 ) . whether the lack of sustained increases in heart rate during mtii in stz - diabetic is because of an inability of chronic mc3/4r activation to cause sustained elevations in cardiac sympathetic activity in a hyperglycemic state or to an inability to increase intrinsic heart rate remains to be determined . in summary , leptin has powerful cns - mediated antidiabetic effects in insulin - deficient diabetic rats that require activation of the cns melanocortin pathway . . however , stimulation of the cns mc3/4r alone does not confer the same long - term metabolic and cardiovascular responses as observed with hyperleptinemia . these observations indicate that although leptin - mediated activation of the melanocortin pathway may be necessary for the antidiabetic and cardiovascular actions of leptin in this model , it is not sufficient to completely mimic leptin 's chronic actions . this suggests that leptin exerts its cns - mediated effects on appetite , blood glucose , map , and heart rate via a complex system that likely involves interaction of multiple pathways . unraveling these interactions would contribute to a better understanding of the cns control of appetite , glucose homeostasis , and cardiovascular function and could lead to the development of novel therapeutic strategies to treated obesity , metabolic syndrome , diabetes , and cardiovascular diseases .

objectivewe recently showed that leptin has powerful central nervous system ( cns)-mediated antidiabetic and cardiovascular actions . this study tested whether the cns melanocortin system mediates these actions of leptin in diabetic rats.research design and methodsa cannula was placed in the lateral ventricle of sprague - dawley rats for intracerebroventricular infusions , and arterial and venous catheters were implanted to measure mean arterial pressure ( map ) and heart rate 24 h / day and for intravenous infusions . after recovery from surgery for 8 days , rats were injected with streptozotocin ( stz ) , and 5 days later , either saline or the melanocortin 3 and 4 receptor ( mc3/4r ) antagonist shu-9119 ( 1 nmol / h ) was infused intracerebroventricularly for 17 days . seven days after starting the antagonist , leptin ( 0.62 g / h ) was added to the intracerebroventricular infusion for 10 days . another group of diabetic rats was infused with the mc3/4r agonist mtii ( 10 ng / h i.c.v . ) for 12 days , followed by 7 days at 50 ng / h.resultsinduction of diabetes caused hyperphagia , hyperglycemia , and decreases in heart rate ( 76 bpm ) and map ( 7 mmhg ) . leptin restored appetite , blood glucose , heart rate , and map back to pre - diabetic values in vehicle - treated rats , whereas it had no effect in shu-9119treated rats . mtii infusions transiently reduced blood glucose and raised heart rate and map , which returned to diabetic values 57 days after starting the infusion.conclusionsalthough a functional melanocortin system is necessary for the cns - mediated antidiabetic and cardiovascular actions of leptin , chronic mc3/4r activation is apparently not sufficient to mimic these actions of leptin that may involve interactions of multiple pathways .

RESEARCH DESIGN AND METHODS Animal surgeries. Intra-arterial and intravenous catheterization. Intracerebroventricular cannulation. Experimental protocols. Induction of diabetes. Chronic intracerebroventricular leptin infusion in diabetic rats ( Chronic intracerebroventricular infusion of a MC3/4R agonist, MTII ( Chronic intracerebroventricular leptin infusion during MC3/4R blockade ( Statistical analysis. RESULTS Effects of induction of diabetes and chronic intracerebroventricular leptin infusion on appetite, blood glucose, and cardiovascular function. Effects of chronic intracerebroventricular infusion of MC3/4R agonist on appetite, blood glucose, and cardiovascular function. Effects of chronic intracerebroventricular leptin infusion on appetite, glucose levels, and cardiovascular function during MC3/4R antagonism. DISCUSSION

male sprague - dawley ( harlan , indianapolis , in ) rats ( 325350 g , n = 20 ) were anesthetized with 50 mg / kg sodium pentobarbital ( nembutal ) , and atropine sulfate ( 0.1 mg / kg ) was administered to prevent excess airway secretions . the arterial catheter was connected to a pressure transducer ( maxxim ) for continuous 24-h measurement of mean arterial pressure ( map ) and heart rate using computerized techniques as previously described ( 7,8 ) . map , heart rate , urine volume , food , and water intake were measured 24 h / day and average values were recorded daily . five days after stz injection , leptin ( 0.62 g / h , 0.5 l / h ) was infused intracerebroventricularly for 10 days via osmotic minipump ( model 2002 , durect ) implanted subcutaneously in the scapular region as previously described ( 20 ) . five days after stz injection , the mc3/4r agonist , mtii ( 10 ng / h , 0.5 l / h , polypeptide laboratories ) , was infused intracerebroventricularly for 10 days via osmotic minipump as described above ( n = 5 ) . on the 10th day of mtii infusion at 10 ng / h , the rats were lightly anesthetized with isofluorane and the osmotic minipump was replaced by another pump to deliver the agonist at a higher concentration ( 50 ng / h ) . in a separate experiment , diabetic rats ( n = 5 ) were treated with mtii at the dose of 10 ng / h and were pair - fed the same amount of food consumed by diabetic rats during chronic leptin intracerebroventricular infusion . five days after stz injection , the mc3/4r antagonist shu-9119 ( 1 nmol / h , 0.5 l / h ; polypeptide laboratories ) was infused intracerebroventricularly for 17 days via osmotic minipump as described above . after the first 7 days of intracerebroventricular shu-9119 infusion , an additional minipump was implanted 12 cm apart from the other minipump and connected to the intracerebroventricular cannula via a y connector to deliver leptin intracerebroventricularly for 10 days at 0.62 g / h . the rate of shu-9119 infusion was based on our previous study showing that this dose effectively blocks the mc3/4r and the chronic dietary and cardiovascular effects of leptin in normal sprague - dawley rats ( 9 ) . male sprague - dawley ( harlan , indianapolis , in ) rats ( 325350 g , n = 20 ) were anesthetized with 50 mg / kg sodium pentobarbital ( nembutal ) , and atropine sulfate ( 0.1 mg / kg ) was administered to prevent excess airway secretions . the arterial catheter was connected to a pressure transducer ( maxxim ) for continuous 24-h measurement of mean arterial pressure ( map ) and heart rate using computerized techniques as previously described ( 7,8 ) . map , heart rate , urine volume , food , and water intake were measured 24 h / day and average values were recorded daily . five days after stz injection , leptin ( 0.62 g / h , 0.5 l / h ) was infused intracerebroventricularly for 10 days via osmotic minipump ( model 2002 , durect ) implanted subcutaneously in the scapular region as previously described ( 20 ) . five days after stz injection , the mc3/4r agonist , mtii ( 10 ng / h , 0.5 l / h , polypeptide laboratories ) , was infused intracerebroventricularly for 10 days via osmotic minipump as described above ( n = 5 ) . on the 10th day of mtii infusion at 10 ng / h , the rats were lightly anesthetized with isofluorane and the osmotic minipump was replaced by another pump to deliver the agonist at a higher concentration ( 50 ng / h ) . in a separate experiment , diabetic rats ( n = 5 ) were treated with mtii at the dose of 10 ng / h and were pair - fed the same amount of food consumed by diabetic rats during chronic leptin intracerebroventricular infusion . five days after stz injection , the mc3/4r antagonist shu-9119 ( 1 nmol / h , 0.5 l / h ; polypeptide laboratories ) was infused intracerebroventricularly for 17 days via osmotic minipump as described above . after the first 7 days of intracerebroventricular shu-9119 infusion , an additional minipump was implanted 12 cm apart from the other minipump and connected to the intracerebroventricular cannula via a y connector to deliver leptin intracerebroventricularly for 10 days at 0.62 g / h . the rate of shu-9119 infusion was based on our previous study showing that this dose effectively blocks the mc3/4r and the chronic dietary and cardiovascular effects of leptin in normal sprague - dawley rats ( 9 ) . food intake ( a ) and blood glucose ( b ) responses to chronic intracerebroventricular infusion of leptin ( , n = 5 ) or the mc3/4r agonist mtii in ad libitum fed ( , n = 5 ) and pair - fed ( , n = 5 ) stz - diabetic rats . blood glucose , water intake , and urine output in stz - diabetic rats treated with leptin alone ( 0.62 g / h i.c.v . ) or during mc3/4r antagonism with shu-9119 ( 1 nmol / h i.c.v . ) and in diabetic rats fed ad libitum or pair - fed that were infused with the mc3/4r agonist mtii ( 10 ng / h i.c.v . ) heart rate ( a ) and map ( b ) responses to chronic intracerebroventricular infusion of leptin ( , n = 5 ) or the mc3/4r agonist mtii in ad libitum fed ( , n = 5 ) and pair - fed ( , n = 5 ) stz - diabetic rats . baseline heart rate and map values for leptin , mtii ad libitum fed , and mtii pair - fed groups were 378 5 bpm and 100 3 mmhg , 366 11 bpm and 90 1 mmhg , and 398 8 bpm and 101 4 mmhg , respectively . chronic intracerebroventricular leptin infusion for 10 days in diabetic rats decreased food intake from 45 2 g / day to an average of 16 1 g / day during the last 5 days of leptin infusion ( fig . 1a ) and reduced blood glucose levels all the way back to pre - diabetic control values ( 118 19 mg/100 ml , fig . in addition , chronic leptin treatment reversed the bradycardia and raised heart rate by 4050 bpm above the pre - diabetic control values ( fig . chronic intracerebroventricular infusion of the mc3/4r agonist , mtii , at a dose of 10 ng / h for 10 days in ad libitum fed diabetic rats caused only a transient reduction in food intake lasting for 34 days , after which food intake returned to values observed before the mtii infusion was started ( fig . chronic mc3/4r activation in ad libitum fed diabetic rats also attenuated the bradycardia associated with induction of diabetes and raised heart rate by 50 bpm during the initial 5 days of infusion ( fig . a fivefold increase in the dose of mtii from 1050 ng / h had no additional effect to prevent the hyperphagia , hyperglycemia , and bradycardia associated with stz - induced diabetes ( fig . these results indicate that chronic mc3/4r activation alone does not recapitulate the long - term effects of leptin on food intake , glucose homeostasis , and cardiovascular function in this model of diabetes . inclusion of a group of pair - fed diabetic rats treated with mtii to match the amount of food consumed by the leptin - treated group showed no additional effects of the pair - feeding on the long - term actions of mc3/4r activation on appetite , glucose homeostasis , or cardiovascular function in stz - diabetic rats ( fig . although chronic mtii intracerebroventricular infusion for 17 days did not mimic the results observed in the leptin - treated group , a functional mc3/4r is required for leptin to exert its metabolic and cardiovascular effects in diabetic rats . food intake remained elevated at 57 2 g / day and blood glucose averaged 436 14 mg/100 ml on day 10 of leptin plus shu-9119 intracerebroventricular infusion ( fig . the inability of leptin to reduce blood glucose levels and food consumption during mc3/4r antagonism was also reflected in the maintenance of markedly elevated water intake and urine output during intracerebroventricular leptin infusion , in contrast to the effects of leptin to completely restore these variables to pre - diabetic levels in rats with intact mc3/4r function ( table 1 ) . food intake ( a ) and blood glucose ( b ) responses to chronic intracerebroventricular infusion of mc3/4r antagonist ( shu-9119 ) and leptin during shu-9119 infusion in stz - diabetic rats ( n = 5 ) . in addition to preventing the antidiabetic and anorexic actions of leptin , shu-9119 treatment also abolished the rise in heart rate during intracerebroventricular leptin infusion ; heart rate continued to fall during leptin treatment ( 137 11 bpm on the last day of intracerebroventricular leptin infusion , fig . heart rate ( a ) and map ( b ) responses to chronic intracerebroventricular infusion of mc3/4r antagonist ( shu-9119 ) and leptin during shu-9119 infusion in stz - diabetic rats ( n = 5 ) . food intake ( a ) and blood glucose ( b ) responses to chronic intracerebroventricular infusion of leptin ( , n = 5 ) or the mc3/4r agonist mtii in ad libitum fed ( , n = 5 ) and pair - fed ( , n = 5 ) stz - diabetic rats . blood glucose , water intake , and urine output in stz - diabetic rats treated with leptin alone ( 0.62 g / h i.c.v . ) or during mc3/4r antagonism with shu-9119 ( 1 nmol / h i.c.v . ) and in diabetic rats fed ad libitum or pair - fed that were infused with the mc3/4r agonist mtii ( 10 ng / h i.c.v . ) heart rate ( a ) and map ( b ) responses to chronic intracerebroventricular infusion of leptin ( , n = 5 ) or the mc3/4r agonist mtii in ad libitum fed ( , n = 5 ) and pair - fed ( , n = 5 ) stz - diabetic rats . baseline heart rate and map values for leptin , mtii ad libitum fed , and mtii pair - fed groups were 378 5 bpm and 100 3 mmhg , 366 11 bpm and 90 1 mmhg , and 398 8 bpm and 101 4 mmhg , respectively . chronic intracerebroventricular leptin infusion for 10 days in diabetic rats decreased food intake from 45 2 g / day to an average of 16 1 g / day during the last 5 days of leptin infusion ( fig . 1a ) and reduced blood glucose levels all the way back to pre - diabetic control values ( 118 19 mg/100 ml , fig . in addition , chronic leptin treatment reversed the bradycardia and raised heart rate by 4050 bpm above the pre - diabetic control values ( fig . chronic intracerebroventricular infusion of the mc3/4r agonist , mtii , at a dose of 10 ng / h for 10 days in ad libitum fed diabetic rats caused only a transient reduction in food intake lasting for 34 days , after which food intake returned to values observed before the mtii infusion was started ( fig . chronic mc3/4r activation in ad libitum fed diabetic rats also attenuated the bradycardia associated with induction of diabetes and raised heart rate by 50 bpm during the initial 5 days of infusion ( fig . a fivefold increase in the dose of mtii from 1050 ng / h had no additional effect to prevent the hyperphagia , hyperglycemia , and bradycardia associated with stz - induced diabetes ( fig . these results indicate that chronic mc3/4r activation alone does not recapitulate the long - term effects of leptin on food intake , glucose homeostasis , and cardiovascular function in this model of diabetes . inclusion of a group of pair - fed diabetic rats treated with mtii to match the amount of food consumed by the leptin - treated group showed no additional effects of the pair - feeding on the long - term actions of mc3/4r activation on appetite , glucose homeostasis , or cardiovascular function in stz - diabetic rats ( fig . although chronic mtii intracerebroventricular infusion for 17 days did not mimic the results observed in the leptin - treated group , a functional mc3/4r is required for leptin to exert its metabolic and cardiovascular effects in diabetic rats . food intake remained elevated at 57 2 g / day and blood glucose averaged 436 14 mg/100 ml on day 10 of leptin plus shu-9119 intracerebroventricular infusion ( fig . the inability of leptin to reduce blood glucose levels and food consumption during mc3/4r antagonism was also reflected in the maintenance of markedly elevated water intake and urine output during intracerebroventricular leptin infusion , in contrast to the effects of leptin to completely restore these variables to pre - diabetic levels in rats with intact mc3/4r function ( table 1 ) . food intake ( a ) and blood glucose ( b ) responses to chronic intracerebroventricular infusion of mc3/4r antagonist ( shu-9119 ) and leptin during shu-9119 infusion in stz - diabetic rats ( n = 5 ) . in addition to preventing the antidiabetic and anorexic actions of leptin , shu-9119 treatment also abolished the rise in heart rate during intracerebroventricular leptin infusion ; heart rate continued to fall during leptin treatment ( 137 11 bpm on the last day of intracerebroventricular leptin infusion , fig . heart rate ( a ) and map ( b ) responses to chronic intracerebroventricular infusion of mc3/4r antagonist ( shu-9119 ) and leptin during shu-9119 infusion in stz - diabetic rats ( n = 5 ) . first , we demonstrated that the chronic antidiabetic , appetite , and cardiovascular actions of leptin in insulin - deficient stz - diabetic rats require a functional cns melanocortin system and ultimately activation of mc3/4r ; blockade of these receptors completely prevented the chronic effects of leptin on food and water intake , blood glucose , heart rate , blood pressure , and urine volume . second , chronic mc3/4r activation alone transiently reduced appetite and blood glucose while raising heart rate in diabetic rats , but these effects gradually waned and did not mimic the responses observed during chronic hyperleptinemia . these results indicate that activation of the mc3/4r is required for leptin to exert its metabolic and cardiovascular actions , but is apparently not sufficient to mimic these actions of leptin that may involve interactions of multiple pathways . the observation that mc3/4r antagonism abolished the antidiabetic and cardiovascular actions of leptin is consistent with our previous finding that an intact hypothalamic mc3/4r is necessary for leptin to reduce food intake and fasting insulin levels and to raise heart rate and blood pressure in normal nondiabetic rats ( 9 ) . however , the long - term responses to mc3/4r activation by mtii infusion in stz - diabetic rats differed from our previous observations in nondiabetic rats where , despite not causing sustained reductions in food intake , the elevations in heart rate and map were maintained through the entire period of mtii treatment ( 22,23 ) . another possible explanation for the inability of chronic mc3/4r activation to mimic the antidiabetic effects of leptin is that mc3/4r may have only a short - term anorectic action and that the initial fall in blood glucose is mainly caused by a reduction in food intake . previous studies have implicated a role for the autonomic nervous system in mediating the acute effects of leptin on glucose regulation by showing that the increased insulin sensitivity observed during intracerebroventricular injection of single doses of leptin can be blocked by adrenergic receptor antagonism ( 26,27 ) , or that leptin 's ability to suppress liver glucose production can be prevented by denervation of the vagal fibers innervating the liver ( 28 ) . we recently showed that chronic blockade of the 1 , 1 , 2 , and 3 adrenergic receptors failed to attenuate leptin 's ability to restore euglycemia in stz - diabetic rats ( 11 ) . although our previous study indicates that adrenergic receptor activation does not mediate the chronic antidiabetic effects of leptin , 4050% of the rise in heart rate and prevention of the bradycardia in stz - diabetic rats during leptin infusion was dependent of adrenergic stimulation ( 11 ) . the mechanisms responsible for the remaining 5060% of leptin - induced rise in heart rate in diabetic rats are not well understood . however , the bradycardia caused by induction of stz - diabetes is associated with marked reductions in the intrinsic heart rate ( 29 ) , which is the heart rate in the absence of sympathetic and parasympathetic inputs to the heart . moreover , chronic intracerebroventricular leptin infusion completely restored intrinsic heart rate back to pre - diabetic values ( 29 ) . whether the lack of sustained increases in heart rate during mtii in stz - diabetic is because of an inability of chronic mc3/4r activation to cause sustained elevations in cardiac sympathetic activity in a hyperglycemic state or to an inability to increase intrinsic heart rate remains to be determined . in summary , leptin has powerful cns - mediated antidiabetic effects in insulin - deficient diabetic rats that require activation of the cns melanocortin pathway . these observations indicate that although leptin - mediated activation of the melanocortin pathway may be necessary for the antidiabetic and cardiovascular actions of leptin in this model , it is not sufficient to completely mimic leptin 's chronic actions . this suggests that leptin exerts its cns - mediated effects on appetite , blood glucose , map , and heart rate via a complex system that likely involves interaction of multiple pathways . unraveling these interactions would contribute to a better understanding of the cns control of appetite , glucose homeostasis , and cardiovascular function and could lead to the development of novel therapeutic strategies to treated obesity , metabolic syndrome , diabetes , and cardiovascular diseases .

this was an interpretive comparative interview study with 20 foreign - born chinese american couples ( n = 40 ) , in which one member was diagnosed with type 2 diabetes . inclusion criteria included having a diabetes diagnosis for at least 1 year , being aged 3575 years , having been married for a minimum of 1 year , self - identifying as chinese american or chinese , having immigrated to the u.s . from mainland china or hong kong , and having a spouse who would agree to participate . exclusion criteria for patients included major diabetes complications ( cerebrovascular accident or myocardial infarction within the last 12 months ; proliferative retinopathy ; renal insufficiency ; or amputations ) because our intent was to study patients who were early enough in the disease to benefit from behavioral and family interventions . a convenience sample was recruited from community clinics , community service organizations , and via public notices . six semistructured interviews with couples in individual , couple , and group contexts focused on illness understandings , perceptions of diabetes care , acculturation histories , and concrete positive , negative , and memorable narratives of diabetes care . couples narrated in each other 's presence ( two couple interviews ) and in group interviews with those who shared their experience as a patient ( two group interviews ) or spouse ( two group interviews ) . a subset of participants ( n = 13 ) was also interviewed individually if extra time was needed to complete interview questions or if nondisclosure in shared interview settings suggested that a private interview would yield more complete data . interviews were conducted in cantonese , and audiotaped text was simultaneously translated from cantonese to english and transcribed verbatim by skilled bilingual staff . each audiofile was then reviewed and checked for accuracy by a separate bilingual bicultural staff member who had conducted the interview . narrative and thematic analyses were conducted by a multicultural and multidisciplinary team of chinese american and caucasian nurses and psychologists ( 18,19 ) . after all text was coded for thematic codes in atlas - ti , codes were selected for review that identified challenging situations in diabetes management by patients and spouses . complete text from three codes was examined : couple dynamics , conflict , and diabetes management . both reflective discussions and narratives of positive , difficult , and meaningful aspects of care were analyzed . the analyzed text comprised > 450 pages of extracted text and represented a broad inclusive portion of narratives that were drawn from interviews conducted in all three contexts ( couple , group , and individual interviews ) . thus , text for this manuscript was analyzed in the context of the holistic analysis of each couple 's dynamics . to address generalizability , findings were presented to separate respondent groups of patients and spouses who met the same inclusion criteria as the original sample . respondents ( n = 19 ) met in groups of patients or spouses for two separate 2-h interviews . they were asked to review themes presented in this manuscript for adequacy and to add personal variations to the presented themes . the sample of informants was 20 foreign - born chinese american couples ( n = 40 ) , with one member with diabetes . on average , informants were ( means sd ) age 62 9.2 years , married for 34 13.4 years , and had immigrated to the u.s . 15 13.4 years ago from mainland china ( 55% ) or hong kong ( 45% ) . patients were 40% male , had been diagnosed 8.4 5.9 years , were treated primarily with oral medications ( 85% ) , and had an average a1c of 6.93 0.96% . members of the respondent group ( n = 19 ; 13 patients , 6 spouses ) representing 16 separate families were similar to the informant group on all measures . they were on average aged 60 9.4 years , married for 32 12.1 years , and had immigrated to the u.s . 11.8 12 years ago from mainland china ( 46% ) and hong kong ( 54% ) . patients in the respondent group were 54% male , had been diagnosed 6.2 4.2 years , and all were treated with oral medications ; a1c was not collected with respondent group participants . cultural and family challenges to diabetes management within foreign - born chinese american families included how 1 ) diabetes symptoms challenged family harmony , 2 ) dietary prescriptions challenged food beliefs and practices , and 3 ) disease management requirements challenged established family role responsibilities . each theme is examined in depth with supportive text . in reporting findings , statements made by patients or spouses are identified as such , and statements endorsed by both groups are identified as participant statements . increased irritability as a symptom of diabetes was frequently described as a challenge to family harmony . participants noted that patients get angry easily and easily throw a temper tantrum emotional variability held particular resonance for chinese immigrants because social ease , avoidance of overt expression of strong negative emotions , and accommodation of family members ' expressed and unexpressed needs were culturally valued . after he was diagnosed , when he became upset , he would yell at his mother or whomever . participants noted situations when disease - related irritability contributed to family disagreements . in an illustrative narrative , the brother requested a pack , but the husband bought a carton to save money and to demonstrate kindness . the patient became extremely angry when she learned of the purchase and sharply reprimanded her husband for supporting the brother 's unhealthy habit . in a patient group interview , she attributed her outburst to diabetes ( diabetes destabilizes a person ) but additionally expressed remorse at a comment that might hurt him . this patient , like many in the study , believed immoderate anger posed harm to herself , by raising her blood glucose , and harm to her husband because he had to tolerate a strongly expressed negative emotion . in this ethnic group , the social rather than physiological aspects of glucose regulation were highlighted . participants seldom remarked upon the physiologic symptoms of glucose disregulation , such as sweating or fatigue . rather , their narratives suggested attunement to social and behavioral symptoms of glucose fluctuation and the social dilemmas these symptoms introduced . patients felt called upon to contain their irritability to promote family harmony , while spouses reciprocally felt responsible to forgive patients ' outbursts as a way of caring for patients . observing a biomedically prescribed diabetes regimen required chinese american patients to distance themselves from familiar and shared cultural food habits and practices within the family and community . participants found that culturally meaningful , familiar , and comforting foods had to be foregone or drastically reduced , new foods had to be accommodated , and food quantity became a source of concern . additionally , social habits , such as eating out , sharing dim sum with family and friends , and easily participating in cultural celebrations and banquets were complicated by perceived disease restrictions . disruptions in meaningful cultural food practices mentioned frequently included rice , restricting amounts of food , and changing the balance of various foods that were thought to be beneficial to general health . the meaning of rice in the chinese family diet was a culturally multifaceted and historically nuanced story about sustaining holistic health and well - being and partaking of a symbolically vital food . patients and families were challenged by being asked to restrict rice and change from familiar white fragrant rice to foreign chewy and tasteless brown , red , or black rice . these challenges were persistently noted by participants who felt called upon to cope with this change in communal meals . participants agreed that the amount of rice provided in institutional food settings like airplanes or hospitals was laughably small . the insufficiency was appreciated as an in - group issue , a cultural thing that westerners were not likely to appreciate . when consumed in limited amounts as prescribed in a diabetic diet , rice was missed not just for its familiarity but as a requirement to health and perhaps survival . if you do n't eat rice can you sustain your daily living ? additionally , participants expressed significant suffering because of restrictions on rice , a symbolically comforting food . a central health metaphor expressed by most participants was the need for balance . many found that disease - related food restrictions disregarded cultural concerns for balancing foods ( e.g. , hot and cold ) understood to have specific medicinal properties according to traditional chinese medicine ( tcm ) . even for those who did not specifically incorporate tcm in their diet management , the metaphor of balance was powerfully invoked . i feel that just purely eating like that with no oil or salt , you may be lacking certain nutrients . none of the participants received guidance from health care providers on balancing their diabetes food restrictions according to cultural concerns for balance , although many recommended foods raised concerns about creating such imbalances . participants additionally feared that diabetic food restrictions , if strictly followed , might lead to emotional imbalance and depression . if i say every time , this and that you ca n't eat some people would develop negative feelings and say , there 's no meaning to life now. restricting food during illness is counterintuitive for many chinese americans ( 16,17 ) . rather , special foods and disease - specific medicinal foods should appropriately be provided for patients as both a means of supporting health and demonstrating family solicitousness . spouses were challenged by being asked to restrict patients ' diets during illness and would have preferred providing abundant foods to comfort and fortify patients ' health . diabetes diets also complicated shared social experiences of outings , meals , and celebrations with family and friends . patients ' difficulties in following an appropriate diet at chinese restaurants led some to withdraw from socializing over meals . spouse : now when i asked her to go to dim sum , she would say , i am not going . ( why ? ) it 's meaningless to go by myself . for many chinese participants , social interaction was an integral part of a meal . attending dim sum or chinese breakfast alone was meaningless because meals were sustaining only if shared . birthdays , weddings , or chinese new year 's banquets , with multiple courses and desserts , were unavoidable , yet socially fraught : gee , i could n't even eat a bowl of sweet dessert soup ! how miserable . the presence of family reminded patients of their responsibility to observe diabetes restrictions , as a duty to family . since they tell you , you do n't dare do it . you are well aware that the bowl of sweet dessert soup may do you a lot of harm . reciprocally , family members felt obliged to care for patients ' disease and yet at the same time to create social ease and pleasure . a final social challenge was standing out socially , requiring special attention , or even stigmatized attention because of diabetes . as in earlier reports ( 16,17 ) , patients varied in their willingness to disclose diabetes to friends , but most were distressed when diabetes was the focus of attention at social gatherings . living with diabetes challenged chinese americans to adapt their family roles to accommodate the disease . beliefs varied about who in the family should be responsible to manage the disease , creating conflicts in negotiating differing role expectations . many spouses believed that the family 's role was to assist and encourage but that ultimately the patient had to be responsible : he has to rely on himself . other spouses believed in and constructed a broader role , which included learning about the disease , assisting with disease management efforts , and offering ongoing advice and encouragement . assistance with diet was most often cited , but help with all aspects of diabetes care were considered appropriate . family role conflicts centered on who should create , observe , and enforce food restrictions ; the degree of understanding each family member should have about the diabetes regimen ; and whose philosophy of treatment should prevail . many spouses suggested that a communal diet should be respectful and accommodating of the patient 's dietary restrictions . however , conflicts arose when the cook was over- or underresponsive to restrictions when cooking . for example , one patient asked her husband to cook more healthfully , but he forgot on a daily basis and continued to cook according to his own tastes . conflicts also arose when a spouse cooked healthfully , but the patient felt unduly restricted : a few participants argued in favor of family restraint because the diabetic diet supported general health and observing restrictions demonstrated camaraderie with the patient . longstanding couple and family dynamics were undoubtedly enacted in food arguments ; diabetes provided a focus for these dynamics to be displayed . one couple , for example , reported a persistent , unresolved argument over the husband 's wish to eat pork buns . he agreed to his wife 's strict restrictions on family dinners but would not relinquish his breakfast pork buns . families were additionally challenged when patients and spouses held differing expectations about what family members should learn about the disease . some patients felt neglected because spouses were not very aware of the risks and demands of the disease . these patients held that spouses had a reciprocal role responsibility to understand their condition and assist with its management . they expected spouses to intuitively understand , to be highly sensitive to , and to anticipate their diabetes management needs . differences were apparent in the degree to which spouses believed in and relied upon western biomedicine and tcm treatments and beliefs about the relative importance of treatment regimens , such as diet and exercise : my wife does n't quite accept the things of modern science she 's been using the method that her mother had taught her before patients role responsibilities encompassed managing diabetes well not only for health benefits but out of respect for their families . reciprocal role responsibilities , or caring for diabetes in reciprocation for the family 's care , reflected an interdependent self - construal and added emotional complexity to patients ' responses to their disease . they may have been angry or upset at the disease requirements , but these responses had to be tempered to be responsive to the family 's care . in addition to reciprocity , an interdependent norm of managing diabetes to protect the family 's well - being was evident . patient : this person by my side ( husband ) has to worry about me and watch my diet . reciprocating care with filial children , those who perform their responsibility to care for a parent , also arose as a key concern for many patients . patient : my daughters ( in china ) are often concerned about me . dad , you need to treasure your health . do n't eat sweets , do n't eat fat , and do n't eat salty food ( chuckle ) . interviewer : how do you feel when they talk to you like this ? patient : this speaks to the family members ' concern about me . finally , some families believed the patient was weakened or made vulnerable by the disease . some spouses doubted the patient 's capacity to work at vigorous activity because restrictions on food or rice were thought to affect the patient 's energy and stamina . family sex roles shifted , with some wives shouldering greater financial support responsibilities to accommodate the perceived decrease in patient capabilities . increased irritability as a symptom of diabetes was frequently described as a challenge to family harmony . participants noted that patients get angry easily and easily throw a temper tantrum emotional variability held particular resonance for chinese immigrants because social ease , avoidance of overt expression of strong negative emotions , and accommodation of family members ' expressed and unexpressed needs were culturally valued . after he was diagnosed , when he became upset , he would yell at his mother or whomever . participants noted situations when disease - related irritability contributed to family disagreements . in an illustrative narrative , the brother requested a pack , but the husband bought a carton to save money and to demonstrate kindness . the patient became extremely angry when she learned of the purchase and sharply reprimanded her husband for supporting the brother 's unhealthy habit . in a patient group interview , she attributed her outburst to diabetes ( diabetes destabilizes a person ) but additionally expressed remorse at a comment that might hurt him . this patient , like many in the study , believed immoderate anger posed harm to herself , by raising her blood glucose , and harm to her husband because he had to tolerate a strongly expressed negative emotion . in this ethnic group , the social rather than physiological aspects of glucose regulation were highlighted . participants seldom remarked upon the physiologic symptoms of glucose disregulation , such as sweating or fatigue . rather , their narratives suggested attunement to social and behavioral symptoms of glucose fluctuation and the social dilemmas these symptoms introduced . patients felt called upon to contain their irritability to promote family harmony , while spouses reciprocally felt responsible to forgive patients ' outbursts as a way of caring for patients . observing a biomedically prescribed diabetes regimen required chinese american patients to distance themselves from familiar and shared cultural food habits and practices within the family and community . participants found that culturally meaningful , familiar , and comforting foods had to be foregone or drastically reduced , new foods had to be accommodated , and food quantity became a source of concern . additionally , social habits , such as eating out , sharing dim sum with family and friends , and easily participating in cultural celebrations and banquets were complicated by perceived disease restrictions . disruptions in meaningful cultural food practices mentioned frequently included rice , restricting amounts of food , and changing the balance of various foods that were thought to be beneficial to general health . the meaning of rice in the chinese family diet was a culturally multifaceted and historically nuanced story about sustaining holistic health and well - being and partaking of a symbolically vital food . patients and families were challenged by being asked to restrict rice and change from familiar white fragrant rice to foreign these challenges were persistently noted by participants who felt called upon to cope with this change in communal meals . participants agreed that the amount of rice provided in institutional food settings like airplanes or hospitals was laughably small . the insufficiency was appreciated as an in - group issue , a cultural thing that westerners were not likely to appreciate . when consumed in limited amounts as prescribed in a diabetic diet , rice was missed not just for its familiarity but as a requirement to health and perhaps survival . if you do n't eat rice can you sustain your daily living ? additionally , participants expressed significant suffering because of restrictions on rice , a symbolically comforting food . a central health metaphor expressed by most participants was the need for balance . many found that disease - related food restrictions disregarded cultural concerns for balancing foods ( e.g. , hot and cold ) understood to have specific medicinal properties according to traditional chinese medicine ( tcm ) . even for those who did not specifically incorporate tcm in their diet management , i feel that just purely eating like that with no oil or salt , you may be lacking certain nutrients . none of the participants received guidance from health care providers on balancing their diabetes food restrictions according to cultural concerns for balance , although many recommended foods raised concerns about creating such imbalances . participants additionally feared that diabetic food restrictions , if strictly followed , might lead to emotional imbalance and depression . if i say every time , this and that you ca n't eat some people would develop negative feelings and say , there 's no meaning to life now. restricting food during illness is counterintuitive for many chinese americans ( 16,17 ) . rather , special foods and disease - specific medicinal foods should appropriately be provided for patients as both a means of supporting health and demonstrating family solicitousness . spouses were challenged by being asked to restrict patients ' diets during illness and would have preferred providing abundant foods to comfort and fortify patients ' health . diabetes diets also complicated shared social experiences of outings , meals , and celebrations with family and friends . patients ' difficulties in following an appropriate diet at chinese restaurants led some to withdraw from socializing over meals . spouse : now when i asked her to go to dim sum , she would say , i am not going . it 's meaningless to go by myself . for many chinese participants , social interaction was an integral part of a meal . attending dim sum or chinese breakfast alone was meaningless because meals were sustaining only if shared . birthdays , weddings , or chinese new year 's banquets , with multiple courses and desserts , were unavoidable , yet socially fraught : gee , i could n't even eat a bowl of sweet dessert soup ! how miserable . the presence of family reminded patients of their responsibility to observe diabetes restrictions , as a duty to family . since they tell you , you do n't dare do it . you are well aware that the bowl of sweet dessert soup may do you a lot of harm . reciprocally , family members felt obliged to care for patients ' disease and yet at the same time to create social ease and pleasure . a final social challenge was standing out socially , requiring special attention , or even stigmatized attention because of diabetes . as in earlier reports ( 16,17 ) , patients varied in their willingness to disclose diabetes to friends , but most were distressed when diabetes was the focus of attention at social gatherings . living with diabetes challenged chinese americans to adapt their family roles to accommodate the disease . beliefs varied about who in the family should be responsible to manage the disease , creating conflicts in negotiating differing role expectations . many spouses believed that the family 's role was to assist and encourage but that ultimately the patient had to be responsible : he has to rely on himself . other spouses believed in and constructed a broader role , which included learning about the disease , assisting with disease management efforts , and offering ongoing advice and encouragement . assistance with diet was most often cited , but help with all aspects of diabetes care were considered appropriate . family role conflicts centered on who should create , observe , and enforce food restrictions ; the degree of understanding each family member should have about the diabetes regimen ; and whose philosophy of treatment should prevail . many spouses suggested that a communal diet should be respectful and accommodating of the patient 's dietary restrictions . however , conflicts arose when the cook was over- or underresponsive to restrictions when cooking . for example , one patient asked her husband to cook more healthfully , but he forgot on a daily basis and continued to cook according to his own tastes . conflicts also arose when a spouse cooked healthfully , but the patient felt unduly restricted : a few participants argued in favor of family restraint because the diabetic diet supported general health and observing restrictions demonstrated camaraderie with the patient . longstanding couple and family dynamics were undoubtedly enacted in food arguments ; diabetes provided a focus for these dynamics to be displayed . one couple , for example , reported a persistent , unresolved argument over the husband 's wish to eat pork buns . he agreed to his wife 's strict restrictions on family dinners but would not relinquish his breakfast pork buns . families were additionally challenged when patients and spouses held differing expectations about what family members should learn about the disease . some patients felt neglected because spouses were not very aware of the risks and demands of the disease . these patients held that spouses had a reciprocal role responsibility to understand their condition and assist with its management . they expected spouses to intuitively understand , to be highly sensitive to , and to anticipate their diabetes management needs . differences were apparent in the degree to which spouses believed in and relied upon western biomedicine and tcm treatments and beliefs about the relative importance of treatment regimens , such as diet and exercise : my wife does n't quite accept the things of modern science she 's been using the method that her mother had taught her before patients role responsibilities encompassed managing diabetes well not only for health benefits but out of respect for their families . reciprocal role responsibilities , or caring for diabetes in reciprocation for the family 's care , reflected an interdependent self - construal and added emotional complexity to patients ' responses to their disease . they may have been angry or upset at the disease requirements , but these responses had to be tempered to be responsive to the family 's care . in addition to reciprocity , an interdependent norm of managing diabetes to protect the family 's well - being was evident . patient : this person by my side ( husband ) has to worry about me and watch my diet . i feel apologetic , so i just eat these things that my husband wants . reciprocating care with filial children , those who perform their responsibility to care for a parent , also arose as a key concern for many patients . patient : my daughters ( in china ) are often concerned about me . dad , you need to treasure your health . do n't eat sweets , do n't eat fat , and do n't eat salty food ( chuckle ) . interviewer : how do you feel when they talk to you like this ? patient : this speaks to the family members ' concern about me . finally , some families believed the patient was weakened or made vulnerable by the disease . some spouses doubted the patient 's capacity to work at vigorous activity because restrictions on food or rice were thought to affect the patient 's energy and stamina . family sex roles shifted , with some wives shouldering greater financial support responsibilities to accommodate the perceived decrease in patient capabilities . foreign - born chinese american patients and their spouses note multiple culturally nuanced dilemmas posed by type 2 diabetes . their narratives suggest that chinese families are integrally involved in interpreting symptoms and constructing disease management responses . prescribed restrictions on symbolically vital foods and disruptions in valued family rituals and practices are sources of suffering and loss . health prescriptions delivered in western biomedical terms directly challenge cultural valuing of balance in emotional , social , and physical realms . interdependencies and reciprocal role responsibilities additionally complicate disease management but differently for patients and spouses . finally , overriding concerns for family well being frequently result in complex and layered decisions about disease management . these data provide insight into the practical disease management of immigrant chinese americans whose perspectives have previously been poorly explored . clinical implications include a need for 1 ) concentrated attention regarding the social and family reverberations of diabetes symptoms , 2 ) consideration given to the central and pivotal role rice likely plays in chinese patients ' diets , 3 ) efforts to reframe diabetes guidelines as a way to increase balance rather than control , 4 ) care that incorporates an awareness that patients ' disease management decisions are tempered by concerns for family well - being , family face , and the reciprocal role responsibilities . teaching foreign - born chinese americans about diabetes requires sensitivity to the social relevance of common symptoms , particularly irritability and emotional lability . open expression of strong emotions , particularly negative emotions , is considered culturally inappropriate ( 14 ) given the potential to disrupt interpersonal harmony . indirect , muted , and contained expressions of emotions are valued yet directly challenged by patients ' perceived lack of control when symptomatic . chinese americans may experience emotional disregulation to be stressful and socially inappropriate and thus should be coached in advance about its possible occurrence . dietary restrictions on carbohydrates can be particularly challenging to a population that has historic , symbolic , and ritualized inclusion of rice in their daily diets . recommended reductions in rice may be highly distressing and may challenge core beliefs about health maintenance . stepwise approximations to an ideal diet may ease the transition and allow patients and families to explore healthy alternatives to highly processed white rice or rice noodles . teaching patients about how rice affects consultation with dietitians who are familiar with chinese food preferences and can offer culturally acceptable food substitutions is warranted . helping chinese families who may eat communally , with dietary guidance , will likely lead to greater observance of recommendations . the central biomedical representation of ideal diabetes management is control or , optimally , intensive control ( 20 ) . prescriptions for a diabetic diet are perceived by many foreign - born chinese to be prescriptions to restrict , control , or limit what they eat . many foreign - born chinese believe that balance and moderation in all things including diet ( hot and cold , yin and yang ) , interpersonal relations , and environmental relations promotes health ( 21 ) . when working with foreign - born chinese americans , greater success may be achieved by adapting the frame for diabetes management from limits or restrictions to balance . actual prescriptions need not change tremendously , but prescribing a diet that balances new with old foods or rice with vegetables and proteins is more culturally suitable . appreciation of the socially embedded nature of health and health practices in immigrant chinese americans is necessary to provide culturally respectful care . patients ' and families ' disease management decisions are seldom made independent of their concerns for family well - being , family face , and the reciprocal responsibilities required by varied family roles . acknowledging the multiple and competing social concerns being managed by patients and families provides an opening for discussion and problem solving with the provider . diet - related distress and family conflict are highly prevalent , and incorporating this social concern into diabetes management planning is warranted . additionally , stress related to social concerns for family well - being and face are frequently factored into patient disease management decisions and should be part of patient - centered care with immigrant chinese americans ( 14,21 ) . study strengths and weaknesses deserve mention . the sample size for an intensive interpretive study is robust , and participants who match the initial sample report in respondent groups that themes derived from the initial sample matched their lived realities . in interpretive work the investigator is the instrument , thus having a bicultural and bilingual team of interpreters strengthens the findings . interviews were conducted only in cantonese ; therefore , cultural variations beyond cantonese - speaking regions of hong kong and china are not represented .

objectivealthough asians demonstrate elevated levels of type 2 diabetes , little attention has been directed to their unique cultural beliefs and practices regarding diabetes . we describe cultural and family challenges to illness management in foreign - born chinese american patients with type 2 diabetes and their spouses.research design and methodsthis was an interpretive comparative interview study with 20 foreign - born chinese american couples ( n = 40 ) living with type 2 diabetes . multiple ( six to seven ) semistructured interviews with each couple in individual , group , and couple settings elicited beliefs about diabetes and narratives of care within the family and community . interpretive narrative and thematic analysis were completed . a separate respondent group of 19 patients and spouses who met the inclusion criteria reviewed and confirmed the themes developed from the initial couples.resultscultural and family challenges to diabetes management within foreign - born chinese american families included how 1 ) diabetes symptoms challenged family harmony , 2 ) dietary prescriptions challenged food beliefs and practices , and 3 ) disease management requirements challenged established family role responsibilities.conclusionsculturally nuanced care with immigrant chinese americans requires attentiveness to the social context of disease management . patients ' and families ' disease management decisions are seldom made independent of their concerns for family well - being , family face , and the reciprocal responsibilities required by varied family roles . framing disease recommendations to include cultural concerns for balance and significant food rituals are warranted .

RESEARCH DESIGN AND METHODS RESULTS Symptoms challenged family harmony Prescribed diet challenged cultural beliefs and practices Challenges to family roles and responsibilities CONCLUSIONS

this was an interpretive comparative interview study with 20 foreign - born chinese american couples ( n = 40 ) , in which one member was diagnosed with type 2 diabetes . inclusion criteria included having a diabetes diagnosis for at least 1 year , being aged 3575 years , having been married for a minimum of 1 year , self - identifying as chinese american or chinese , having immigrated to the u.s . from mainland china or hong kong , and having a spouse who would agree to participate . exclusion criteria for patients included major diabetes complications ( cerebrovascular accident or myocardial infarction within the last 12 months ; proliferative retinopathy ; renal insufficiency ; or amputations ) because our intent was to study patients who were early enough in the disease to benefit from behavioral and family interventions . a convenience sample was recruited from community clinics , community service organizations , and via public notices . six semistructured interviews with couples in individual , couple , and group contexts focused on illness understandings , perceptions of diabetes care , acculturation histories , and concrete positive , negative , and memorable narratives of diabetes care . couples narrated in each other 's presence ( two couple interviews ) and in group interviews with those who shared their experience as a patient ( two group interviews ) or spouse ( two group interviews ) . a subset of participants ( n = 13 ) was also interviewed individually if extra time was needed to complete interview questions or if nondisclosure in shared interview settings suggested that a private interview would yield more complete data . interviews were conducted in cantonese , and audiotaped text was simultaneously translated from cantonese to english and transcribed verbatim by skilled bilingual staff . each audiofile was then reviewed and checked for accuracy by a separate bilingual bicultural staff member who had conducted the interview . narrative and thematic analyses were conducted by a multicultural and multidisciplinary team of chinese american and caucasian nurses and psychologists ( 18,19 ) . after all text was coded for thematic codes in atlas - ti , codes were selected for review that identified challenging situations in diabetes management by patients and spouses . complete text from three codes was examined : couple dynamics , conflict , and diabetes management . both reflective discussions and narratives of positive , difficult , and meaningful aspects of care were analyzed . the analyzed text comprised > 450 pages of extracted text and represented a broad inclusive portion of narratives that were drawn from interviews conducted in all three contexts ( couple , group , and individual interviews ) . thus , text for this manuscript was analyzed in the context of the holistic analysis of each couple 's dynamics . to address generalizability , findings were presented to separate respondent groups of patients and spouses who met the same inclusion criteria as the original sample . respondents ( n = 19 ) met in groups of patients or spouses for two separate 2-h interviews . they were asked to review themes presented in this manuscript for adequacy and to add personal variations to the presented themes . the sample of informants was 20 foreign - born chinese american couples ( n = 40 ) , with one member with diabetes . on average , informants were ( means sd ) age 62 9.2 years , married for 34 13.4 years , and had immigrated to the u.s . patients were 40% male , had been diagnosed 8.4 5.9 years , were treated primarily with oral medications ( 85% ) , and had an average a1c of 6.93 0.96% . members of the respondent group ( n = 19 ; 13 patients , 6 spouses ) representing 16 separate families were similar to the informant group on all measures . they were on average aged 60 9.4 years , married for 32 12.1 years , and had immigrated to the u.s . 11.8 12 years ago from mainland china ( 46% ) and hong kong ( 54% ) . patients in the respondent group were 54% male , had been diagnosed 6.2 4.2 years , and all were treated with oral medications ; a1c was not collected with respondent group participants . cultural and family challenges to diabetes management within foreign - born chinese american families included how 1 ) diabetes symptoms challenged family harmony , 2 ) dietary prescriptions challenged food beliefs and practices , and 3 ) disease management requirements challenged established family role responsibilities . each theme is examined in depth with supportive text . in reporting findings , statements made by patients or spouses are identified as such , and statements endorsed by both groups are identified as participant statements . increased irritability as a symptom of diabetes was frequently described as a challenge to family harmony . participants noted that patients get angry easily and easily throw a temper tantrum emotional variability held particular resonance for chinese immigrants because social ease , avoidance of overt expression of strong negative emotions , and accommodation of family members ' expressed and unexpressed needs were culturally valued . in a patient group interview , she attributed her outburst to diabetes ( diabetes destabilizes a person ) but additionally expressed remorse at a comment that might hurt him . this patient , like many in the study , believed immoderate anger posed harm to herself , by raising her blood glucose , and harm to her husband because he had to tolerate a strongly expressed negative emotion . in this ethnic group , the social rather than physiological aspects of glucose regulation were highlighted . rather , their narratives suggested attunement to social and behavioral symptoms of glucose fluctuation and the social dilemmas these symptoms introduced . patients felt called upon to contain their irritability to promote family harmony , while spouses reciprocally felt responsible to forgive patients ' outbursts as a way of caring for patients . observing a biomedically prescribed diabetes regimen required chinese american patients to distance themselves from familiar and shared cultural food habits and practices within the family and community . participants found that culturally meaningful , familiar , and comforting foods had to be foregone or drastically reduced , new foods had to be accommodated , and food quantity became a source of concern . additionally , social habits , such as eating out , sharing dim sum with family and friends , and easily participating in cultural celebrations and banquets were complicated by perceived disease restrictions . disruptions in meaningful cultural food practices mentioned frequently included rice , restricting amounts of food , and changing the balance of various foods that were thought to be beneficial to general health . the meaning of rice in the chinese family diet was a culturally multifaceted and historically nuanced story about sustaining holistic health and well - being and partaking of a symbolically vital food . patients and families were challenged by being asked to restrict rice and change from familiar white fragrant rice to foreign chewy and tasteless brown , red , or black rice . a central health metaphor expressed by most participants was the need for balance . many found that disease - related food restrictions disregarded cultural concerns for balancing foods ( e.g. none of the participants received guidance from health care providers on balancing their diabetes food restrictions according to cultural concerns for balance , although many recommended foods raised concerns about creating such imbalances . restricting food during illness is counterintuitive for many chinese americans ( 16,17 ) . spouses were challenged by being asked to restrict patients ' diets during illness and would have preferred providing abundant foods to comfort and fortify patients ' health . diabetes diets also complicated shared social experiences of outings , meals , and celebrations with family and friends . patients ' difficulties in following an appropriate diet at chinese restaurants led some to withdraw from socializing over meals . for many chinese participants , social interaction was an integral part of a meal . the presence of family reminded patients of their responsibility to observe diabetes restrictions , as a duty to family . reciprocally , family members felt obliged to care for patients ' disease and yet at the same time to create social ease and pleasure . living with diabetes challenged chinese americans to adapt their family roles to accommodate the disease . beliefs varied about who in the family should be responsible to manage the disease , creating conflicts in negotiating differing role expectations . many spouses believed that the family 's role was to assist and encourage but that ultimately the patient had to be responsible : he has to rely on himself . other spouses believed in and constructed a broader role , which included learning about the disease , assisting with disease management efforts , and offering ongoing advice and encouragement . family role conflicts centered on who should create , observe , and enforce food restrictions ; the degree of understanding each family member should have about the diabetes regimen ; and whose philosophy of treatment should prevail . longstanding couple and family dynamics were undoubtedly enacted in food arguments ; diabetes provided a focus for these dynamics to be displayed . families were additionally challenged when patients and spouses held differing expectations about what family members should learn about the disease . they expected spouses to intuitively understand , to be highly sensitive to , and to anticipate their diabetes management needs . differences were apparent in the degree to which spouses believed in and relied upon western biomedicine and tcm treatments and beliefs about the relative importance of treatment regimens , such as diet and exercise : my wife does n't quite accept the things of modern science she 's been using the method that her mother had taught her before patients role responsibilities encompassed managing diabetes well not only for health benefits but out of respect for their families . reciprocal role responsibilities , or caring for diabetes in reciprocation for the family 's care , reflected an interdependent self - construal and added emotional complexity to patients ' responses to their disease . they may have been angry or upset at the disease requirements , but these responses had to be tempered to be responsive to the family 's care . in addition to reciprocity , an interdependent norm of managing diabetes to protect the family 's well - being was evident . reciprocating care with filial children , those who perform their responsibility to care for a parent , also arose as a key concern for many patients . do n't eat sweets , do n't eat fat , and do n't eat salty food ( chuckle ) . patient : this speaks to the family members ' concern about me . increased irritability as a symptom of diabetes was frequently described as a challenge to family harmony . participants noted that patients get angry easily and easily throw a temper tantrum emotional variability held particular resonance for chinese immigrants because social ease , avoidance of overt expression of strong negative emotions , and accommodation of family members ' expressed and unexpressed needs were culturally valued . in a patient group interview , she attributed her outburst to diabetes ( diabetes destabilizes a person ) but additionally expressed remorse at a comment that might hurt him . this patient , like many in the study , believed immoderate anger posed harm to herself , by raising her blood glucose , and harm to her husband because he had to tolerate a strongly expressed negative emotion . in this ethnic group , the social rather than physiological aspects of glucose regulation were highlighted . rather , their narratives suggested attunement to social and behavioral symptoms of glucose fluctuation and the social dilemmas these symptoms introduced . patients felt called upon to contain their irritability to promote family harmony , while spouses reciprocally felt responsible to forgive patients ' outbursts as a way of caring for patients . observing a biomedically prescribed diabetes regimen required chinese american patients to distance themselves from familiar and shared cultural food habits and practices within the family and community . participants found that culturally meaningful , familiar , and comforting foods had to be foregone or drastically reduced , new foods had to be accommodated , and food quantity became a source of concern . additionally , social habits , such as eating out , sharing dim sum with family and friends , and easily participating in cultural celebrations and banquets were complicated by perceived disease restrictions . disruptions in meaningful cultural food practices mentioned frequently included rice , restricting amounts of food , and changing the balance of various foods that were thought to be beneficial to general health . the meaning of rice in the chinese family diet was a culturally multifaceted and historically nuanced story about sustaining holistic health and well - being and partaking of a symbolically vital food . patients and families were challenged by being asked to restrict rice and change from familiar white fragrant rice to foreign these challenges were persistently noted by participants who felt called upon to cope with this change in communal meals . when consumed in limited amounts as prescribed in a diabetic diet , rice was missed not just for its familiarity but as a requirement to health and perhaps survival . a central health metaphor expressed by most participants was the need for balance . many found that disease - related food restrictions disregarded cultural concerns for balancing foods ( e.g. none of the participants received guidance from health care providers on balancing their diabetes food restrictions according to cultural concerns for balance , although many recommended foods raised concerns about creating such imbalances . restricting food during illness is counterintuitive for many chinese americans ( 16,17 ) . spouses were challenged by being asked to restrict patients ' diets during illness and would have preferred providing abundant foods to comfort and fortify patients ' health . diabetes diets also complicated shared social experiences of outings , meals , and celebrations with family and friends . patients ' difficulties in following an appropriate diet at chinese restaurants led some to withdraw from socializing over meals . spouse : now when i asked her to go to dim sum , she would say , i am not going . for many chinese participants , social interaction was an integral part of a meal . the presence of family reminded patients of their responsibility to observe diabetes restrictions , as a duty to family . you are well aware that the bowl of sweet dessert soup may do you a lot of harm . reciprocally , family members felt obliged to care for patients ' disease and yet at the same time to create social ease and pleasure . a final social challenge was standing out socially , requiring special attention , or even stigmatized attention because of diabetes . living with diabetes challenged chinese americans to adapt their family roles to accommodate the disease . beliefs varied about who in the family should be responsible to manage the disease , creating conflicts in negotiating differing role expectations . many spouses believed that the family 's role was to assist and encourage but that ultimately the patient had to be responsible : he has to rely on himself . other spouses believed in and constructed a broader role , which included learning about the disease , assisting with disease management efforts , and offering ongoing advice and encouragement . family role conflicts centered on who should create , observe , and enforce food restrictions ; the degree of understanding each family member should have about the diabetes regimen ; and whose philosophy of treatment should prevail . longstanding couple and family dynamics were undoubtedly enacted in food arguments ; diabetes provided a focus for these dynamics to be displayed . families were additionally challenged when patients and spouses held differing expectations about what family members should learn about the disease . they expected spouses to intuitively understand , to be highly sensitive to , and to anticipate their diabetes management needs . differences were apparent in the degree to which spouses believed in and relied upon western biomedicine and tcm treatments and beliefs about the relative importance of treatment regimens , such as diet and exercise : my wife does n't quite accept the things of modern science she 's been using the method that her mother had taught her before patients role responsibilities encompassed managing diabetes well not only for health benefits but out of respect for their families . reciprocal role responsibilities , or caring for diabetes in reciprocation for the family 's care , reflected an interdependent self - construal and added emotional complexity to patients ' responses to their disease . they may have been angry or upset at the disease requirements , but these responses had to be tempered to be responsive to the family 's care . in addition to reciprocity , an interdependent norm of managing diabetes to protect the family 's well - being was evident . reciprocating care with filial children , those who perform their responsibility to care for a parent , also arose as a key concern for many patients . do n't eat sweets , do n't eat fat , and do n't eat salty food ( chuckle ) . patient : this speaks to the family members ' concern about me . foreign - born chinese american patients and their spouses note multiple culturally nuanced dilemmas posed by type 2 diabetes . their narratives suggest that chinese families are integrally involved in interpreting symptoms and constructing disease management responses . prescribed restrictions on symbolically vital foods and disruptions in valued family rituals and practices are sources of suffering and loss . health prescriptions delivered in western biomedical terms directly challenge cultural valuing of balance in emotional , social , and physical realms . interdependencies and reciprocal role responsibilities additionally complicate disease management but differently for patients and spouses . finally , overriding concerns for family well being frequently result in complex and layered decisions about disease management . these data provide insight into the practical disease management of immigrant chinese americans whose perspectives have previously been poorly explored . clinical implications include a need for 1 ) concentrated attention regarding the social and family reverberations of diabetes symptoms , 2 ) consideration given to the central and pivotal role rice likely plays in chinese patients ' diets , 3 ) efforts to reframe diabetes guidelines as a way to increase balance rather than control , 4 ) care that incorporates an awareness that patients ' disease management decisions are tempered by concerns for family well - being , family face , and the reciprocal role responsibilities . teaching foreign - born chinese americans about diabetes requires sensitivity to the social relevance of common symptoms , particularly irritability and emotional lability . open expression of strong emotions , particularly negative emotions , is considered culturally inappropriate ( 14 ) given the potential to disrupt interpersonal harmony . indirect , muted , and contained expressions of emotions are valued yet directly challenged by patients ' perceived lack of control when symptomatic . chinese americans may experience emotional disregulation to be stressful and socially inappropriate and thus should be coached in advance about its possible occurrence . dietary restrictions on carbohydrates can be particularly challenging to a population that has historic , symbolic , and ritualized inclusion of rice in their daily diets . recommended reductions in rice may be highly distressing and may challenge core beliefs about health maintenance . stepwise approximations to an ideal diet may ease the transition and allow patients and families to explore healthy alternatives to highly processed white rice or rice noodles . helping chinese families who may eat communally , with dietary guidance , will likely lead to greater observance of recommendations . the central biomedical representation of ideal diabetes management is control or , optimally , intensive control ( 20 ) . prescriptions for a diabetic diet are perceived by many foreign - born chinese to be prescriptions to restrict , control , or limit what they eat . many foreign - born chinese believe that balance and moderation in all things including diet ( hot and cold , yin and yang ) , interpersonal relations , and environmental relations promotes health ( 21 ) . when working with foreign - born chinese americans , greater success may be achieved by adapting the frame for diabetes management from limits or restrictions to balance . appreciation of the socially embedded nature of health and health practices in immigrant chinese americans is necessary to provide culturally respectful care . patients ' and families ' disease management decisions are seldom made independent of their concerns for family well - being , family face , and the reciprocal responsibilities required by varied family roles . acknowledging the multiple and competing social concerns being managed by patients and families provides an opening for discussion and problem solving with the provider . diet - related distress and family conflict are highly prevalent , and incorporating this social concern into diabetes management planning is warranted . additionally , stress related to social concerns for family well - being and face are frequently factored into patient disease management decisions and should be part of patient - centered care with immigrant chinese americans ( 14,21 ) . the sample size for an intensive interpretive study is robust , and participants who match the initial sample report in respondent groups that themes derived from the initial sample matched their lived realities .

a first step in the prevention of stress - induced diseases could be to quantify anxieties that individuals have , although quantification per se may not prevent the diseases . while a variety of written self - report anxiety measures provide valid and reliable measures of anxiety , these indices tend to be time consuming to acquire . for medical patients , completing a written report can be burdensome if they are weak , in pain , or in acute anxiety states . thus , simple and fast non - invasive methods for assessing stress response from neurophysiological data are essential . in , we used a near - infrared spectroscopy ( nirs ) device to investigate neurophysiological mechanisms of stress responses . one of our findings was that the prefrontal cortex ( pfc ) plays an important role in stress response as the asymmetry of pfc activity measured by nirs correlated with behavioral and somatic responses to mental stress . it has been observed that there is left / right asymmetry of oxy hemoglobin concentrations in pfc activities during tasks involving mental stress . we measured these activity patterns using a two - channel nirs device instead of fmri or eeg and found that they correlated with the systemic stress responses of the autonomic nervous system and the hypothalamic - pituitary - adrenal ( hpa ) axis system . our present study attempted to go beyond the study of such asymmetries of pfc activities in two directions : a)the first direction was to consider the resting state of a subject instead of the subject being under a stress task . this makes task design easier because it is nontrivial and often difficult to design a stress task that is effective for every subject . it should be noted that research on the brain under resting conditions is an active study area , and it has been reported that the brain at rest consumes a significant amount of energy and much activities with distinctive patterns are reported.b)the other direction was to make predictions about anxiety index from oxy and deoxy hemoglobin concentration changes of pfc using a two - channel portable nirs device . we achieved this by constructing a machine learning algorithm within a bayesian framework and implemented it with markov chain monte carlo ( mcmc ) . a brief overview of mcmc is given in appendix b. the first direction was to consider the resting state of a subject instead of the subject being under a stress task . this makes task design easier because it is nontrivial and often difficult to design a stress task that is effective for every subject . it should be noted that research on the brain under resting conditions is an active study area , and it has been reported that the brain at rest consumes a significant amount of energy and much activities with distinctive patterns are reported . the other direction was to make predictions about anxiety index from oxy and deoxy hemoglobin concentration changes of pfc using a two - channel portable nirs device . we achieved this by constructing a machine learning algorithm within a bayesian framework and implemented it with markov chain monte carlo ( mcmc ) . a brief overview of mcmc is given in appendix b. specifically the study proceeded in the following steps : i)acquire the anxiety index via the state - trait anxiety inventory ( stai ) test.ii)use a two - channel portable nirs device to measure oxy and deoxy hemoglobin concentration changes of pfc in four different groups , where two of them comprise young subjects , whereas the other two comprise elderly subjects.iii)place each subject in a resting state in which no task is performed.iv)build a hierarchical bayesian machine learning algorithm for anxiety index predictions by taking into account both oxy and deoxy hemoglobin concentration changes . because the amount of data was limited , the predictive capability of the algorithm was evaluated by excluding one set of data and using the remaining set for learning . following the learning phase , the set of data previously withheld was used to examine the predictive capability of the algorithm . the hierarchical bayesian algorithm does not need a validation set since it does not utilize leave - one - out cross validation.v)evaluate the prediction capabilities of the algorithm against four different datasets from each of which one set of data was withheld for testing . acquire the anxiety index via the state - trait anxiety inventory ( stai ) test . use a two - channel portable nirs device to measure oxy and deoxy hemoglobin concentration changes of pfc in four different groups , where two of them comprise young subjects , whereas the other two comprise elderly subjects . place each subject in a resting state in which no task is performed . build a hierarchical bayesian machine learning algorithm for anxiety index predictions by taking into account both oxy and deoxy hemoglobin concentration changes . because the amount of data was limited , the predictive capability of the algorithm was evaluated by excluding one set of data and using the remaining set for learning . following the learning phase , the set of data previously withheld was used to examine the predictive capability of the algorithm . the hierarchical bayesian algorithm does not need a validation set since it does not utilize leave - one - out cross validation . evaluate the prediction capabilities of the algorithm against four different datasets from each of which one set of data was withheld for testing . the remainder of this paper is organized as follows : section ii reviews work related to our study . section iii describes the materials and methods used as well as the experimental settings , subjects , machine learning algorithm , and feature extraction method . nirs is a relatively well established method of measuring oxy hemoglobin ( oxy - hb ) and deoxy hemoglobin ( deoxy - hb ) concentrations in cerebral vessels . changes in oxy - hb are correlated with changes in regional cerebral blood flow ( rcbf ) , whereas changes in total hemoglobin ( sum of oxy - hb and deoxy - hb ; t - hb ) reflect regional cerebral blood volume ( rcbv ) changes . the results of a number of studies indicate that correlations exist between the electrical neural activity and changes in the blood oxygenation levels measured by nirs . simultaneous recordings of nirs and visual evoked potential signals in humans during visual stimulation reveal a linear correlation between hemodynamic changes and evoked potential amplitude . further , simultaneous measurements of nirs and eeg at rest indicate that an increase in oxy - hb is associated with an increase in neuronal activity and vice versa , . a verbal evaluation method attempted in proved faster than the written method and demonstrated a high correlation with stai . in , we conclude this section by stating that this paper is a significantly improved and expanded version of in that four datasets from different age groups are analyzed here , whereas only one target dataset was used in . we acquired four datasets from different locations and on different occasions in 2010 and 2011 . i)dataset 1population : 19 subjects(six women ; 13 men ) , age range : 1926 years.ii)dataset 2population : 19 subjects(13 women ; six men ) , age range : 2024 years.iii)dataset 3population : 17 subjects(10 women ; seven men ) , age range : 6179 years.iv)dataset 4population : 20 subjects(16 women ; four men ) , age range : 6079 years . population : 19 subjects ( six women ; 13 men ) , age range : 1926 years . population : 19 subjects ( 13 women ; six men ) , age range : 2024 years . population : 17 subjects ( 10 women ; seven men ) , age range : 6179 years . population : 20 subjects ( 16 women ; four men ) , age range : 6079 years . they all gave written informed consent on forms approved by the ethical committees of the nihon university school of medicine and waseda university . in our experiments , each subject was seated in a comfortable chair in a dimly lit room ( fig . 1(a ) and we measured the oxy- and deoxy - hb concentration changes using a portable two - channel nirs system ( pnirs-10 , hamamatsu photonics k.k . the nirs probes were set symmetrically on the forehead of each subject ; the positioning is similar to the midpoint between the electrode positions fp1/f3 ( left ) and fp2/f4 ( right ) of the international 10 - 20 system , as demonstrated in fig . 1(c ) . figure 1.(a ) experimental setup ; ( b ) two - channel pocket nirs ; ( c ) international 1020 system red circles indicate the measurement areas . ( a ) experimental setup ; ( b ) two - channel pocket nirs ; ( c ) international 1020 system red circles indicate the measurement areas . the sensor part ( with weight approximately 100 g , resulting in only a very small burden on the subject ) communicated with a pc via bluetooth ( class 2 ) . in general , the machine learning paradigm comprises the following three ingredients : i)preprocessingii)feature extractioniii)learning / prediction algorithm . the nature of nirs data is not well understood ; consequently , the procedure that needs to be followed in each step is nontrivial . this paper reports on our attempts to carry out ( ii ) and ( iii ) , with ( i ) , which is no less important , being left for future work . although the results are not included in this paper , we attempted to devise third and fifth order butterworth bandpass filters ( passband 0.011.0 hz ) and lof outlier detection / deletion on the nirs raw data for ( i ) . we will report on the effects of preprocessing in a future paper ; in this paper , our focus is on ( ii ) and ( iii ) . using all the available features is information rich ; however , less relevant features often degrade predictive performance . this is due to the fact that as the number of features increases , the number of unknown parameters also increases , such that poorly learned parameters degrade prediction capabilities . note that each channel of the target device acquires the concentration changes of oxy- and deoxy - hb so that the acquired data is a four - dimensional vector . figure 3.typical nirs data consisting of four quantities : right / left \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } \(\bigtriangleup \ ) \end{document } oxy and right / left \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } \(\bigtriangleup \ ) \end{document } deoxy . note that the measured quantities from the device used in this study are the concentration changes \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } \(\bigtriangleup \ ) \end{document } oxy and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } \(\bigtriangleup \ ) \end{document } deoxy , instead of absolute quantities . typical nirs data consisting of four quantities : right / left \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } \(\bigtriangleup \ ) \end{document } oxy and right / left \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } \(\bigtriangleup \ ) \end{document } deoxy . note that the measured quantities from the device used in this study are the concentration changes \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } \(\bigtriangleup \ ) \end{document } oxy and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } \(\bigtriangleup \ ) \end{document } deoxy , instead of absolute quantities . in extracting appropriate features , we first noted that the measured quantities from the device used in the study were the concentration changes \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } \(\bigtriangleup \ ) \end{document } oxy and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } \(\bigtriangleup \ ) \end{document } deoxy , instead of absolute quantities , ( see the appendix also ) . therefore , the first order statistics , the mean values , should be avoided for prediction purposes . in the study , we used the following three pearson correlation coefficients that were reported as effective in : i)\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } \(\bigtriangleup \ ) \end{document } oxy ( left)/\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } \(\bigtriangleup \ ) \end{document } deoxy ( left)ii)\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } \(\bigtriangleup \ ) \end{document } oxy ( left)/ \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } \(\bigtriangleup \ ) \end{document } deoxy ( right)iii)\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } \(\bigtriangleup \ ) \end{document } deoxy ( left)/\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } \(\bigtriangleup \ ) \end{document } deoxy ( right)across the four datasets . \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } \(\bigtriangleup \ ) \end{document } oxy ( left)/\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } \(\bigtriangleup \ ) \end{document } deoxy ( left ) \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } \(\bigtriangleup \ ) \end{document } oxy ( left)/ \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } \(\bigtriangleup \ ) \end{document } deoxy ( right ) \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } \(\bigtriangleup \ ) \end{document } deoxy ( left)/\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } \(\bigtriangleup \ ) \end{document } deoxy ( right ) the three quantities are illustrated in fig . 4 in a schematic manner , with an arrow indicating pearson correlation coefficient between the two associated quantities . note that pearson correlation coefficients are normalized in the range [ 1 , + 1 ] . all the arguments in the ensuing section are based on these three second - order features . since the acquired nirs data was four - dimensional , there are six pearson correlation coefficients so that there were \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } \(\sum \nolimits _ { n=1}^{6 } { { } _ { 6}c_{n } } = 63\ ) \end{document } possibly combinations . by exhaustive search let \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } \(x^{(i)}:=(x_{1}^{(i)},\ldots , x_{k}^{(i)})\in r^{k},\kern 1pt \;\kern 1pt i\,=\,1,\ldots , n\ ) \end{document } be the nirs feature vector of the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } \(i\ ) \end{document}-th individual . for our selected features , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } \(k = 3\ ) \end{document } , as described in fig . 4 . let \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } \(y^{(i)},\kern 1pt \;\kern 1pt i\,=\,1,\ldots , n \ ) \end{document } be the stai state index obtained from the questionnaire , where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } \(n\ ) \end{document } is the number of subjects in an experiment . of the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } \(n\ ) \end{document } available pieces of data , we reserved one \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } \((x_{reserved } , \;y_{reserved } ) \ ) \end{document } and attempted to fit the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } \(n-1\ ) \end{document } remaining pieces of data with the following machine learning model:\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } \begin{align } p(y^{(i)}\vert x^{(i)};\omega , \beta ) = & \sqrt { \frac { \beta } { 2\pi } } \exp ( -\frac { \beta } { 2}(y^{(i)}-f(x^{(i)};\omega ) ) ^{2 } ) \notag \\ i=&1,\ldots , n-1 \end{align } \end{document } where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } \(f\ ) \end{document } is a basis function for data fitting , and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } \(\omega \ ) \end{document } denotes the associated unknown parameter to be learned . \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } \(\beta \ ) \end{document } is another parameter to be learned that is often called the hyperparameter , and corresponds to the uncertainty level associated with the acquired stai value . in order to capture the potential nonlinear relationship between the nirs data and the stai , we considered the following nonlinear basis function:\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } \begin{align } f(x^{(i)};\omega ) : = \sum \limits _ { h=1}^{h } { \big ( \omega _ { ( k+1)h } \sigma \big ( \sum \limits _ { k=1}^{k } { \omega _ { kh } x_{k}^{(i ) } + \omega _ { 0h } } \big ) \big ) } + \omega _ { 0(k+1)}\hspace { -1pc}\notag \\\text{}\end{align } \end{document } where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } \(\sigma \ ) \end{document } is a sigmoidal function . this basis function is nonlinear with respect not only to \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } \(x\ ) \end{document } but also \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } \(\omega \ ) \end{document } , which makes the learning algorithm nontrivial . it is known that this basis function ( perceptron ) can approximate any nonlinear function with arbitrary precision . as in , the unknown parameter vector \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } \(\omega \ ) \end{document } is decomposed as \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } \(\omega = \left \ { { \{\omega _ { k } \}_{k=1}^{k } , \;\omega _ { ( k+1 ) , } \;\omega _ { 0 , } \;\omega _ { 0(k+1 ) } } \right \ } \ ) \end{document } with \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } \(\omega _ { k } : = ( \omega _ { k1 } , \ldots , \omega _ { kh } ) \in r^{h}\ ) \end{document } , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } \(\vphantom { \sum ^{r^{r^{r}}}}\omega _ { ( k+1 ) } : = ( \omega _ { ( k+1)1 } , \ldots , \omega _ { ( k+1)h } ) \in r^{h}\ ) \end{document } , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } \(\omega _ { 0 } : = ( \omega _ { 01 } , \ldots , \omega _ { 0h } ) \in r^{h}\ ) \end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } \(\omega _ { 0(k+1 ) } \in r\ ) \end{document}. this decomposition amounts to the fact that each \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } \(\omega _ { k } \ ) \end{document } is the first - layer weights , associated with each feature \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } \(x^{(i)}_{k},~k\,=\,1,\ldots , k\ ) \end{document}. \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } \(\omega _ { ( k+1 ) } \ ) the remaining two parameters \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } \(\ { { \omega _ { 0 , } \;\omega _ { 0(k+1 ) } } \}\ ) \end{document } were the bias parameters . the number of hidden units \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } \(h\ ) \end{document } can be estimated within the present hierarchical bayesian framework . this endeavor , however , requires a more complicated algorithm and much more time to execute . from our own experiences , as well as those reported by other researchers , we used eight as the choice for \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } \(h\ ) \end{document}. our study formulated the prediction problem within a bayesian framework , where a prior distribution was assumed about the unknown parameters and that information incorporated intothe data fitting model given by ( 1 ) . the prior distribution for \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } \(\omega _ { k } \ ) \end{document } was assumed to be specified by \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } \begin{equation * } p({\omega } _ { k}|{\alpha } _ { k})=n\left ( 0,\left ( \frac { 1}{{\alpha } _ { k}}\right ) i\right ) \end{equation*}\end{document } which is the gaussian distribution with a zero mean vector and covariance matrix ( \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } \(1/{\alpha } _ { k})i;{\alpha } _ { k}\ ) \end{document } is the hyperparameter , associated with first - layer weights \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } \({\omega } _ { k}\ ) \end{document } , and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } \(i\ ) \end{document } denotes the identity matrix , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } \(k=1,\ldots , k\ ) \end{document}. this prior distribution often prevents overfitting . \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } \({\alpha } _ { ( k+1)}\ ) \end{document } is associated with second - layer weights \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } \({\omega } _ { ( k+1)}\ ) \end{document}. there were two other hyperparameters \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } \(\ { { \alpha _ { 0 } , \alpha _ { 0(k+1 ) } } \}\ ) \end{document } , which were associated with the bias parameters \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } \(\ { { \omega _ { 0 } , \omega _ { 0(k+1 ) } } \}\ ) \end{document}. the prior distributions for \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } \(\beta \ ) \end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } \(\alpha \ ) \end{document } were assumed to follow the gamma distribution . let \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } \begin{align } \alpha : = & \{\{\alpha _ { k } \}_{k=1}^{k } , \alpha _ { ( k-1 ) } , \alpha _ { 0 } , \alpha _ { 0(k-1 ) } \},\,x:=\{x^{(i)}\}_{i=1}^{n-1 } , \notag \\ y:=&\{y^{(i)}\}_{i=1}^{n-1}. \end{align } \end{document } the rationale behind the gamma distribution for prior is explained in appendix c. assuming the data from each individual to be independent , the bayes formula gives the posterior distribution:\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } \begin{align } & \hspace { -1.9pc}p(\omega , \alpha , \beta |x , y)\notag \\ \!\!=\!\!&\frac { \prod \limits _ { i=1}^{n-1}p(y^{(i)}|x^{(i)};\omega , \beta ) p(\omega |\alpha , \beta ) p(\alpha , \beta ) } { \int \!\!\!\int \!\!\!\int \prod \limits _ { i=1}^{n-1}p(y^{(i)}|x^{(i)};\omega , \beta ) p(\omega |\alpha , \beta ) p(\alpha , \beta ) d\omega d\alpha d\beta } \end{align } \end{document } prior distribution for hyperparameter \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } \(\alpha \ ) \end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } \(\beta \ ) \end{document } are not explicitly written in ( 5 ) , for the purpose of clarity . given the reserved nirs data \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } \(x_{reserved } \ ) \end{document } , the prediction of the stai index associated with the reserved data \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } \(y_{reserved } \ ) \end{document } was performed by computing the predictive distribution:\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } \begin{align } & \hspace { -1.3pc } p(y_{reserved } \vert x_{reserved } , x , y ) \notag \\ = & \int \!\!\!\int \!\!\!\int { p(y_{reserved } \vert x_{reserved } , \omega , \beta ) p(\omega , \alpha , \beta \vert x , y)d\omega d\alpha d\beta . } \notag \\\text{}\end{align } \end{document } as described in subsection iiid , one piece of data \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } \((x_{reserved } , \;y_{reserved } ) \ ) \end{document } out of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } \(n\ ) \end{document } was reserved , whereas the remaining \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } \(n-1\ ) \end{document } pieces of data were used for learning the unknown parameters . after the parameters were learned , the reserved data were input to the algorithm for stai prediction . because one stai value was also reserved , we were able to compute the difference between the predicted and the reserved stai values , which can signify prediction error . since hyperparameters ( \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } \(\alpha \ ) \end{document } , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } \(\beta ) \ ) \end{document } can also be learned via ( 5 ) , the method is called the hierarchical bayesian algorithm . we performed four prediction experiments , one experiment for each of the four datasets . within each dataset , one set of data was reserved for testing while the rest were used for learning . the reserved data were not used in the learning process . it should be noted that in the stai prediction of each subject , the algorithm had never seen the data from the particular subject to be tested . it should also be noted that this is not leave - one - out cross validation . the hierarchical bayesian method used in this study only the state anxiety index was used in the present study , even though the trait anxiety index was available . we will attempt to make predictions of the trait anxiety index in our future project . depicts scatter plots of the actual stai values and the predicted values with the nonlinear model . figs . it should be noted that the figures are not the results of linear regression between the quantities of the horizontal and the vertical axes ( see subsection iiid ) . the blue lines signify the rmse of 10 , which is an empirical value proposed by one of the coauthors of this paper who is a clinician in neurosurgery in the sense that an rmse of 10 for stai could be tolerable in a clinical practice where the stai score is between 20 and 80 . we note that only a limited amount of data was available for stai values near 20 and 80 . as a result , the algorithm had very little chance of seeing very low / high stai scores and it was difficult to make good predictions ; however , the results appear to be reasonable , including the low / high stai values . figure 6.scatter plots of actual stai and predicted stai values with the proposed algorithm based on the nonlinear model : ( a ) dataset 1 ; ( b ) dataset 2 ; ( c ) dataset 3 ; ( d ) dataset 4 . scatter plots of actual stai and predicted stai values with the proposed algorithm based on the nonlinear model : ( a ) dataset 1 ; ( b ) dataset 2 ; ( c ) dataset 3 ; ( d ) dataset 4 . table 1 summarizes the results with multiple evaluation indices : rmse , pearson correlation coefficients , and the p - values of two tailed t - tests between the actual stai values and the predicted stai values . note that datasets 1 and 2 were acquired from young subjects ( 1926 years and 2024 years , respectively ) whereas datasets 3 and 4 were obtained from elderly subjects ( 6179 years and 6079 years , respectively ) . the effect of aging did not appear to be critical within this prediction framework . performance indices are rmse , pearson correlation , and the associated p - value of two tailed t - test between the actual stai and the predicted values . performance indices are rmse , pearson correlation , and the associated p - value of two tailed t - test between the actual stai and the predicted values . it can be seen that the linear predictions were much less accurate than those with the proposed nonlinear predictions . table 2.summary of the proportions of rmse for differing upper bounds with the proposed algorithm based on the nonlinear model . summary of the proportions of rmse for differing upper bounds with the proposed algorithm based on the nonlinear model . to assess the intra - subject variation of the proposed algorithm , we used the following multiple nirs measurement data . following acquisition of dataset 1 , we asked the 19 subjects to repeat three more trials subsequent on the trial described in iiia the bayesian learning was conducted using the ( 18 out of 19 ) first set of trial data , which was in fact the same as those used in iiia and tested against the remaining data . using the parameters learned from the first set of trial data , tests were performed against the second , third , and fourth set of trial data . learning was performed with the data from the first set of trial data for 18 subjects other than subject 1 , whereas prediction capability was tested against the withheld data for subject 1 . with the same learned data , tests were performed on the second , third , and fourth sets of data from subject 1 . in 10 of the 19 subjects , the prediction error of the fourth trial was greater than that of the first trial . learning was performed with the data from the first set of trial data for 18 subjects other than subject 1 , whereas prediction capability was tested against the withheld data for subject 1 . with the same learned data , tests were performed on the second , third , and fourth sets of data from subject 1 . in 10 of the 19 subjects , the prediction error of the fourth trial was greater than that of the first trial . the prediction error for the fourth trial was greater than that for the first trial in 10 subjects out of 19 . however , in nine subjects , the prediction error for the fourth trial was less than that for the first trial . 8 shows the results for subject 10 . figure 8.intra-subject variability of the prediction capability of subject 10 over four trials . learning and prediction are similar to those in fig . the prediction error for the fourth trial was less than that for the first trial . the prediction error for the fourth trial was less than that for the first trial . the overall average intra - subject prediction error was 7.638 , which is greater than those listed in table 1 . the brain at rest has been an active research area and numerous activities with distinctive patterns have been reported . our study considered the nirs data in the resting condition . the method used in the study did not need task design , which made experimentation easier . with nirs measurements for stress task , a subject s nirs data started changing when the subject was told that the task would begin in 30 s , even before the actual stress task began . in the resting condition assumed in our study , that effect was less than in the stress task ; however , we could not exclude the potential effect of the information on the subject when the subject was informed of the beginning of the measurement 30 s before the measurements began . the present study went beyond investigating the left / right asymmetry of oxy - hb concentrations in the pfc activity during tasks involving mental stress such that the stai index predictions were performed based on the four - dimensional nirs values instead of the mere oxy - hb concentration changes . the three - dimensional features were extracted from the oxy- and deoxy - hb concentrations changes acquired from a two - channel nirs device placed on the pfc . note that the prediction experiments were performed on four different datasets that were acquired from different individuals on different occasions . since the stai values varied between 20 and 80 , the rmse values 6.20 , 6.62 , 4.50 , and 6.38 appear reasonable . table 2 summarizes the proportions of the rmse values that were less than 10 as well as those less than five in each dataset . there are three facts that should be mentioned here : i)the algorithm had never seen the nirs values as well as the associated stai of a particular individual before . the parameters were learned from the data of other subjects only , suggesting that the stai values of unknown subjects may be predictable once the machine learned.ii)there were several subjects whose stai values could be predicted by the proposed algorithm with reasonable accuracy . table 2 , for instance , shows that there were 84.2100% of the young as well as elderly subjects whose stai prediction errors were less than 10 , which appears reasonable since the stai varied between 20 and 80 . the rightmost column of table 2 gives the minimum rmse for each dataset.iii)in machine learning prediction problems , it is often critical to choose appropriate features . the algorithm had never seen the nirs values as well as the associated stai of a particular individual before . the parameters were learned from the data of other subjects only , suggesting that the stai values of unknown subjects may be predictable once the machine learned . there were several subjects whose stai values could be predicted by the proposed algorithm with reasonable accuracy . table 2 , for instance , shows that there were 84.2100% of the young as well as elderly subjects whose stai prediction errors were less than 10 , which appears reasonable since the stai varied between 20 and 80 . 4 throughout the four datasets , irrespective of age . recall that our basis function ( 2 ) is nonlinear not only with respect to feature vector \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } \(x\ ) \end{document } , but also with respect to unknown parameters . we tested a linear model instead of the nonlinear model and found that the linear model was much less accurate than the proposed nonlinear model , as summarized in table 1 . two groups comprised young subjects ( 1926 years and 2024 years , respectively ) , whereas the remaining two comprised elderly subjects ( 6179 years and 6079 years , respectively ) . the results in table 1 appear to indicate that age does not play any significant role in the stai prediction problem . we note that a gender difference is discernible in that the highest rmse was 6.62 , followed by 6.38 in datasets 2 and 4 , respectively , where the result for the female subjects dominates that for the male subjects . potential differences in the results due to gender difference are not surprising because it is a known fact that gender differences exist in brain functions . in language processing tasks , for example , the right pfc is often activated in male subjects , whereas both the right and left pfc are activated in female subjects . fig.7 appears to suggest that the subject could have started becoming tired as the trial proceeded . however , there were other cases where the prediction error decreased instead of increased over the four trials , as was shown in fig.8 . on comparing our experiences with eeg - based experiments , both portable and non - portable , we determined that the two - channel portable nirs device is easier to equip than eeg devices . this is mainly because when conducting eeg measurements , one needs to carefully reduce the contact impedance between the head and the probe , whereas there is no such intricacy associated with the nirs device used in our study . the burden on the part of the subjects was also light compared with eeg measurements because the subjects could blink and carry out minor movements , whereas blinking and head movement significantly distort eeg signals . the fact that the nirs device was portable and lightweight also helped subjects to participate in the experiments with relative ease . the results of our prediction experiments , which we report in section iv , appear to indicate that the proposed algorithm is functional at least in the target datasets because the average prediction error is relatively small . the device is portable , weighs 100 g , and is easy to attach ; therefore , there could be potential applications in practical situations . one of the coauthors of this study , who is a neurosurgeon and routinely examines the stai index of patients before major operations , found that it helps when reasonable anxiety is predictable with this kind of simple device without a written self - report . the proposed method can be utilized in cases where patients are weak , in pain , or in acute anxiety states such that it is difficult for them to complete written questionnaires . one of the coauthors of this paper is a neurosurgery clinician and routinely acquires stai questionnaires from patients prior to major operations . sometimes , however , a patient is not able to answer the questionnaire in a credible manner . if the prediction method reported in this paper is functional , it could help in such scenarios . our predictions are based on a bayesian machine learning algorithm using the features extracted in terms of prediction errors . the fact that our predictions were reasonable means that the acquired nirs data contained information about the stai index via a complicated nonlinear manner provided appropriate features are found . this endeavor , however , necessitates a significant amount of effort and will therefore be left for future research . another limitation is that nirs measures the blood oxygen changes within illuminated pathways such that measured values of hemoglobin changes are also affected by extra - cranial tissues , not only intracranial tissues . in addition , it was suggested that the experiment itself could affect pfc activity at rest since the subject might feel the strain during nirs measurements , depending on their anxiety levels . the following possible future research projects are under consideration : i)it would be interesting to study if the capability of the proposed prediction algorithm depends on gender and handedness , among other characteristics.ii)it would also be worth investigating the prediction capability of the stai trait index , which is the feelings of stress , worry , discomfort , etc . that one experiences on a daily basis , instead of the stai state index , which is the feeling solely at the time of the experiment.iii)nirs measurements are the building blocks for the proposed algorithm ; therefore , we need to eliminate possible uncertainties and examine its prediction capabilities . for instance , a project worth pursuing would be to consider a particular subject performing the experiment over several time intervals , e.g. , one or several weeks or several months , and examine the prediction capabilities of the proposed method . environmental factors worth considering include room temperature , humidity , lighting , time of a day , among others.iv)an advantage of the present method is that it is not restricted to the stai data and the nirs data , as long as targeted written test data and associated sensor data are available.v)providing experimental data considering both cases , i.e. , answering the questions before or after acquisition , could reveal useful information about nirs measurement sensitivity at resting protocols . it would be interesting to study if the capability of the proposed prediction algorithm depends on gender and handedness , among other characteristics . it would also be worth investigating the prediction capability of the stai trait index , which is the feelings of stress , worry , discomfort , etc . that one experiences on a daily basis , instead of the stai state index , which is the feeling solely at the time of the experiment . nirs measurements are the building blocks for the proposed algorithm ; therefore , we need to eliminate possible uncertainties and examine its prediction capabilities . for instance , a project worth pursuing would be to consider a particular subject performing the experiment over several time intervals , e.g. , one or several weeks or several months , and examine the prediction capabilities of the proposed method . environmental factors worth considering include room temperature , humidity , lighting , time of a day , among others . an advantage of the present method is that it is not restricted to the stai data and the nirs data , as long as targeted written test data and associated sensor data are available . providing experimental data considering both cases , i.e. , answering the questions before or after acquisition , could reveal useful information about nirs measurement sensitivity at resting protocols . in this paper , we reported on a study in which we attempted to make predictions of the anxiety index of subjects from data acquired by a two - channel nirs device placed on the pfc . the method utilized comprised several steps : i)data acquisitionii)feature extractioniii)construction of machine learning algorithmiv)prediction construction of machine learning algorithm data were acquired from four different groups two comprising young subjects and the remaining two comprising elderly subjects . care had to be taken in constructing the machine learning algorithm because the amount of data acquired was highly limited to 1720 subjects in each dataset , in addition to the potentially significant amount of uncertainties contained . since the stai score varies between 20 and 80 , the prediction accuracies appeared reasonably accurate . there appeared no significant difference in prediction capabilities between the young and the elderly subjects . the device is portable , weighs only 100 g , and is easy to position ; therefore , the method can be used in practical situations . in one of our ongoing research projects , a new algorithm for improving prediction accuracies is being developed .

stress - induced psychological and somatic diseases are virtually endemic nowadays . written self - report anxiety measures are available ; however , these indices tend to be time consuming to acquire . for medical patients , completing written reports can be burdensome if they are weak , in pain , or in acute anxiety states . consequently , simple and fast non - invasive methods for assessing stress response from neurophysiological data are essential . in this paper , we report on a study that makes predictions of the state - trait anxiety inventory ( stai ) index from oxyhemoglobin and deoxyhemoglobin concentration changes of the prefrontal cortex using a two - channel portable near - infrared spectroscopy device . predictions are achieved by constructing machine learning algorithms within a bayesian framework with nonlinear basis function together with markov chain monte carlo implementation . in this paper , prediction experiments were performed against four different data sets , i.e. , two comprising young subjects , and the remaining two comprising elderly subjects . the number of subjects in each data set varied between 17 and 20 and each subject participated only once . they were not asked to perform any task ; instead , they were at rest . the root mean square errors for the four groups were 6.20 , 6.62 , 4.50 , and 6.38 , respectively . there appeared to be no significant distinctions of prediction accuracies between age groups and since the stai are defined between 20 and 80 , the predictions appeared reasonably accurate . the results indicate potential applications to practical situations such as stress management and medical practice .

Introduction Related Work Materials and Methods Experimental Results Discussion Conclusion

while a variety of written self - report anxiety measures provide valid and reliable measures of anxiety , these indices tend to be time consuming to acquire . for medical patients , completing a written report can be burdensome if they are weak , in pain , or in acute anxiety states . thus , simple and fast non - invasive methods for assessing stress response from neurophysiological data are essential . in , we used a near - infrared spectroscopy ( nirs ) device to investigate neurophysiological mechanisms of stress responses . one of our findings was that the prefrontal cortex ( pfc ) plays an important role in stress response as the asymmetry of pfc activity measured by nirs correlated with behavioral and somatic responses to mental stress . we measured these activity patterns using a two - channel nirs device instead of fmri or eeg and found that they correlated with the systemic stress responses of the autonomic nervous system and the hypothalamic - pituitary - adrenal ( hpa ) axis system . it should be noted that research on the brain under resting conditions is an active study area , and it has been reported that the brain at rest consumes a significant amount of energy and much activities with distinctive patterns are reported.b)the other direction was to make predictions about anxiety index from oxy and deoxy hemoglobin concentration changes of pfc using a two - channel portable nirs device . we achieved this by constructing a machine learning algorithm within a bayesian framework and implemented it with markov chain monte carlo ( mcmc ) . the other direction was to make predictions about anxiety index from oxy and deoxy hemoglobin concentration changes of pfc using a two - channel portable nirs device . we achieved this by constructing a machine learning algorithm within a bayesian framework and implemented it with markov chain monte carlo ( mcmc ) . a brief overview of mcmc is given in appendix b. specifically the study proceeded in the following steps : i)acquire the anxiety index via the state - trait anxiety inventory ( stai ) test.ii)use a two - channel portable nirs device to measure oxy and deoxy hemoglobin concentration changes of pfc in four different groups , where two of them comprise young subjects , whereas the other two comprise elderly subjects.iii)place each subject in a resting state in which no task is performed.iv)build a hierarchical bayesian machine learning algorithm for anxiety index predictions by taking into account both oxy and deoxy hemoglobin concentration changes . because the amount of data was limited , the predictive capability of the algorithm was evaluated by excluding one set of data and using the remaining set for learning . the hierarchical bayesian algorithm does not need a validation set since it does not utilize leave - one - out cross validation.v)evaluate the prediction capabilities of the algorithm against four different datasets from each of which one set of data was withheld for testing . acquire the anxiety index via the state - trait anxiety inventory ( stai ) test . use a two - channel portable nirs device to measure oxy and deoxy hemoglobin concentration changes of pfc in four different groups , where two of them comprise young subjects , whereas the other two comprise elderly subjects . because the amount of data was limited , the predictive capability of the algorithm was evaluated by excluding one set of data and using the remaining set for learning . evaluate the prediction capabilities of the algorithm against four different datasets from each of which one set of data was withheld for testing . section iii describes the materials and methods used as well as the experimental settings , subjects , machine learning algorithm , and feature extraction method . in , we conclude this section by stating that this paper is a significantly improved and expanded version of in that four datasets from different age groups are analyzed here , whereas only one target dataset was used in . 1(a ) and we measured the oxy- and deoxy - hb concentration changes using a portable two - channel nirs system ( pnirs-10 , hamamatsu photonics k.k . the nature of nirs data is not well understood ; consequently , the procedure that needs to be followed in each step is nontrivial . although the results are not included in this paper , we attempted to devise third and fifth order butterworth bandpass filters ( passband 0.011.0 hz ) and lof outlier detection / deletion on the nirs raw data for ( i ) . we will report on the effects of preprocessing in a future paper ; in this paper , our focus is on ( ii ) and ( iii ) . this is due to the fact that as the number of features increases , the number of unknown parameters also increases , such that poorly learned parameters degrade prediction capabilities . note that each channel of the target device acquires the concentration changes of oxy- and deoxy - hb so that the acquired data is a four - dimensional vector . let \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } \(y^{(i)},\kern 1pt \;\kern 1pt i\,=\,1,\ldots , n \ ) \end{document } be the stai state index obtained from the questionnaire , where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } \(n\ ) \end{document } is the number of subjects in an experiment . of the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } \(n\ ) \end{document } available pieces of data , we reserved one \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } \((x_{reserved } , \;y_{reserved } ) \ ) \end{document } and attempted to fit the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } \(n-1\ ) \end{document } remaining pieces of data with the following machine learning model:\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } \begin{align } p(y^{(i)}\vert x^{(i)};\omega , \beta ) = & \sqrt { \frac { \beta } { 2\pi } } \exp ( -\frac { \beta } { 2}(y^{(i)}-f(x^{(i)};\omega ) ) ^{2 } ) \notag \\ i=&1,\ldots , n-1 \end{align } \end{document } where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } \(f\ ) \end{document } is a basis function for data fitting , and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } \(\omega \ ) \end{document } denotes the associated unknown parameter to be learned . in order to capture the potential nonlinear relationship between the nirs data and the stai , we considered the following nonlinear basis function:\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } \begin{align } f(x^{(i)};\omega ) : = \sum \limits _ { h=1}^{h } { \big ( \omega _ { ( k+1)h } \sigma \big ( \sum \limits _ { k=1}^{k } { \omega _ { kh } x_{k}^{(i ) } + \omega _ { 0h } } \big ) \big ) } + \omega _ { 0(k+1)}\hspace { -1pc}\notag \\\text{}\end{align } \end{document } where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } \(\sigma \ ) \end{document } is a sigmoidal function . the number of hidden units \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } \(h\ ) \end{document } can be estimated within the present hierarchical bayesian framework . our study formulated the prediction problem within a bayesian framework , where a prior distribution was assumed about the unknown parameters and that information incorporated intothe data fitting model given by ( 1 ) . given the reserved nirs data \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } \(x_{reserved } \ ) \end{document } , the prediction of the stai index associated with the reserved data \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } \(y_{reserved } \ ) \end{document } was performed by computing the predictive distribution:\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } \begin{align } & \hspace { -1.3pc } p(y_{reserved } \vert x_{reserved } , x , y ) \notag \\ = & \int \!\!\!\int \!\!\!\int { p(y_{reserved } \vert x_{reserved } , \omega , \beta ) p(\omega , \alpha , \beta \vert x , y)d\omega d\alpha d\beta . } we performed four prediction experiments , one experiment for each of the four datasets . it should be noted that in the stai prediction of each subject , the algorithm had never seen the data from the particular subject to be tested . the hierarchical bayesian method used in this study only the state anxiety index was used in the present study , even though the trait anxiety index was available . we will attempt to make predictions of the trait anxiety index in our future project . it should be noted that the figures are not the results of linear regression between the quantities of the horizontal and the vertical axes ( see subsection iiid ) . the blue lines signify the rmse of 10 , which is an empirical value proposed by one of the coauthors of this paper who is a clinician in neurosurgery in the sense that an rmse of 10 for stai could be tolerable in a clinical practice where the stai score is between 20 and 80 . as a result , the algorithm had very little chance of seeing very low / high stai scores and it was difficult to make good predictions ; however , the results appear to be reasonable , including the low / high stai values . table 1 summarizes the results with multiple evaluation indices : rmse , pearson correlation coefficients , and the p - values of two tailed t - tests between the actual stai values and the predicted stai values . using the parameters learned from the first set of trial data , tests were performed against the second , third , and fourth set of trial data . in 10 of the 19 subjects , the prediction error of the fourth trial was greater than that of the first trial . in 10 of the 19 subjects , the prediction error of the fourth trial was greater than that of the first trial . however , in nine subjects , the prediction error for the fourth trial was less than that for the first trial . in the resting condition assumed in our study , that effect was less than in the stress task ; however , we could not exclude the potential effect of the information on the subject when the subject was informed of the beginning of the measurement 30 s before the measurements began . the present study went beyond investigating the left / right asymmetry of oxy - hb concentrations in the pfc activity during tasks involving mental stress such that the stai index predictions were performed based on the four - dimensional nirs values instead of the mere oxy - hb concentration changes . the three - dimensional features were extracted from the oxy- and deoxy - hb concentrations changes acquired from a two - channel nirs device placed on the pfc . note that the prediction experiments were performed on four different datasets that were acquired from different individuals on different occasions . since the stai values varied between 20 and 80 , the rmse values 6.20 , 6.62 , 4.50 , and 6.38 appear reasonable . table 2 , for instance , shows that there were 84.2100% of the young as well as elderly subjects whose stai prediction errors were less than 10 , which appears reasonable since the stai varied between 20 and 80 . table 2 , for instance , shows that there were 84.2100% of the young as well as elderly subjects whose stai prediction errors were less than 10 , which appears reasonable since the stai varied between 20 and 80 . two groups comprised young subjects ( 1926 years and 2024 years , respectively ) , whereas the remaining two comprised elderly subjects ( 6179 years and 6079 years , respectively ) . we note that a gender difference is discernible in that the highest rmse was 6.62 , followed by 6.38 in datasets 2 and 4 , respectively , where the result for the female subjects dominates that for the male subjects . on comparing our experiences with eeg - based experiments , both portable and non - portable , we determined that the two - channel portable nirs device is easier to equip than eeg devices . the results of our prediction experiments , which we report in section iv , appear to indicate that the proposed algorithm is functional at least in the target datasets because the average prediction error is relatively small . the device is portable , weighs 100 g , and is easy to attach ; therefore , there could be potential applications in practical situations . one of the coauthors of this study , who is a neurosurgeon and routinely examines the stai index of patients before major operations , found that it helps when reasonable anxiety is predictable with this kind of simple device without a written self - report . the proposed method can be utilized in cases where patients are weak , in pain , or in acute anxiety states such that it is difficult for them to complete written questionnaires . our predictions are based on a bayesian machine learning algorithm using the features extracted in terms of prediction errors . that one experiences on a daily basis , instead of the stai state index , which is the feeling solely at the time of the experiment.iii)nirs measurements are the building blocks for the proposed algorithm ; therefore , we need to eliminate possible uncertainties and examine its prediction capabilities . environmental factors worth considering include room temperature , humidity , lighting , time of a day , among others.iv)an advantage of the present method is that it is not restricted to the stai data and the nirs data , as long as targeted written test data and associated sensor data are available.v)providing experimental data considering both cases , i.e. that one experiences on a daily basis , instead of the stai state index , which is the feeling solely at the time of the experiment . an advantage of the present method is that it is not restricted to the stai data and the nirs data , as long as targeted written test data and associated sensor data are available . in this paper , we reported on a study in which we attempted to make predictions of the anxiety index of subjects from data acquired by a two - channel nirs device placed on the pfc . the method utilized comprised several steps : i)data acquisitionii)feature extractioniii)construction of machine learning algorithmiv)prediction construction of machine learning algorithm data were acquired from four different groups two comprising young subjects and the remaining two comprising elderly subjects . care had to be taken in constructing the machine learning algorithm because the amount of data acquired was highly limited to 1720 subjects in each dataset , in addition to the potentially significant amount of uncertainties contained . since the stai score varies between 20 and 80 , the prediction accuracies appeared reasonably accurate . there appeared no significant difference in prediction capabilities between the young and the elderly subjects . the device is portable , weighs only 100 g , and is easy to position ; therefore , the method can be used in practical situations .

alkylphenols ( e.g. , 4-tert - octylphenol , op ; 4-tert - nonylphenol , np ) and alkylphenol ethoxylates are a group of endocrine - disrupting chemicals that accumulate at high concentrations in air , soil and aquatic environment from the use of detergents , paints , pesticides , and plastic manufacturing . they are also found in the fluids and body fat of animals and humans . these compounds are classified as xenoestrogens , since they are able to induce inappropriate estrogenic action or interfere with the actions of endogenous estrogens affecting reproductive development and functions of laboratory and wild animals , and most likely humans . alkylphenols bind to the nuclear estrogen receptors ( ers ) , but their binding affinity and ability to activate er - mediated functions is 10010,000 times less potent than that of 17-estradiol . however , they exhibit quite robust nongenomic activity . since 2000 , european union restricted use of alkylphenols as priority hazardous substances by directive 2000/60/ec . recent analyses indicate that although alkylphenol ethoxylates levels have been decreasing in the last years , europe is much more contaminated than north america and developing countries . spermatogenesis , a process by which spermatogonia undergo a series of divisions and differentiation to become spermatozoa , is tightly controlled by sertoli cells . direct interactions between sertoli cells and between sertoli and germ cells are mediated by proteins that form specialized cell junctions . they form complexes localized between sertoli cells at the site of blood - testis barrier ( btb ) and between sertoli cells and elongated spermatids , in the apical ectoplasmic specializations . cadherin / catenin complexes play a determining role in stabilizing cell - cell contacts and their restructuring during movement of preleptotene spermatocytes across the btb . it was reported that the altered expression or loss of the protein - protein interactions between n - cadherin and -catenin induces germ cell detachment from the seminiferous epithelium [ 8 , 9 ] . connexin 43 ( cx43 ) , the predominant gap junction protein in seminiferous epithelium , is of absolute requirement for normal testicular development and spermatogenesis . in adult testis cx43 is a component of the junctional complex enabling direct communication and exchange of small molecules between adjacent sertoli cells , sertoli and germ cells , and between leydig cells . recent studies provided clear evidence that cx43 can also control spermatogenesis through regulation of tight and anchoring junctions that are closely intermingled with each other at the site of the btb . recent papers have reported that xenoestrogens can affect spermatogenesis by perturbing direct cell - cell interactions in the seminiferous epithelium . extensively studied xenoestrogen bisphenol a was demonstrated to cause a dose - dependent reduction in several junction proteins ( including n - cadherin , -catenin , and cx43 ) in sertoli cells in vitro and to disrupt the btb when administered to immature rats [ 12 , 13 ] . the estrogenic actions of alkylphenols on the testis are less studied when compared with bisphenol a. although it was demonstrated that exposure to op or np induces changes in multiple gene transcription , cell proliferation , and hormone production in vitro and in vivo , scarce data are available on their influence on junction proteins in the testes [ 1416 ] . in our previous study bank voles , seasonally breeding rodents , were used to investigate the effects of op on male reproductive organs , depending on the length of exposure and reproductive status of animals . we found that long - term exposure of adult males affected expressions of 3-hydroxysteroid dehydrogenase , aromatase , estrogen receptor , and androgen receptor as well as sex steroids levels in the testes and seminal vesicles . interestingly , a subtle difference in the sensitivity to op between voles kept in different light conditions was noted . to better understand the mechanism of op - induced alterations in the testis , the present study was aimed to examine changes in the distribution and expression of cell junction proteins , n - cadherin , -catenin , and connexin 43 , in bank vole following op exposure in vivo . importantly , to our knowledge this is the first in vivo study on the effect of op on adherens and gap junction proteins in the testes . in addition , to evaluate potential direct effects of op on junction protein expressions in seminiferous epithelium and to examine whether these effects are mediated through binding op to ers , organ culture model was used . bank voles ( clethrionomys glareolus , schreber ) were obtained from our own colony ( department of endocrinology , institute of zoology , jagiellonian university , krakow , poland ) which have been reared under long light cycles ( 18 h light and 6 h darkness ; 18l : 6d ) or short light cycles ( 6 h light and 18 h darkness ; 6l : 18d ) . the animal rooms were maintained at a temperature of 18c and a relative humidity of 55 5% . voles were housed in polyethylene cages ( 42 27 18 cm ) furnished with sawdust and wood shavings for bedding . a standard pelleted diet ( lsm diet , agropol , motycz , poland ; total isoflavone content below 450 mg / kg diet ) supplemented with seeds of wheat or oat , red beet , apples , and water was provided ad libitum . twenty - four mature male bank voles ( 6070-day - old ) kept in long light cycles ( 18l : 6d , n = 12 ) and short light cycles ( 6l : 18d , n = 12 ) were dosed orally with 4-tert - octylphenol ( op , 200 mg / kg body weight ; sigma - aldrich , st louis , mo , usa ) every monday , wednesday , and friday for 30 ( op30 ) or 60 days ( op60 ) . 4-tert - octylphenol was dissolved in a minimum amount of absolute ethanol and then a measured amount of sesame oil ( sigma - aldrich , st louis , mo , usa ) was added in 1 : 14 ( v / v ) , as previously described in detail . a dose of exposure to op was based on the literature [ 15 , 18 ] and it was finally selected upon our preliminary study in which lower doses ( 50 and 100 mg / kg body weight ) had no effects on bank vole reproductive organs weight and morphology . the control animals ( n = 24 ) for each experimental group ( n = 6/each group ) were given a vehicle only ( ethanol + sesame oil ) . after the animals were sacrificed by cervical dislocation , testes were immediately excised and serum was collected and frozen in 20c . experiments were performed in accordance with polish legal requirements , under the license given by the local ethics committee at the jagiellonian university , krakow , poland ( no . 88/iv/2010 ) . for immunohistochemical analysis one testis from each animal was immersed immediately in 4% formaldehyde and embedded in paraplast ( sigma - aldrich , st louis , mo , usa ) . sections of 5 m in thickness were mounted on slides coated with 3-aminopropyltriethoxysilane ( sigma - aldrich , st louis , mo , usa ) , deparaffinized , and rehydrated through decreasing alcoholic solutions . for western blot and elisa analysis , contralateral testis from each animal was frozen and stored in 80c . twenty - four untreated male bank voles ( 6070-day - old ) reared under long light cycles or short light cycles were sacrificed and testes were immediately removed and trimmed free of excess fat and connective tissue . after the capsule had been removed , the testes were cut into small pieces of 2 - 3 mm in diameter and were placed on millipore filters ( pore size 0.4 m ) ( millipore corporation , billerica , ma , usa ) . the filters were floated on 1.5 ml culture waymouth 's media ( gibco , grand island , ny , usa ) supplemented with 5% fetal bovine serum ( sigma - aldrich , st louis , mo , usa ) and l - glutamine containing 50 u / ml penicillin , 50 g / ml streptomycin in tissue culture dishes and incubated at 37c , in a humidified atmosphere containing 95% air : 5% co2 , for 24 h. 4-tert - octylphenol and ici 182,780 ( sigma - aldrich , st louis , mo , usa ) were dissolved in absolute ethanol and the final concentration of the solvent in culture medium was 0.1% ( v / v ) . in the first experiment the response to op was measured by comparing the testes from 18l : 6d and 6l : 18d males cultured in medium containing op ( at doses 10 , 100 , and 500 mg / l ) with the testes cultured in medium containing solvent ( control ) . in the second experiment ( to examine the mechanism of op action ) testes were cultured in medium containing op ( at doses 10 , 100 , and 500 mg / l ) or ici ( 6 mg / l ) , or combination of each dose of op plus 6 mg / l ici . as a control , testes were exposed to medium containing solvent . lower doses of op ( 0.1 , 1 mg / l ) and ici ( 2 mg since they appeared to have no effect on junction proteins , the data were not presented herein . after culture , the media and testes were collected and stored at 20c and 80c , respectively . to optimize immunohistochemical staining , slices were immersed for 4 min in 10 mm citrate buffer ( ph 6.0 ) and heated in a microwave oven ( 600 w ) . in short , nonspecific staining was blocked twice , first with 3% h2o2 in methanol for 15 min , to inhibit endogenous peroxidase activity , and second with 10% normal goat or horse serum ( sigma - aldrich , st louis , mo , usa ) for 30 min at room temperature to block nonspecific binding sites . thereafter , sections were incubated overnight at 4c in a humidified chamber in the presence of primary antibodies : ( 1 ) a mouse monoclonal antibody against n - cadherin ( 1 : 100 ; invitrogen , eugene , or , usa ) ; ( 2 ) a rabbit polyclonal antibody against -catenin ( 1 : 300 ; invitrogen , eugene , or , usa ) ; ( 3 ) a rabbit polyclonal antibody against cx43 ( 1 : 2,000 ; sigma - aldrich , st louis , mo , usa ) . next , biotinylated secondary antibody , goat anti - rabbit igg or horse anti - mouse igg , respectively ( 1 : 400 ; vector labs . , burlingame , ca , usa ) was applied for 1 h. finally , avidin - biotinylated horseradish peroxidase complex ( vectastain abc kit ; 1 : 100 ; vector labs . , burlingame , ca , usa ) for a further 30 min was used . after each step in these procedures , sections were carefully rinsed with tris - buffered saline ( tbs ; 0.05 m tris - hcl plus 0.15 m nacl , ph 7.6 ) . bound antibody was visualized with tbs containing 0.05% 3,3-diaminobenzidine ( sigma - aldrich , st louis , mo , usa ) and 0.07% imidazole ( sigma - aldrich , st louis , mo , usa ) for 3 - 4 min . the slides were processed immunohistochemically at the same time with the same treatment so that the staining intensities among testicular cells could be compared . the sections were examined with a leica dmr microscope ( leica microsystems , wetzlar , germany ) . tissues were homogenized on ice with a cold ripa buffer ( sigma - aldrich , st louis , mo , usa ) , sonicated , and centrifuged at 15,000 g for 20 min at 4c . the protein concentration for each sample was estimated using bradford dye - binding procedure with bovine serum albumin ( sigma - aldrich , st louis , mo , usa ) as a standard . homogenates containing 100 g of protein were solubilized in a sample buffer ( bio - rad labs . separated proteins were transferred onto a polyvinylidene difluoride ( pvdf ) membrane ( millipore corporation , billerica , ma , usa ) using a wet blotter in the genie transfer buffer ( ph 8.4 ) for 90 min at a constant voltage of 135 v. then blots were blocked with 5% nonfat dry milk in tbs , 0.1% tween 20 for 1 h with shaking , followed by an incubation with appropriate primary antibody ( as for immunohistochemistry ; anti - n - cadherin , dilution 1 : 250 ; anti--catenin , dilution 1 : 800 ; anti - cx43 , dilution 1 : 10,000 ) overnight at 4c . the membranes were washed and incubated with the horseradish - peroxidase labeled goat anti - rabbit igg or horse anti - mouse igg ( vector labs . , burlingame , ca , usa ) at a dilution 1 : 3,000 , for 1 h at room temperature . immunoreactive proteins were detected by chemiluminescence with western blotting luminol reagent ( santa cruz biotechnology , santa cruz , ca , usa ) , and images were captured with a chemidoc xrs+ system ( bio - rad labs . all immunoblots were stripped with stripping buffer containing 62.5 mm tris - hcl , 100 mm 2-mercaptoethanol and 2% sds ( wt / vol ) ( ph 6.7 ) at 50c for 30 min , and incubated in a rabbit polyclonal antibody against -actin ( dilution , 1 : 3,000 ; sigma - aldrich ) which served as a loading control . molecular masses were estimated by reference to standard proteins ( fermentas , gmbh , st . plasma fsh concentrations were measured with commercially available mouse follitropin subunit beta elisa kit ( eiaab , wuhan , china ) according to the manufacturer 's instructions . all statistical analyses were performed using one - way analysis of variance ( anova ) . tukey multiple comparison test was used to determine which values differed significantly from controls ( * p < 0.05 , * * p < 0.01 , * * * p < 0.001 ) . in control males expression and distribution of n - cadherin , -catenin , and cx43 were dependent on the length of the photoperiod . in seminiferous tubules of 18l : 6d animals strong , linear staining for n - cadherin and -catenin was localized at the region of btb , running parallel to the basement membrane . additionally , discrete punctuate staining was observed in the adluminal compartment , mainly at sertoli cell - elongated spermatid interface ( figures 1(a ) and 1(e ) ) . cx43 was detected predominantly at the base of the tubules , between sertoli cells and spermatogonia or pachytene spermatocytes as well as in the cytoplasm of some sertoli cells . a moderate staining the localization and intensity of n - cadherin , -catenin , and cx43 immunostaining displayed some evidence of stage - specificity . in the leydig cells , a very strong linear cx43 signal on the plasma membrane was found as the prevalent staining pattern ( figure 1(i ) ) . in tubules of 6l : 18d animals the intensity of n - cadherin and -catenin staining in the basal region of the epithelium was clearly reduced when compared to 18l : 6d voles ; weak , punctuate signal extended through much of the epithelium ( figures 1(c ) and 1(g ) ) . similarly as in 18l : 6d animals , cx43 was present predominantly in the basal compartment , however its distribution at the region of btb was irregular and discontinuous . in the adluminal compartment weak staining was frequently dispersed in the cytoplasm of sertoli or germ cells ( figure 1(k ) ) . cytoplasmic staining was also detected in leydig cells of 6l : 18d males ( insert in figure 1(k ) ) . exposure to op for 30 days did not alter the distribution of n - cadherin , -catenin , and cx43 neither in 18l : 6d nor 6l : 18d males ( not shown ) . in males treated with op for 60 days distribution pattern of these proteins was changed in tubules with altered spermatogenesis , as well as in some morphologically normal tubules . n - cadherin and cx43 were frequently localized in the form of irregular lines or distinct foci between the cells ( figures 1(b ) and 1(j ) ) , in the cytoplasm of sertoli cells ( figures 1(b ) , 1(d ) , and 1(j ) ) , or at the entire surfaces of sertoli and germ cells ( figure 1(l ) ) . -catenin reactivity remained at the region of btb , whereas loss of the staining was detected in the apical compartment of seminiferous epithelium ( figures 1(f ) and 1(h ) ) . in 18l : 6d group signal intensities of all studied proteins were diminished following op treatment when compared to the respective controls . in leydig cells of op60 no staining was detected when the antibody against n - cadherin , -catenin , or cx43 was omitted ( inserts in figures 1(c ) , 1(g ) , and 1(l ) ) . immunodetectable n - cadherin , -catenin , and cx43 proteins were observed as single bands near the 127 , 92 , and 43 kda positions of the sds gel , respectively , in testicular homogenates of the control voles and those treated with op ( figure 2 ) . in the testes of 18l : 6d males expression levels of n - cadherin and -catenin were significantly decreased only in op60 animals when compared to the respective controls ( figures 2(a ) and 2(b ) ) , whereas cx43 was reduced in both op30 and op60 groups ( figure 2(c ) ) . in 6l : 18d group an increase in the expression levels of all studied proteins was found . however , the increase was statistically significant only in case of cx43 ( figure 2(c ) ) . serum fsh concentration was higher in 18l : 6d animals when compared with 6l : 18d males . no significant influence of op was seen on fsh concentrations in both 18l : 6d and 6l : 18d groups ( figure 3 ) . when op was present in the culture medium at concentrations of 10 , 100 , and 500 mg / l , in homogenates of testicular pieces from 18l : 6d animals a progressive decline in n - cadherin and cx43 expression levels was obtained at all doses after 24 h of culture ( figures 4(b ) and 4(d ) ) ; however only higher concentrations ( 100 and 500 mg / l ) elicited statistically significant effect . expression of -catenin decreased following exposure to 500 mg / l op , whereas lower doses had no effect . in testis explants of 6l : 18d males protein expression levels were increased , but a relatively large variation in the expressions of n - cadherin and -catenin between the individual testes exposed to op meant the differences were not statistically significant ( figures 4(b)4(d ) ) . explants from 18l : 6d males were tested with 6 mg / l ici alone for 24 h ; 100 and 500 mg / l op alone for 24 h ; or combination of each dose of op plus 6 mg / l ici . since ici showed no discernible effect on proteins ' expression , solvent - treated explants served as controls in this experiment . coadministration of ici did not block the effects of op on n - cadherin expression ( figure 5(a ) ) . on the other hand , ici partially reversed the op - induced decrease in -catenin and cx43 in testicular fragments cultured in vitro ( figures 5(b ) and 5(c ) ) . to assess the effects of op on junction proteins expression in bank vole testis in vivo and in vitro methods were used . in vivo experiments are required for understanding the action of tested chemical in the internal environment of the organism , where it undergoes various biochemical transformations , affecting their bioavailability and activity . it was reported that in mammals op is metabolized by the liver to two glucuronide conjugates . glucuronidation of op eliminates its estrogen - like activity ; however glucuronides may be hydrolysed back to active compounds . at doses that exceed the metabolization level , op accumulates in various organs , particularly , in adipose tissue , liver , muscles , and brain [ 23 , 24 ] . therefore , the effects of op action are noted following administration of high doses or after chronic exposure , as it was demonstrated by data obtained in our and other laboratories [ 17 , 25 ] . in addition , the influence of op on endogenous estrogens production might contribute to the effects observed in vivo . in contrast , during in vitro organ culture of the testis the influence of extratesticular factors is avoided , while relationship among the cells remains intact and the interactions between the cells can be accurately evaluated . therefore , short - term organ culture was used to examine whether op could exert direct and immediate effects on junction proteins expression in the cells of the seminiferous tubule . the results presented herein demonstrate that exposure of male bank voles to op has a potential to induce adverse effects on junction proteins in the testes . interestingly , alterations in the expressions and localizations of these proteins appeared to be dependent on the length of the photoperiod . our results are the first reported on the influence of the length of the photoperiod on n - cadherin and -catenin proteins in the testis of seasonally breeding rodents . it should be noted that bank voles kept under long photoperiod ( 18l : 6d ) show similar reproductive characteristics as those observed in the wild in reproductively active males during spring and summer , whereas animals exposed to short light regime ( 6l : 18d ) show regressive phase of reproduction that occurs in the wild voles during late autumn and winter . in the testes of males kept in short light cycles most of seminiferous tubules exhibit small or no lumen with not fully differentiated germ cells . decrease in the level of plasma gonadotropins during the short photoperiod results in reduced testosterone production decreased expression and activity of steroidogenic enzymes and reduced expression of androgen receptor when compared to long - day animals [ 17 , 27 , 28 ] . the present study provides clear evidence that in bank voles also direct cell - cell interactions are regulated by length of the light cycle , since the expression and localization of protein markers of adherens and gap junctions are different in animals reared under different light conditions . we noticed that organization of n - cadherin/-catenin and cx43-based junctions in the seminiferous epithelium of 18l : 6d males was consistent with the organization of morphologically mature junctions in the adult testes of continual breeders such as mice and rats [ 11 , 13 , 29 , 30 ] . on the contrary , in 6l : 18d animals distribution of junction proteins was similar to those observed in immature or prepubertal males , in which weaker , punctuate , or cytoplasmic staining was dispersed in the basal and adluminal compartment of the epithelium [ 13 , 31 ] . this is in line with the results of tarulli et al . , who reported that in short - day males of seasonally breeding djungarian hamster sertoli cells have characteristics of both adult and immature phenotypes , suggesting that they take on an intermediate or transitional state . it should be mentioned that early study by pelletier demonstrated the localization of cx43 in another seasonal breeder , mink . in contrast to our observations , the author reported that during winter testicular regression immunoexpression of cx43 is present only in the basal third of the epithelium . such a result was presumably a consequence of performing less sensitive immunohistochemical method with the use of secondary antibody conjugated directly to peroxidase . disorganization and decreased expressions of n - cadherin , -catenin , and cx43 proteins in 6l : 18d bank vole males were accompanied with reduced fsh level in these animals . there is evidence that fsh stimulates the formation of extensive inter - sertoli cell adherens junctions ; in the absence of fsh adherens junction puncta were observed in rat sertoli cells in vitro , whereas the addition of fsh induced the reorganization of these puncta into adherens junction belts . also gap junction coupling and organization of cx43 gap junction plaques between sertoli cells in vitro appeared to be regulated by fsh and camp [ 35 , 36 ] . moreover , recent data on djungarian hamsters indicate that gonadotropin suppression induced by short photoperiod disrupted distribution and expressions of tight junction proteins , and fsh replacement led to a rapid reorganization of these proteins . based on these data and our results we believe that in bank voles fsh could be a factor controlling adherens and gap junctions arrangement in the seminiferous epithelium during transition from reproductive quiescence to reproductive activity . despite numerous studies on testis histopathology of males treated with op , very little is known about the alterations induced by this xenoestrogen on cell cell junction molecules . in the only paper reporting the effects of op on sertoli cell junction proteins , fiorini et al . observed reduction in the levels of occludin , n - cadherin , and cx43 in the serw3 sertoli cell line treated with 0.2 m op for 24 h. in addition to its effect on protein levels , op was able to delocalize the proteins from the membrane to the cytoplasmic compartment . in the present study , it was shown that long - term in vivo op treatment of adult bank voles also resulted in the reduction of n - cadherin , -catenin , and cx43 proteins expressions and their delocalization , but only in the testes of males kept in long photoperiod . surprisingly , in short - day animals slight increase of cx43 ( p < 0.05 ) , n - cadherin , and -catenin ( statistically nonsignificant ) levels was found . these differences appeared to be independent of fsh , since op treatment did not change fsh concentrations neither in long - day nor in short - day animals . thus , we hypothesize that op affects junction proteins expression acting directly on the testis . to test this hypothesis we used organ culture model . we found that in testis explants cultured with op for 24 h expression levels of all studied proteins were reduced in long - day voles and slightly elevated in short - day animals . photoperiod - dependent effect of op on junction proteins in bank vole testes was therefore maintained in in vitro conditions , indicating that hypothalamic - pituitary axis is not involved in this effect . the reason for diverse responses of males kept in different light conditions to op remains to be elucidated . nevertheless , since in our previous studies on bank vole testis we found elevated estradiol concentrations and aromatase expression following op exposure , it is possible that in short - day voles op directly acting ( as estrogen - like compound ) , or more likely through induction of local estradiol production , restores the expression levels of junction proteins . these hypothesis is based on our earlier observations that exposure to low dose of exogenous estradiol induced acceleration of the onset of spermatogenesis in voles kept under short light cycle conditions and on the finding that estrogens have fsh - independent stimulatory effect on spermatogenesis in photoregressed siberian hamster [ 38 , 39 ] . on the other hand , in long - day males , op exerts negative effect on the expression of junction proteins , presumably by inducing supraphysiological estrogen level or action . indeed , our previous studies revealed that in bank vole males treated with a high dose of estradiol disruption of testicular structure and tubular atrophy occurred . moreover , in rats exogenous estrogens appeared to affect cell - cell junctions in the testis ; 17-estradiol was shown to be disruptive to the btb integrity , while 17-ethinylestradiol altered intercellular communication by disrupting gap junction functionality and cx43 trafficking in cultured sertoli cells as well as in isolated rat seminiferous tubules [ 12 , 40 ] . it should be noted that in our study op had the most rapid effects on cx43 protein expression in the testes of 18l : 6d males ( visible just after 30-day exposure ) . this observation argues for the recent hypothesis that cx43 is one of the main targets for endocrine disruptors and that impaired cx43 expression affects other proteins of the btb . the question arises as to the mechanism of op action on junction proteins in the seminiferous tubules . to determine whether this actions was mediated through binding to er or er , testicular explants from 18l : 6d males were treated concomitantly with a pure estrogen antagonist , ici 187 780 ( 6 mg / l ) , and increasing op concentrations ( 10 to 500 mg we found that -catenin and cx43 op - induced decrease was partially blocked by ici , suggesting that the biological effects of op on the expression of these proteins involve an er - mediated response . on the other hand , the influence of op on n - cadherin does not appear to be mediated through the classic nuclear ers , since ici did not alter the effects of op . several earlier papers reported that also some other effects of op were not mediated through the er , possibly involving interactions with membrane components or receptors . for example , murono et al . [ 42 , 43 ] described the effects of op on the conversion of progesterone to testosterone and hcg - stimulated testosterone biosynthesis as independent of er and er. it can not be , however , excluded that after the relatively short incubation period ( 24 h ) some amounts of the proteins are not degraded and still exist in the cultured tissue , even if the mrna level was downregulated . therefore , further studies including sequencing of bank vole genes and the application of rt - pcr method are needed to verify data regarding mechanisms of op action on junction proteins expression in the bank vole testes . in this study we demonstrated that in seasonally breeding bank vole organization of adherens and gap junctions as well as the expression of junction proteins is related to the length of photoperiod and , in consequence , to the reproductive status of the animal . finally , we found that alterations in cadherin / catenin and cx43-based junction in the testes following op exposure may partially result from activation of er and/or er signaling pathway .

in the present study we evaluated in vivo and in vitro effects of 4-tert - octylphenol ( op ) on the expression and distribution of adherens and gap junction proteins , n - cadherin , -catenin , and connexin 43 ( cx43 ) , in testes of seasonally breeding rodents , bank voles . we found that in bank vole testes expression and distribution of n - cadherin , -catenin , and cx43 were photoperiod dependent . long - term treatment with op ( 200 mg / kg b.w . ) resulted in the reduction of junction proteins expressions ( p < 0.05 , p < 0.01 ) and their delocalization in the testes of males kept in long photoperiod , whereas in short - day animals slight increase of cx43 ( p < 0.05 ) , n - cadherin , and -catenin ( statistically nonsignificant ) levels was observed . effects of op appeared to be independent of fsh and were maintained during in vitro organ culture , indicating that op acts directly on adherens and gap junction proteins in the testes . an experiment performed using an antiestrogen ici 182,780 demonstrated that the biological effects of op on -catenin and cx43 involve an estrogen receptor - mediated response . taken together , in bank vole organization of adherens and gap junctions and their susceptibility to op are related to the length of photoperiod . alterations in cadherin / catenin and cx43-based junction may partially result from activation of estrogen receptor and/or signaling pathway .

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

, 4-tert - octylphenol , op ; 4-tert - nonylphenol , np ) and alkylphenol ethoxylates are a group of endocrine - disrupting chemicals that accumulate at high concentrations in air , soil and aquatic environment from the use of detergents , paints , pesticides , and plastic manufacturing . alkylphenols bind to the nuclear estrogen receptors ( ers ) , but their binding affinity and ability to activate er - mediated functions is 10010,000 times less potent than that of 17-estradiol . they form complexes localized between sertoli cells at the site of blood - testis barrier ( btb ) and between sertoli cells and elongated spermatids , in the apical ectoplasmic specializations . cadherin / catenin complexes play a determining role in stabilizing cell - cell contacts and their restructuring during movement of preleptotene spermatocytes across the btb . it was reported that the altered expression or loss of the protein - protein interactions between n - cadherin and -catenin induces germ cell detachment from the seminiferous epithelium [ 8 , 9 ] . connexin 43 ( cx43 ) , the predominant gap junction protein in seminiferous epithelium , is of absolute requirement for normal testicular development and spermatogenesis . extensively studied xenoestrogen bisphenol a was demonstrated to cause a dose - dependent reduction in several junction proteins ( including n - cadherin , -catenin , and cx43 ) in sertoli cells in vitro and to disrupt the btb when administered to immature rats [ 12 , 13 ] . the estrogenic actions of alkylphenols on the testis are less studied when compared with bisphenol a. although it was demonstrated that exposure to op or np induces changes in multiple gene transcription , cell proliferation , and hormone production in vitro and in vivo , scarce data are available on their influence on junction proteins in the testes [ 1416 ] . in our previous study bank voles , seasonally breeding rodents , were used to investigate the effects of op on male reproductive organs , depending on the length of exposure and reproductive status of animals . we found that long - term exposure of adult males affected expressions of 3-hydroxysteroid dehydrogenase , aromatase , estrogen receptor , and androgen receptor as well as sex steroids levels in the testes and seminal vesicles . to better understand the mechanism of op - induced alterations in the testis , the present study was aimed to examine changes in the distribution and expression of cell junction proteins , n - cadherin , -catenin , and connexin 43 , in bank vole following op exposure in vivo . importantly , to our knowledge this is the first in vivo study on the effect of op on adherens and gap junction proteins in the testes . in addition , to evaluate potential direct effects of op on junction protein expressions in seminiferous epithelium and to examine whether these effects are mediated through binding op to ers , organ culture model was used . twenty - four mature male bank voles ( 6070-day - old ) kept in long light cycles ( 18l : 6d , n = 12 ) and short light cycles ( 6l : 18d , n = 12 ) were dosed orally with 4-tert - octylphenol ( op , 200 mg / kg body weight ; sigma - aldrich , st louis , mo , usa ) every monday , wednesday , and friday for 30 ( op30 ) or 60 days ( op60 ) . a dose of exposure to op was based on the literature [ 15 , 18 ] and it was finally selected upon our preliminary study in which lower doses ( 50 and 100 mg / kg body weight ) had no effects on bank vole reproductive organs weight and morphology . the filters were floated on 1.5 ml culture waymouth 's media ( gibco , grand island , ny , usa ) supplemented with 5% fetal bovine serum ( sigma - aldrich , st louis , mo , usa ) and l - glutamine containing 50 u / ml penicillin , 50 g / ml streptomycin in tissue culture dishes and incubated at 37c , in a humidified atmosphere containing 95% air : 5% co2 , for 24 h. 4-tert - octylphenol and ici 182,780 ( sigma - aldrich , st louis , mo , usa ) were dissolved in absolute ethanol and the final concentration of the solvent in culture medium was 0.1% ( v / v ) . in the first experiment the response to op was measured by comparing the testes from 18l : 6d and 6l : 18d males cultured in medium containing op ( at doses 10 , 100 , and 500 mg / l ) with the testes cultured in medium containing solvent ( control ) . in the second experiment ( to examine the mechanism of op action ) testes were cultured in medium containing op ( at doses 10 , 100 , and 500 mg / l ) or ici ( 6 mg / l ) , or combination of each dose of op plus 6 mg / l ici . lower doses of op ( 0.1 , 1 mg / l ) and ici ( 2 mg since they appeared to have no effect on junction proteins , the data were not presented herein . thereafter , sections were incubated overnight at 4c in a humidified chamber in the presence of primary antibodies : ( 1 ) a mouse monoclonal antibody against n - cadherin ( 1 : 100 ; invitrogen , eugene , or , usa ) ; ( 2 ) a rabbit polyclonal antibody against -catenin ( 1 : 300 ; invitrogen , eugene , or , usa ) ; ( 3 ) a rabbit polyclonal antibody against cx43 ( 1 : 2,000 ; sigma - aldrich , st louis , mo , usa ) . separated proteins were transferred onto a polyvinylidene difluoride ( pvdf ) membrane ( millipore corporation , billerica , ma , usa ) using a wet blotter in the genie transfer buffer ( ph 8.4 ) for 90 min at a constant voltage of 135 v. then blots were blocked with 5% nonfat dry milk in tbs , 0.1% tween 20 for 1 h with shaking , followed by an incubation with appropriate primary antibody ( as for immunohistochemistry ; anti - n - cadherin , dilution 1 : 250 ; anti--catenin , dilution 1 : 800 ; anti - cx43 , dilution 1 : 10,000 ) overnight at 4c . tukey multiple comparison test was used to determine which values differed significantly from controls ( * p < 0.05 , * * p < 0.01 , * * * p < 0.001 ) . in control males expression and distribution of n - cadherin , -catenin , and cx43 were dependent on the length of the photoperiod . in seminiferous tubules of 18l : 6d animals strong , linear staining for n - cadherin and -catenin was localized at the region of btb , running parallel to the basement membrane . additionally , discrete punctuate staining was observed in the adluminal compartment , mainly at sertoli cell - elongated spermatid interface ( figures 1(a ) and 1(e ) ) . a moderate staining the localization and intensity of n - cadherin , -catenin , and cx43 immunostaining displayed some evidence of stage - specificity . in tubules of 6l : 18d animals the intensity of n - cadherin and -catenin staining in the basal region of the epithelium was clearly reduced when compared to 18l : 6d voles ; weak , punctuate signal extended through much of the epithelium ( figures 1(c ) and 1(g ) ) . exposure to op for 30 days did not alter the distribution of n - cadherin , -catenin , and cx43 neither in 18l : 6d nor 6l : 18d males ( not shown ) . n - cadherin and cx43 were frequently localized in the form of irregular lines or distinct foci between the cells ( figures 1(b ) and 1(j ) ) , in the cytoplasm of sertoli cells ( figures 1(b ) , 1(d ) , and 1(j ) ) , or at the entire surfaces of sertoli and germ cells ( figure 1(l ) ) . -catenin reactivity remained at the region of btb , whereas loss of the staining was detected in the apical compartment of seminiferous epithelium ( figures 1(f ) and 1(h ) ) . in leydig cells of op60 no staining was detected when the antibody against n - cadherin , -catenin , or cx43 was omitted ( inserts in figures 1(c ) , 1(g ) , and 1(l ) ) . immunodetectable n - cadherin , -catenin , and cx43 proteins were observed as single bands near the 127 , 92 , and 43 kda positions of the sds gel , respectively , in testicular homogenates of the control voles and those treated with op ( figure 2 ) . in the testes of 18l : 6d males expression levels of n - cadherin and -catenin were significantly decreased only in op60 animals when compared to the respective controls ( figures 2(a ) and 2(b ) ) , whereas cx43 was reduced in both op30 and op60 groups ( figure 2(c ) ) . when op was present in the culture medium at concentrations of 10 , 100 , and 500 mg / l , in homogenates of testicular pieces from 18l : 6d animals a progressive decline in n - cadherin and cx43 expression levels was obtained at all doses after 24 h of culture ( figures 4(b ) and 4(d ) ) ; however only higher concentrations ( 100 and 500 mg / l ) elicited statistically significant effect . in testis explants of 6l : 18d males protein expression levels were increased , but a relatively large variation in the expressions of n - cadherin and -catenin between the individual testes exposed to op meant the differences were not statistically significant ( figures 4(b)4(d ) ) . coadministration of ici did not block the effects of op on n - cadherin expression ( figure 5(a ) ) . on the other hand , ici partially reversed the op - induced decrease in -catenin and cx43 in testicular fragments cultured in vitro ( figures 5(b ) and 5(c ) ) . to assess the effects of op on junction proteins expression in bank vole testis in vivo and in vitro methods were used . in addition , the influence of op on endogenous estrogens production might contribute to the effects observed in vivo . in contrast , during in vitro organ culture of the testis the influence of extratesticular factors is avoided , while relationship among the cells remains intact and the interactions between the cells can be accurately evaluated . therefore , short - term organ culture was used to examine whether op could exert direct and immediate effects on junction proteins expression in the cells of the seminiferous tubule . the results presented herein demonstrate that exposure of male bank voles to op has a potential to induce adverse effects on junction proteins in the testes . interestingly , alterations in the expressions and localizations of these proteins appeared to be dependent on the length of the photoperiod . our results are the first reported on the influence of the length of the photoperiod on n - cadherin and -catenin proteins in the testis of seasonally breeding rodents . it should be noted that bank voles kept under long photoperiod ( 18l : 6d ) show similar reproductive characteristics as those observed in the wild in reproductively active males during spring and summer , whereas animals exposed to short light regime ( 6l : 18d ) show regressive phase of reproduction that occurs in the wild voles during late autumn and winter . in the testes of males kept in short light cycles most of seminiferous tubules exhibit small or no lumen with not fully differentiated germ cells . decrease in the level of plasma gonadotropins during the short photoperiod results in reduced testosterone production decreased expression and activity of steroidogenic enzymes and reduced expression of androgen receptor when compared to long - day animals [ 17 , 27 , 28 ] . the present study provides clear evidence that in bank voles also direct cell - cell interactions are regulated by length of the light cycle , since the expression and localization of protein markers of adherens and gap junctions are different in animals reared under different light conditions . we noticed that organization of n - cadherin/-catenin and cx43-based junctions in the seminiferous epithelium of 18l : 6d males was consistent with the organization of morphologically mature junctions in the adult testes of continual breeders such as mice and rats [ 11 , 13 , 29 , 30 ] . on the contrary , in 6l : 18d animals distribution of junction proteins was similar to those observed in immature or prepubertal males , in which weaker , punctuate , or cytoplasmic staining was dispersed in the basal and adluminal compartment of the epithelium [ 13 , 31 ] . , who reported that in short - day males of seasonally breeding djungarian hamster sertoli cells have characteristics of both adult and immature phenotypes , suggesting that they take on an intermediate or transitional state . disorganization and decreased expressions of n - cadherin , -catenin , and cx43 proteins in 6l : 18d bank vole males were accompanied with reduced fsh level in these animals . there is evidence that fsh stimulates the formation of extensive inter - sertoli cell adherens junctions ; in the absence of fsh adherens junction puncta were observed in rat sertoli cells in vitro , whereas the addition of fsh induced the reorganization of these puncta into adherens junction belts . also gap junction coupling and organization of cx43 gap junction plaques between sertoli cells in vitro appeared to be regulated by fsh and camp [ 35 , 36 ] . moreover , recent data on djungarian hamsters indicate that gonadotropin suppression induced by short photoperiod disrupted distribution and expressions of tight junction proteins , and fsh replacement led to a rapid reorganization of these proteins . based on these data and our results we believe that in bank voles fsh could be a factor controlling adherens and gap junctions arrangement in the seminiferous epithelium during transition from reproductive quiescence to reproductive activity . in the only paper reporting the effects of op on sertoli cell junction proteins , fiorini et al . observed reduction in the levels of occludin , n - cadherin , and cx43 in the serw3 sertoli cell line treated with 0.2 m op for 24 h. in addition to its effect on protein levels , op was able to delocalize the proteins from the membrane to the cytoplasmic compartment . in the present study , it was shown that long - term in vivo op treatment of adult bank voles also resulted in the reduction of n - cadherin , -catenin , and cx43 proteins expressions and their delocalization , but only in the testes of males kept in long photoperiod . surprisingly , in short - day animals slight increase of cx43 ( p < 0.05 ) , n - cadherin , and -catenin ( statistically nonsignificant ) levels was found . these differences appeared to be independent of fsh , since op treatment did not change fsh concentrations neither in long - day nor in short - day animals . thus , we hypothesize that op affects junction proteins expression acting directly on the testis . we found that in testis explants cultured with op for 24 h expression levels of all studied proteins were reduced in long - day voles and slightly elevated in short - day animals . photoperiod - dependent effect of op on junction proteins in bank vole testes was therefore maintained in in vitro conditions , indicating that hypothalamic - pituitary axis is not involved in this effect . the reason for diverse responses of males kept in different light conditions to op remains to be elucidated . nevertheless , since in our previous studies on bank vole testis we found elevated estradiol concentrations and aromatase expression following op exposure , it is possible that in short - day voles op directly acting ( as estrogen - like compound ) , or more likely through induction of local estradiol production , restores the expression levels of junction proteins . on the other hand , in long - day males , op exerts negative effect on the expression of junction proteins , presumably by inducing supraphysiological estrogen level or action . moreover , in rats exogenous estrogens appeared to affect cell - cell junctions in the testis ; 17-estradiol was shown to be disruptive to the btb integrity , while 17-ethinylestradiol altered intercellular communication by disrupting gap junction functionality and cx43 trafficking in cultured sertoli cells as well as in isolated rat seminiferous tubules [ 12 , 40 ] . it should be noted that in our study op had the most rapid effects on cx43 protein expression in the testes of 18l : 6d males ( visible just after 30-day exposure ) . the question arises as to the mechanism of op action on junction proteins in the seminiferous tubules . to determine whether this actions was mediated through binding to er or er , testicular explants from 18l : 6d males were treated concomitantly with a pure estrogen antagonist , ici 187 780 ( 6 mg / l ) , and increasing op concentrations ( 10 to 500 mg we found that -catenin and cx43 op - induced decrease was partially blocked by ici , suggesting that the biological effects of op on the expression of these proteins involve an er - mediated response . on the other hand , the influence of op on n - cadherin does not appear to be mediated through the classic nuclear ers , since ici did not alter the effects of op . [ 42 , 43 ] described the effects of op on the conversion of progesterone to testosterone and hcg - stimulated testosterone biosynthesis as independent of er and er. therefore , further studies including sequencing of bank vole genes and the application of rt - pcr method are needed to verify data regarding mechanisms of op action on junction proteins expression in the bank vole testes . in this study we demonstrated that in seasonally breeding bank vole organization of adherens and gap junctions as well as the expression of junction proteins is related to the length of photoperiod and , in consequence , to the reproductive status of the animal . finally , we found that alterations in cadherin / catenin and cx43-based junction in the testes following op exposure may partially result from activation of er and/or er signaling pathway .

from march 2003 to february 2005 , 10 patients ( 3 males and 7 females : mean age , 58.7 years [ range , 43 - 76 years ] ) with alavgs were referred to our interventional radiology practice for evaluation of graft patency or upper arm swelling . this study was approved by our institutional review board , and all the patients gave us their informed written consent . the patients had no central venous stenosis or obstruction on venography before creation of the hemodialysis grafts . all patients had lost their optimal sites for a hemodialysis fistula or graft in both upper extremities and they had undergone an operation for creating alavgs with a 4 - 6 mm or 4 - 7 mm ringed - tapered polytetrafluoroethylene graft ( ptfe , vascugraft b braun , berlin , germany ) . eight patients were referred for evaluation and treatment of graft dysfunction or graft failure , which was clinically suggested on the basis of clot aspiration , decreased or impalpable thrill , and a decreased flow rate during hemodialysis . the other two patients had swelling of the ipsilateral upper arm ( table 1 ) . the interval between the creation of the alavgs and the first intervention was 7 to 1,184 days ( average , 272.8 329.1 ) . when thrombus was present , 100,000 iu of urokinase was administrated into the graft , and aspiration thrombectomy was done 1 or 2 hours later ( the ' lyse and wait ' technique ) ( 6 ) . the apex of the graft was punctured using a 21 gauge needle , and contrast media was then slowly injected . an 8 fr hoffman sheath ( cook , bloomington , in ) was inserted through the apex of the graft , and manual aspiration was then performed . the thrombus in the venous limb was aspirated first and , if present , any thrombus in the arterial limb was then removed after changing the direction of the sheath into the arterial limb . fistulography was repeated after aspiration thrombectomy by using a 5 fr multiside - hole or cobra catheters ( cook , bloomington , in ) to evaluate the precise location of any stenosis or central venous stenosis , and to determine the diameters of the graft and vein . percutaneous transluminal angioplasty ( pta ) was performed after the stenosis was diagnosed , 6 to 8 mm pta balloon dilatation catheters were used for the intragraft and anastomotic stenoses , and 12 - 14 mm pta balloon dilatation catheters were used for the central venous stenoses . a 6 to 8 mm cutting balloon catheter ( boston scientifics , in ) was applied when necessary to abolish the waist of a tight stenosis . a small amount of residual thrombus , if present , was pushed out into the central veins after dilatation of the stenotic segment . a stent was applied in the case of elastic recoil after pta with using the usual balloon catheter or a cutting balloon catheter . the basilic vein was punctured under ultrasonographic guidance and a 6 - 8 fr introducing sheath was inserted . a larger size introducing sheath a 5 fr multisidehole catheter ( cook , bloomington , in ) was introduced into the basilic vein for venography . when a central vein stenosis was found , a 5 fr cobra catheter ( cook , bloomington , in ) was advanced into the subclavian artery via the graft to evaluate the precise location of the anastomotic sites and the anastomotic stenosis . after the interventional procedures , the puncture sites were closed with a symmetric purse - string suture arrangement using 4 - 0 nylon monofilament ( ethicon ; johnson & johnson , somerville , nj ) . follow - up was ended on june 2006 and a retrospective review of the patients ' charts was done . the procedural success rate , the post - intervention primary and secondary patency rates and the complications were defined according to ' reporting standards for percutaneous interventions in dialysis access ' ( 8) . the procedural success rate was defined as an anatomic success with at least one indicator of either hemodynamic or clinical success . primary patency was defined as the interval following intervention until the next access thrombosis or repeated intervention . secondary patency meant the interval after intervention until the access was surgically declotted , revised or abandoned because of the inability to treat the original lesion , the choice of the surgeon , transplant , the patient was lost to follow - up etc . the 6 month and 12 month post - intervention primary and secondary patency rates were calculated by the kaplan - meier method using the spss for windows , version 12.0 ( spss inc , chicago , il ) . major complications were defined as those resulting in admission to a hospital for therapy , an unplanned increase in the level of care , prolonged hospitalization , permanent adverse sequela or death . when thrombus was present , 100,000 iu of urokinase was administrated into the graft , and aspiration thrombectomy was done 1 or 2 hours later ( the ' lyse and wait ' technique ) ( 6 ) . the apex of the graft was punctured using a 21 gauge needle , and contrast media was then slowly injected . an 8 fr hoffman sheath ( cook , bloomington , in ) was inserted through the apex of the graft , and manual aspiration was then performed . the thrombus in the venous limb was aspirated first and , if present , any thrombus in the arterial limb was then removed after changing the direction of the sheath into the arterial limb . fistulography was repeated after aspiration thrombectomy by using a 5 fr multiside - hole or cobra catheters ( cook , bloomington , in ) to evaluate the precise location of any stenosis or central venous stenosis , and to determine the diameters of the graft and vein . percutaneous transluminal angioplasty ( pta ) was performed after the stenosis was diagnosed , 6 to 8 mm pta balloon dilatation catheters were used for the intragraft and anastomotic stenoses , and 12 - 14 mm pta balloon dilatation catheters were used for the central venous stenoses . a 6 to 8 mm cutting balloon catheter ( boston scientifics , in ) was applied when necessary to abolish the waist of a tight stenosis . a small amount of residual thrombus , if present , was pushed out into the central veins after dilatation of the stenotic segment . a stent was applied in the case of elastic recoil after pta with using the usual balloon catheter or a cutting balloon catheter . the basilic vein was punctured under ultrasonographic guidance and a 6 - 8 fr introducing sheath was inserted . a larger size introducing sheath a 5 fr multisidehole catheter ( cook , bloomington , in ) was introduced into the basilic vein for venography . when a central vein stenosis was found , a 5 fr cobra catheter ( cook , bloomington , in ) was advanced into the subclavian artery via the graft to evaluate the precise location of the anastomotic sites and the anastomotic stenosis . after the interventional procedures , the puncture sites were closed with a symmetric purse - string suture arrangement using 4 - 0 nylon monofilament ( ethicon ; johnson & johnson , somerville , nj ) . follow - up was ended on june 2006 and a retrospective review of the patients ' charts was done . the procedural success rate , the post - intervention primary and secondary patency rates and the complications were defined according to ' reporting standards for percutaneous interventions in dialysis access ' ( 8) . the procedural success rate was defined as an anatomic success with at least one indicator of either hemodynamic or clinical success . primary patency was defined as the interval following intervention until the next access thrombosis or repeated intervention . secondary patency meant the interval after intervention until the access was surgically declotted , revised or abandoned because of the inability to treat the original lesion , the choice of the surgeon , transplant , the patient was lost to follow - up etc . the 6 month and 12 month post - intervention primary and secondary patency rates were calculated by the kaplan - meier method using the spss for windows , version 12.0 ( spss inc , chicago , il ) . major complications were defined as those resulting in admission to a hospital for therapy , an unplanned increase in the level of care , prolonged hospitalization , permanent adverse sequela or death . in all the patients , the outflow vein was the infraclavicular proximal axillary vein , and the inflow artery was proximal axillary artery just beneath the infraclavicular proximal axillary vein . all the arterial and venous anastomoses were located near the pinch - off region and they overlapped each other ( fig . 1 ) . on the initial fistulographies , six patients ( 60% ) had graft - related stenosis ; the stenosis was at the arterial anastomosis in all six patients ( 100% ) , the stenosis was at the venous anastomosis in four ( 67% ) , the stenosis was at both the arterial and venous anastomosis in four ( 67% ) and there was an intragraft stenosis in four patients ( table 1 ) . of the six patients with a graft - related stenosis , four patients also had a subclavian or proximal axillary vein stenosis . the other four patients showed subclavian or proximal axillary vein stenosis without a graft - related stenosis , so there was a total of eight patients who showed subclavian or proximal axillary vein stenoses . of these eight patients , only four patients ( 50% ) had difficult hemodialysis or hemodialysis failure and they showed graft - related stenoses ; both arterial and venous anastomotic stenosis were found in three patients ( 3 of 4 patients , 75% ) , and arterial anastomotic stenosis was found in one patient . the other four patients had neither hemodialysis failure nor graft - related stenosis on their fistulograms . the other two patients were referred for clot aspiration or decreased thrill ; nevertheless , they did not have difficult hemodialysis or hemodialysis failure . sixteen sessions of interventional procedures were performed in eight patients ( an average of 2 sessions / patient ) until the end of follow - up , and six patients experienced two or more interventional procedures : there were eight sessions of pta with thrombolysis , five sessions of pta only and three sessions of stent placement ( figs . 2 , 3 ) . in one female patient , 6 mm 4 cm and 6 mm 8 cm sized zilver vascular stents ( cook , bloomington , in ) were placed for relieving a residual stenosis at a venous anastomosis , but complete failure of the graft occurred . the avg was movable in accordance with standing and lying down , and also according to movement of the shoulder joint . so the cephalic vein was folded at the point of the proximal end of the stent and the graft was completely occluded . in one case of subclavian venous stenosis , a 14 mm 6 cm self - expandable hercules stent ( s&g biotech , seoul , korea ) among the eight patients with subclavian venous stenosis , only four patients with simultaneous graft - related stenosis had problems during hemodialysis . on the other hand , the other four patients without simultaneous graft - related stenosis did well ; nevertheless , their subclavian veins were stenotic . among all six patients with graft - related stenosis , four patients showed subclavian venous stenosis and so balloon angioplasties were done . however , hemodialysis failure or difficult hemodialysis did not occur , even though the residual stenosis of the subclavian vein was over 30% in diameter after pta . follow - up was possible in nine patients with an average follow - up period of 415.5 days ( range : 1 - 700 days ) . six alavgs were functioning at the end of follow - up and three alavgs were terminated . in the case of termination of the alavg , one patient died two weeks after fistulography due to acute myocardial infarction ( table 2 ) . the post - intervention primary and secondary patency rates of our study were 50% and 63% at three months , 38% and 63% at six months and 25% and 63% at one year , respectively . distal arterial embolization occurred at the bifurcation level of the brachial artery , and this was successfully treated with thrombolysis and aspiration of the residual embolus . yet hospitalization was not prolonged in spite of thrombolysis therapy and no other complication was noted . in all the patients , the outflow vein was the infraclavicular proximal axillary vein , and the inflow artery was proximal axillary artery just beneath the infraclavicular proximal axillary vein . all the arterial and venous anastomoses were located near the pinch - off region and they overlapped each other ( fig . 1 ) . on the initial fistulographies , six patients ( 60% ) had graft - related stenosis ; the stenosis was at the arterial anastomosis in all six patients ( 100% ) , the stenosis was at the venous anastomosis in four ( 67% ) , the stenosis was at both the arterial and venous anastomosis in four ( 67% ) and there was an intragraft stenosis in four patients ( table 1 ) . of the six patients with a graft - related stenosis , four patients also had a subclavian or proximal axillary vein stenosis . the other four patients showed subclavian or proximal axillary vein stenosis without a graft - related stenosis , so there was a total of eight patients who showed subclavian or proximal axillary vein stenoses . of these eight patients , only four patients ( 50% ) had difficult hemodialysis or hemodialysis failure and they showed graft - related stenoses ; both arterial and venous anastomotic stenosis were found in three patients ( 3 of 4 patients , 75% ) , and arterial anastomotic stenosis was found in one patient . the other four patients had neither hemodialysis failure nor graft - related stenosis on their fistulograms . the other two patients were referred for clot aspiration or decreased thrill ; nevertheless , they did not have difficult hemodialysis or hemodialysis failure . sixteen sessions of interventional procedures were performed in eight patients ( an average of 2 sessions / patient ) until the end of follow - up , and six patients experienced two or more interventional procedures : there were eight sessions of pta with thrombolysis , five sessions of pta only and three sessions of stent placement ( figs . 2 , 3 ) . in one female patient , 6 mm 4 cm and 6 mm 8 cm sized zilver vascular stents ( cook , bloomington , in ) were placed for relieving a residual stenosis at a venous anastomosis , but complete failure of the graft occurred . the avg was movable in accordance with standing and lying down , and also according to movement of the shoulder joint . so the cephalic vein was folded at the point of the proximal end of the stent and the graft was completely occluded . in one case of subclavian venous stenosis , a 14 mm 6 cm self - expandable hercules stent ( s&g biotech , seoul , korea ) the overall procedure success rate was 69% ( 11 of 16 sessions ) . among the eight patients with subclavian venous stenosis , only four patients with simultaneous graft - related stenosis had problems during hemodialysis . on the other hand , the other four patients without simultaneous graft - related stenosis did well ; nevertheless , their subclavian veins were stenotic . among all six patients with graft - related stenosis , four patients showed subclavian venous stenosis and so balloon angioplasties were done . however , hemodialysis failure or difficult hemodialysis did not occur , even though the residual stenosis of the subclavian vein was over 30% in diameter after pta . follow - up was possible in nine patients with an average follow - up period of 415.5 days ( range : 1 - 700 days ) . six alavgs were functioning at the end of follow - up and three alavgs were terminated . in the case of termination of the alavg , one patient died two weeks after fistulography due to acute myocardial infarction ( table 2 ) . the post - intervention primary and secondary patency rates of our study were 50% and 63% at three months , 38% and 63% at six months and 25% and 63% at one year , respectively . distal arterial embolization occurred at the bifurcation level of the brachial artery , and this was successfully treated with thrombolysis and aspiration of the residual embolus . yet hospitalization was not prolonged in spite of thrombolysis therapy and no other complication was noted . when all the upper extremity sites for creating an arteriovenous fistula in a chronic hemodialysis patient are exhausted , the secondary site that 's most frequently selected is the leg or permanent venous catheters are used . however , the femoral vessels are frequently diseased and the femoral avg has a high rate of complications such as infection ( 20% ) , thrombosis , extremity loss ( 22% ) and even a high mortality rate ( 20% ) ( 9 , 10 ) . permanent central venous catheters also show a high rate of septic complications , low patency rates and discomfort ( 11 ) . the use of the axillary artery and vein as dialysis access sites is not a new idea , and various types of avgs in the anterior chest wall have been described in the literature since manning et al . ( 12 ) first reported on an experimental case in 1975 ( 2 , 12 - 16 ) . ( 2 ) described only three cases of ipsilateral alavgs among 27 avgs in the anterior chest wall , and jean - baptiste et al . ( 3 ) reported the midterm results of 27 cases of ipsilateral alavg and they suggested that alavg construction may be preferable to lower extremity vascular access . although we had not reviewed all the cases of alavgs in our institute , the results of our study also suggest that construction of an alavg may be preferable to lower extremity arteriovenous hemodialysis access . in a large series of avg stenoses , maya et al . ( 17 ) found that venous anastomosis was the most common site of stenosis in an avg , followed by stenoses of the venous outlet , intragraft stenoses , central vein stenoses and arterial anastomosis stenoses . in our present study , the distal subclavian or proximal axillary vein adjacent to the venous anastomosis was the most common site of stenosis , while arterial anastomotic stenosis was the second most common , followed by venous anastomotic and intragraft stenoses . ( 3 ) reported two cases of stenosis in the postanastomotic outflow vein with ipsilateral axillary loop grafts . in most cases of avg , a subclavian vein stenosis is a consequence of previous subclavian catheterizations or high flow from an arteriovenous fistula ( 18 ) . yet in our series , no one had a history of ipsilateral subclavian vein catheterization or central vein stenosis on the preoperative venography . the anastomotic sites are very close to the pinch - off region and they are under the pectoralis major muscle . the space for anastomoses is narrow and surrounded by bone and muscle , which is in contrast to the conventional avg in the upper extremity . furthermore , the arterial and venous anastomoses overlap each other in that narrow space , and it is mandatory to dissect the pectoralis major muscle to expose the vessels and anastomose the graft to the vessels . therefore , formation of granulation tissue and fibrosis is expected to be more abundant and to have a more severe affect than that in a conventional avg . as a consequence , stenoses of the subclavian and axillary veins and arterial anastomosis might occur more frequently . high - velocity , turbulent flow from the nearby graft might also affect the formation of intimal hyperplasia ( 18 , 19 ) . for a ptfe avg to function properly , there must be an adequate venous outflow , and stenosis of the venous outflow may cause dysfunction of a ptfe avg . in the present study , a stenosis at the central vein , just proximal to the venous anastomosis , showed a suboptimal post - interventional result . these lesions might be more fibrous and have a greater chance of demonstrating elastic recoil than an intragraft or graft - related stenosis . yet although a 50% degree of narrowing or more was noted in a stenotic subclavian vein , the subclavian vein is a large vessel and it usually has abundant collaterals . therefore , optimal flow for hemodialysis may be sustained . in the present study , only one patient needed stent placement in the subclavian vein due to elastic recoil after pta and hemodialysis disturbance . ( 20 ) concluded that pta of an asymptomatic central venous stenosis greater than 50% in the setting of hemodialysis access maintenance procedures is associated with more rapid progression of stenosis and escalation of lesions , as compared with a nontreatment approach , and kanterman et al . ( 21 ) also reported that central vein stenosis rarely compromises the hemodialysis access function and that treatment with pta may not be a necessary intervention . however , flow disturbance in the venous outflow tract and repeated thrombosis may occur due to changes of a patient 's condition . this was the reason why we performed interventional procedures more frequently than that reported in the literature . 22 ) reported that 1.22 interventions per graft year are necessary for maintaining access patency in the natural history of ptfe grafts . elastic recoil after conventional balloon angioplasty is not uncommon in a stenosis at a venous anastomosis , and stent placement in a rigid stenosis is an effective procedure ( 23 - 26 ) . we performed two sessions of stent placement at a stenosis of the venous anastomosis in one female patient . however , the graft completely failed by kinking of the cephalic vein , which was due to the gravitational movement of the patient 's breast . another case of stent placement was performed in a subclavian vein stenosis and this showed good patency during the follow - up . the post - intervention primary and secondary patency rates of our study were 50% and 63% at three months , 38% and 63% at six months and 25% and 63% at one year , respectively . the primary patency rate at three months in our study was better than the minimum requirement recommended by the national kidney foundation ( 40% primary patency at three months after percutaneous thrombectomy ) . on the other hand , the primary and secondary patency rates at six months and one year were not so excellent . it is quite possible that these results might be due to the location of the graft near the pinch - off region as well as the configuration of the arterial and venous anastomoses overlapping each other . the limitations of our study are the small sample size and the relatively lower primary and secondary patency rates at six month and one year than the results of percutaneous intervention in the usual avg in the upper extremity . other reports that have focused on an avg in the anterior chest wall ( 4 , 5 ) showed that percutaneous intervention was performed in three and five cases and this was successful in 66% ( 2 of 3 ) and 80% ( 4 of 5 ) . but no available data about the primary and secondary patency rates after percutaneous intervention was noted . further experiences with percutaneous intervention in failed alavgs will be needed in a larger population . despite of the small sample size , our study showed that an alavg is an alternative for patients who lost an opportunity for creating an autologous fistula and graft in their forearm and upper arm .

objectivethe purpose of this study was to evaluate the fistulographic features of malfunctioning axillary loop - configured arteriovenous grafts and the efficacy of percutaneous interventions in failed axillary loop - configured arteriovenous grafts.materials and methodsten patients with axillary loop - configured arteriovenous grafts were referred for evaluation of graft patency or upper arm swelling . fistulography and percutaneous intervention , including thrombolysis , percutaneous transluminal angioplasty and stent placement , were performed . statistical analysis of the procedure success rate and the primary and secondary patency rates was done.resultsfour patients had graft related and subclavian venous stenosis , two patients had graft related stenosis and another four patients had subclavian venous stenosis only . sixteen sessions of interventional procedures were performed in eight patients ( average : 2 sessions / patient ) until the end of follow - up . an interventional procedure was not done in two patients with central venous stenosis . the overall procedure success rate was 69% ( 11 of 16 sessions ) . the post - intervention primary and secondary patency rates were 50% and 63% at three months , 38% and 63% at six months and 25% and 63% at one year , respectively.conclusiondysfunctional axillary loop - configured arteriovenous grafts almost always had subclavian venous and graft - related stenosis . interventional treatments are helpful to overcome this and these treatments are expected to play a major role in restoring and maintaining the axillary loop - configured arteriovenous loop grafts .

MATERIALS AND METHODS Fistulography and the Interventional Procedures Definitions and the Study Endpoints RESULTS Fistulographic Features Interventional Procedures DISCUSSION

from march 2003 to february 2005 , 10 patients ( 3 males and 7 females : mean age , 58.7 years [ range , 43 - 76 years ] ) with alavgs were referred to our interventional radiology practice for evaluation of graft patency or upper arm swelling . this study was approved by our institutional review board , and all the patients gave us their informed written consent . the patients had no central venous stenosis or obstruction on venography before creation of the hemodialysis grafts . all patients had lost their optimal sites for a hemodialysis fistula or graft in both upper extremities and they had undergone an operation for creating alavgs with a 4 - 6 mm or 4 - 7 mm ringed - tapered polytetrafluoroethylene graft ( ptfe , vascugraft b braun , berlin , germany ) . eight patients were referred for evaluation and treatment of graft dysfunction or graft failure , which was clinically suggested on the basis of clot aspiration , decreased or impalpable thrill , and a decreased flow rate during hemodialysis . the other two patients had swelling of the ipsilateral upper arm ( table 1 ) . the interval between the creation of the alavgs and the first intervention was 7 to 1,184 days ( average , 272.8 329.1 ) . when thrombus was present , 100,000 iu of urokinase was administrated into the graft , and aspiration thrombectomy was done 1 or 2 hours later ( the ' lyse and wait ' technique ) ( 6 ) . the apex of the graft was punctured using a 21 gauge needle , and contrast media was then slowly injected . an 8 fr hoffman sheath ( cook , bloomington , in ) was inserted through the apex of the graft , and manual aspiration was then performed . the thrombus in the venous limb was aspirated first and , if present , any thrombus in the arterial limb was then removed after changing the direction of the sheath into the arterial limb . fistulography was repeated after aspiration thrombectomy by using a 5 fr multiside - hole or cobra catheters ( cook , bloomington , in ) to evaluate the precise location of any stenosis or central venous stenosis , and to determine the diameters of the graft and vein . percutaneous transluminal angioplasty ( pta ) was performed after the stenosis was diagnosed , 6 to 8 mm pta balloon dilatation catheters were used for the intragraft and anastomotic stenoses , and 12 - 14 mm pta balloon dilatation catheters were used for the central venous stenoses . a small amount of residual thrombus , if present , was pushed out into the central veins after dilatation of the stenotic segment . when a central vein stenosis was found , a 5 fr cobra catheter ( cook , bloomington , in ) was advanced into the subclavian artery via the graft to evaluate the precise location of the anastomotic sites and the anastomotic stenosis . after the interventional procedures , the puncture sites were closed with a symmetric purse - string suture arrangement using 4 - 0 nylon monofilament ( ethicon ; johnson & johnson , somerville , nj ) . follow - up was ended on june 2006 and a retrospective review of the patients ' charts was done . the procedural success rate , the post - intervention primary and secondary patency rates and the complications were defined according to ' reporting standards for percutaneous interventions in dialysis access ' ( 8) . the procedural success rate was defined as an anatomic success with at least one indicator of either hemodynamic or clinical success . primary patency was defined as the interval following intervention until the next access thrombosis or repeated intervention . secondary patency meant the interval after intervention until the access was surgically declotted , revised or abandoned because of the inability to treat the original lesion , the choice of the surgeon , transplant , the patient was lost to follow - up etc . the 6 month and 12 month post - intervention primary and secondary patency rates were calculated by the kaplan - meier method using the spss for windows , version 12.0 ( spss inc , chicago , il ) . when thrombus was present , 100,000 iu of urokinase was administrated into the graft , and aspiration thrombectomy was done 1 or 2 hours later ( the ' lyse and wait ' technique ) ( 6 ) . the apex of the graft was punctured using a 21 gauge needle , and contrast media was then slowly injected . an 8 fr hoffman sheath ( cook , bloomington , in ) was inserted through the apex of the graft , and manual aspiration was then performed . fistulography was repeated after aspiration thrombectomy by using a 5 fr multiside - hole or cobra catheters ( cook , bloomington , in ) to evaluate the precise location of any stenosis or central venous stenosis , and to determine the diameters of the graft and vein . percutaneous transluminal angioplasty ( pta ) was performed after the stenosis was diagnosed , 6 to 8 mm pta balloon dilatation catheters were used for the intragraft and anastomotic stenoses , and 12 - 14 mm pta balloon dilatation catheters were used for the central venous stenoses . when a central vein stenosis was found , a 5 fr cobra catheter ( cook , bloomington , in ) was advanced into the subclavian artery via the graft to evaluate the precise location of the anastomotic sites and the anastomotic stenosis . after the interventional procedures , the puncture sites were closed with a symmetric purse - string suture arrangement using 4 - 0 nylon monofilament ( ethicon ; johnson & johnson , somerville , nj ) . follow - up was ended on june 2006 and a retrospective review of the patients ' charts was done . the procedural success rate , the post - intervention primary and secondary patency rates and the complications were defined according to ' reporting standards for percutaneous interventions in dialysis access ' ( 8) . the procedural success rate was defined as an anatomic success with at least one indicator of either hemodynamic or clinical success . primary patency was defined as the interval following intervention until the next access thrombosis or repeated intervention . secondary patency meant the interval after intervention until the access was surgically declotted , revised or abandoned because of the inability to treat the original lesion , the choice of the surgeon , transplant , the patient was lost to follow - up etc . the 6 month and 12 month post - intervention primary and secondary patency rates were calculated by the kaplan - meier method using the spss for windows , version 12.0 ( spss inc , chicago , il ) . in all the patients , the outflow vein was the infraclavicular proximal axillary vein , and the inflow artery was proximal axillary artery just beneath the infraclavicular proximal axillary vein . on the initial fistulographies , six patients ( 60% ) had graft - related stenosis ; the stenosis was at the arterial anastomosis in all six patients ( 100% ) , the stenosis was at the venous anastomosis in four ( 67% ) , the stenosis was at both the arterial and venous anastomosis in four ( 67% ) and there was an intragraft stenosis in four patients ( table 1 ) . of the six patients with a graft - related stenosis , four patients also had a subclavian or proximal axillary vein stenosis . the other four patients showed subclavian or proximal axillary vein stenosis without a graft - related stenosis , so there was a total of eight patients who showed subclavian or proximal axillary vein stenoses . of these eight patients , only four patients ( 50% ) had difficult hemodialysis or hemodialysis failure and they showed graft - related stenoses ; both arterial and venous anastomotic stenosis were found in three patients ( 3 of 4 patients , 75% ) , and arterial anastomotic stenosis was found in one patient . the other four patients had neither hemodialysis failure nor graft - related stenosis on their fistulograms . the other two patients were referred for clot aspiration or decreased thrill ; nevertheless , they did not have difficult hemodialysis or hemodialysis failure . sixteen sessions of interventional procedures were performed in eight patients ( an average of 2 sessions / patient ) until the end of follow - up , and six patients experienced two or more interventional procedures : there were eight sessions of pta with thrombolysis , five sessions of pta only and three sessions of stent placement ( figs . the avg was movable in accordance with standing and lying down , and also according to movement of the shoulder joint . so the cephalic vein was folded at the point of the proximal end of the stent and the graft was completely occluded . in one case of subclavian venous stenosis , a 14 mm 6 cm self - expandable hercules stent ( s&g biotech , seoul , korea ) among the eight patients with subclavian venous stenosis , only four patients with simultaneous graft - related stenosis had problems during hemodialysis . on the other hand , the other four patients without simultaneous graft - related stenosis did well ; nevertheless , their subclavian veins were stenotic . among all six patients with graft - related stenosis , four patients showed subclavian venous stenosis and so balloon angioplasties were done . however , hemodialysis failure or difficult hemodialysis did not occur , even though the residual stenosis of the subclavian vein was over 30% in diameter after pta . follow - up was possible in nine patients with an average follow - up period of 415.5 days ( range : 1 - 700 days ) . six alavgs were functioning at the end of follow - up and three alavgs were terminated . in the case of termination of the alavg , one patient died two weeks after fistulography due to acute myocardial infarction ( table 2 ) . the post - intervention primary and secondary patency rates of our study were 50% and 63% at three months , 38% and 63% at six months and 25% and 63% at one year , respectively . distal arterial embolization occurred at the bifurcation level of the brachial artery , and this was successfully treated with thrombolysis and aspiration of the residual embolus . yet hospitalization was not prolonged in spite of thrombolysis therapy and no other complication was noted . in all the patients , the outflow vein was the infraclavicular proximal axillary vein , and the inflow artery was proximal axillary artery just beneath the infraclavicular proximal axillary vein . on the initial fistulographies , six patients ( 60% ) had graft - related stenosis ; the stenosis was at the arterial anastomosis in all six patients ( 100% ) , the stenosis was at the venous anastomosis in four ( 67% ) , the stenosis was at both the arterial and venous anastomosis in four ( 67% ) and there was an intragraft stenosis in four patients ( table 1 ) . of the six patients with a graft - related stenosis , four patients also had a subclavian or proximal axillary vein stenosis . the other four patients showed subclavian or proximal axillary vein stenosis without a graft - related stenosis , so there was a total of eight patients who showed subclavian or proximal axillary vein stenoses . of these eight patients , only four patients ( 50% ) had difficult hemodialysis or hemodialysis failure and they showed graft - related stenoses ; both arterial and venous anastomotic stenosis were found in three patients ( 3 of 4 patients , 75% ) , and arterial anastomotic stenosis was found in one patient . the other four patients had neither hemodialysis failure nor graft - related stenosis on their fistulograms . the other two patients were referred for clot aspiration or decreased thrill ; nevertheless , they did not have difficult hemodialysis or hemodialysis failure . sixteen sessions of interventional procedures were performed in eight patients ( an average of 2 sessions / patient ) until the end of follow - up , and six patients experienced two or more interventional procedures : there were eight sessions of pta with thrombolysis , five sessions of pta only and three sessions of stent placement ( figs . in one female patient , 6 mm 4 cm and 6 mm 8 cm sized zilver vascular stents ( cook , bloomington , in ) were placed for relieving a residual stenosis at a venous anastomosis , but complete failure of the graft occurred . the avg was movable in accordance with standing and lying down , and also according to movement of the shoulder joint . so the cephalic vein was folded at the point of the proximal end of the stent and the graft was completely occluded . in one case of subclavian venous stenosis , a 14 mm 6 cm self - expandable hercules stent ( s&g biotech , seoul , korea ) the overall procedure success rate was 69% ( 11 of 16 sessions ) . among the eight patients with subclavian venous stenosis , only four patients with simultaneous graft - related stenosis had problems during hemodialysis . on the other hand , the other four patients without simultaneous graft - related stenosis did well ; nevertheless , their subclavian veins were stenotic . among all six patients with graft - related stenosis , four patients showed subclavian venous stenosis and so balloon angioplasties were done . however , hemodialysis failure or difficult hemodialysis did not occur , even though the residual stenosis of the subclavian vein was over 30% in diameter after pta . follow - up was possible in nine patients with an average follow - up period of 415.5 days ( range : 1 - 700 days ) . six alavgs were functioning at the end of follow - up and three alavgs were terminated . in the case of termination of the alavg , one patient died two weeks after fistulography due to acute myocardial infarction ( table 2 ) . the post - intervention primary and secondary patency rates of our study were 50% and 63% at three months , 38% and 63% at six months and 25% and 63% at one year , respectively . distal arterial embolization occurred at the bifurcation level of the brachial artery , and this was successfully treated with thrombolysis and aspiration of the residual embolus . yet hospitalization was not prolonged in spite of thrombolysis therapy and no other complication was noted . however , the femoral vessels are frequently diseased and the femoral avg has a high rate of complications such as infection ( 20% ) , thrombosis , extremity loss ( 22% ) and even a high mortality rate ( 20% ) ( 9 , 10 ) . permanent central venous catheters also show a high rate of septic complications , low patency rates and discomfort ( 11 ) . the use of the axillary artery and vein as dialysis access sites is not a new idea , and various types of avgs in the anterior chest wall have been described in the literature since manning et al . in our present study , the distal subclavian or proximal axillary vein adjacent to the venous anastomosis was the most common site of stenosis , while arterial anastomotic stenosis was the second most common , followed by venous anastomotic and intragraft stenoses . ( 3 ) reported two cases of stenosis in the postanastomotic outflow vein with ipsilateral axillary loop grafts . as a consequence , stenoses of the subclavian and axillary veins and arterial anastomosis might occur more frequently . for a ptfe avg to function properly , there must be an adequate venous outflow , and stenosis of the venous outflow may cause dysfunction of a ptfe avg . in the present study , a stenosis at the central vein , just proximal to the venous anastomosis , showed a suboptimal post - interventional result . these lesions might be more fibrous and have a greater chance of demonstrating elastic recoil than an intragraft or graft - related stenosis . ( 20 ) concluded that pta of an asymptomatic central venous stenosis greater than 50% in the setting of hemodialysis access maintenance procedures is associated with more rapid progression of stenosis and escalation of lesions , as compared with a nontreatment approach , and kanterman et al . this was the reason why we performed interventional procedures more frequently than that reported in the literature . elastic recoil after conventional balloon angioplasty is not uncommon in a stenosis at a venous anastomosis , and stent placement in a rigid stenosis is an effective procedure ( 23 - 26 ) . we performed two sessions of stent placement at a stenosis of the venous anastomosis in one female patient . however , the graft completely failed by kinking of the cephalic vein , which was due to the gravitational movement of the patient 's breast . another case of stent placement was performed in a subclavian vein stenosis and this showed good patency during the follow - up . the post - intervention primary and secondary patency rates of our study were 50% and 63% at three months , 38% and 63% at six months and 25% and 63% at one year , respectively . the primary patency rate at three months in our study was better than the minimum requirement recommended by the national kidney foundation ( 40% primary patency at three months after percutaneous thrombectomy ) . on the other hand , the primary and secondary patency rates at six months and one year were not so excellent . it is quite possible that these results might be due to the location of the graft near the pinch - off region as well as the configuration of the arterial and venous anastomoses overlapping each other . the limitations of our study are the small sample size and the relatively lower primary and secondary patency rates at six month and one year than the results of percutaneous intervention in the usual avg in the upper extremity . other reports that have focused on an avg in the anterior chest wall ( 4 , 5 ) showed that percutaneous intervention was performed in three and five cases and this was successful in 66% ( 2 of 3 ) and 80% ( 4 of 5 ) . but no available data about the primary and secondary patency rates after percutaneous intervention was noted . further experiences with percutaneous intervention in failed alavgs will be needed in a larger population . despite of the small sample size , our study showed that an alavg is an alternative for patients who lost an opportunity for creating an autologous fistula and graft in their forearm and upper arm .

fatty liver is the most common and earliest response of the liver to heavy alcohol consumption and may develop into alcoholic hepatitis and fibrosis . it has been traditionally held that , during alcohol metabolism , the increase in the cellular nadh concentration generated by alcohol and aldehyde dehydrogenases impairs -oxidation and tricarboxylic acid cycle activity in the liver , which in turn leads to severe ffa overload and enhanced synthesis of triacylglycerol in the liver . more recently , two transcription factors , the peroxisome proliferator - activated receptor ( ppar ) and ppar , have been discovered as new mechanisms that control hepatic fatty acid oxidation and synthesis , respectively [ 3 , 4 ] . ethanol feeding impairs fatty acid catabolism in the liver partly by blocking ppar-mediated responses in c57bl/6j mice . in the meantime , dietary supplementation of ethanol - fed animals with a ppar agonist induces the expression of ppar target genes and stimulates the rate of fatty acid -oxidation , which prevents alcoholic fatty liver . in contrast to ppar , hepatic ppar contributes to triglyceride homeostasis , which regulates both triglyceride clearance from the circulation and the lipogenic program . ethanol increases the mrna expressions of ppar and lipid synthetic enzymes , atp citrate lyase ( acl ) , and fatty acid synthase ( fas ) in mice liver . ethanol is well known to stimulate increased extracellular adenosine concentration through its action on the nucleoside transporter . ethanol ingestion increases purine release into the bloodstream and urine in normal volunteers [ 79 ] and into the extracellular space in liver slices obtained from mice [ 10 , 11 ] . extracellular adenosine regulates various physical processes , including hepatic fibrosis [ 10 , 11 ] , ureagenesis [ 13 , 14 ] , and glycogen metabolism [ 15 , 16 ] , as well as peripheral lipid metabolism [ 17 , 18 ] . these physiological effects of adenosine are mediated by a family of four g - protein - coupled receptors : a1 , a2a , a2b , and a3 ( a1r , a2ar , a2br , and a3r ) , each of which has a unique pharmacological profile , tissue distribution , and effector coupling . recently , peng et al . reported that ethanol - mediated increases in extracellular adenosine , which act via adenosine a1r and a2br , link the ingestion and metabolism of ethanol to the development of hepatic steatosis . thus , they suggested that targeting adenosine receptors may be effective in the prevention of alcohol - induced fatty liver . although fatty liver was thought to be relatively benign , more recent studies show that fat accumulation makes the liver more susceptible to injury from other agents such as drugs and toxins , especially endotoxins , which are believed to be involved in the pathogenesis of alcoholic hepatitis and fibrosis [ 21 , 22 ] . alcohol consumption induces a state of a leaky gut that increases plasma and liver endotoxin levels and leads to the production of the tumor necrosis factor ( tnf ) by kupffer cells via the toll - like receptor ( tlr ) 4 , which is known to mediate lipopolysaccharide- ( lps- ) induced signal transduction and to eventually contribute to liver injury . ethanol - fed mice exhibited an oxidative stress that was dependent on the upregulation of multiple tlrs ( tlr1 , 2 , 4 , 6 , 7 , 8 , and 9 ) in the liver and was sensitive to liver inflammation induced by multiple bacterial products recognized by the tlrs . codonopsis lanceolata ( c. lanceolata ) , which belongs to the campanulaceae family , is a perennial herb that grows naturally in moist places in woods , low mountains , and hills . it is commonly found in east asia , particularly in china . c. lanceolata , which is composed of various active components including tannins , saponins , polyphenolics , alkaloids , essential oils , and steroids , has long been prescribed in traditional folk medicine in korea , japan , and china [ 2628 ] . the dried roots of c. lanceolata have been used as a traditional remedy for lung inflammatory diseases such as asthma , tonsillitis , and pharyngitis . the total methanol extracts of the fresh leaves or roots of c. lanceolata significantly suppress the production of tnf and nitric oxide , the expression of interleukin ( il)-3 and il-6 , and lps - mediated phagocytic uptake in raw 264.7 cells . in a rat model , c. lanceolata water extract significantly improved the hepatic accumulation of triglyceride and cholesterol induced by a high - fat diet . although the lipid - lowering effect of c. lanceolata extract has been demonstrated in a rodent model with diet - induced obesity , the protective effects of this plant against alcoholic fatty liver diseases have not yet been explored . in this study , the potent bioactivities of the c. lanceolata methanol extract that can be used as natural compound or pharmaceutical supplements for the prevention and/or treatment of alcoholic fatty liver were demonstrated . the mechanisms by which the c. lanceolata methanol extract performs its antilipogenic and anti - inflammatory actions were investigated in the hepatic tissues of mice with chronic ethanol consumption . c. lanceolata , which is cultivated in gangwon - do , korea , was purchased and freeze dried . freshly dried material was ground into fine powder in an electric grinder and stored in dessicator . 200 g plant powder was refluxed with 95% methyl alcohol ( meoh ) in a round bottom flask on a water bath for 3 h. crude meoh extract was filtered out and evaporated to dryness for the preparation of the c. lanceolata methanol extract ( cme ) . the residual methanol extract was again refluxed with distilled water for 5 h and filtered . the filtrate , thus obtained , was again added with ethanol at the ratio of 1 : 4 and then stirred for 1 h at 4c . following their centrifugation at 5,000 g for 30 min at 4c , the supernatant was concentrated in a rotary vacuum evaporator and freeze dried for the preparation of the c. lanceolata ethanol supernatant ( ces ) , and the pellets were air dried and freeze dried for the preparation of the c. lanceolata ethanol precipitate ( cep ) . the yields of these three extracts were 3.84% ( cme ) , 5.05% ( ces ) , and 3.30% ( cep ) of the wet c. lanceolata root , respectively . specific pathogen - free male c57bl/6n mice ( eight weeks old ) were purchased ( orient , gyeonggi - do , korea ) and acclimatized to the authors ' animal facility for one week prior to the experimentation . the mice were kept individually in sterilized animal quarters with controlled temperature ( at 21 2c ) and humidity ( at 50 5% ) and with 12 h light and dark cycles and were allowed free access to standard chow and tap water during the acclimatization period . the animals were then randomly divided into five groups ( n = 8) and fed the normal diet ( nd ) , the ethanol diet ( ed ) , the cme - supplemented ethanol diet ( med ) , the ces - supplemented ethanol diet ( esd ) , or the cep - supplemented ethanol diet ( epd ) for nine weeks . the mice in the ed group consumed a liquid diet wherein ethanol provided 36% of the energy , as described by lieber et al . . ethanol was introduced into the diet by gradually mixing it with distilled water , from 0% ( wt / v ) to 5% ( wt / v ) , over a one - week period for adaptation , and was provided at 5% ( wt / v ) for the next eight weeks . the nd mice received the same diet but with isocaloric amounts of dextrin - maltose instead of ethanol , and the med , esd , and epd mice were fed the ethanol diet that contained 0.091% ( wt / v ) cme , ces , and cep , respectively . the nd , med , des , and epd mice were pair fed so that their consumption would be isocalorically equivalent to the consumption on the previous day of the ed mice that were individually paired with them . the animals that were used in this study were treated in accordance with the guide for the care and use of laboratory animals ( institute of laboratory animal resources , commission on life sciences , national research council , 1996 ) , as approved by the institutional animal care and use committee of yonsei university . the liver tissue specimens were fixed in 10% buffered formalin and embedded in paraffin , cut at thicknesses of 5 m , and later stained with hematoxylin and eosin ( h&e ) for the histological examination of fat droplets . the serum concentrations of total cholesterol , hdl cholesterol , triglyceride , and free fatty acid were determined using commercial kits ( bioclinical system , gyeonggi - do , korea ) . , using a chloroform - methanol mixture ( 2 : 1 , v / v ) . the dried lipid residues were dissolved in 1 ml of ethanol for the cholesterol and triglyceride measurements . the hepatic cholesterol , triglyceride , and free fatty acid concentrations were analyzed using the same enzymatic kit that was used in the serum analyses . the serum alanine aminotransferase ( alt ) and aspartate aminotransferase ( ast ) activities were measured using commercial reagents ( bayer , usa ) . the total rna was extracted from the liver using a trizol reagent ( invitrogen , usa ) and was reverse transcribed using the superscript ii kit ( invitrogen , usa ) according to the manufacturer 's recommendations . the primers for the pcr analysis were synthesized by bioneer ( korea ) . the forward ( f ) and reverse ( r ) primer sequences for the genes that were involved in the experiment are shown in the supplementary material available online at doi : 10.1155/2012/141395 . the pcr for the glyceraldehyde-3-phosphate dehydrogenase ( gapdh ) was performed on each sample as an internal positive - control standard . the number of cycles and the annealing temperature were optimized for each primer pair . amplification of the gapdh , a1r , a2ar , a2br , a3r , ppar , carnitine palmitoyltransferase ( cpt-1 ) , microsomal triglyceride transfer protein ( mtp ) , long - chain acyl - coa dehydrogenase ( lcad ) , medium - chain acyl - coa dehydrogenase ( mcad ) , ppar , retinoid x receptor ( rxr ) , ccaat - enhancer - binding protein- ( c / ebp ) , lipoprotein lipase ( lpl ) , adipocyte protein 2 or fatty acid binding protein 4 ( ap2 ) , fas , tlrs , lps binding protein ( lbp ) , cluster of differentiation 14 ( cd14 ) , myeloid differentiation primary response gene 88 ( myd88 ) , myelin and lymphocyte protein ( mal ) , tlr adaptor molecule ( trif ) , tnf - receptor - associated factor 6 ( traf6 ) , interferon regulatory factors ( irfs ) , nuclear factor kappa b - p50 ( p50 ) , p65 , interferon ( ifn ) , ifn , il-12-p40 , chemokine ( c - x - c motif ) ligand 2 ( cxcl2 ) , adiponectin receptor 1 ( adipor1 ) , adipor2 , silent - mating - type information regulation 2 homolog 1 ( sirt1 ) , peroxisome proliferative activated receptor gamma coactivator 1 alpha ( pgc1 ) , and sterol regulatory element binding transcription factor 1 ( srebp-1c ) was initiated via 5 min of denaturation at 94c for 1 cycle , followed by 30 or 35 cycles at 94c for 30 s , 55 or 60c for 30 s , and 72c for 1 min and via 10 min of incubation at 72c . the sequences of the primer pairs and pcr conditions ( annealing temperatures , cycles , and product sizes ) that were used in the rt - pcr experiments are shown in the supplementary material . the pcr products were then separated in a 2% agarose gel and visualized in a gel documentation system ( alpha innotech , usa ) . the intensity of the bands on the gels was converted into a digital image with a gel analyzer . for the western blot studies , protein extracts were obtained from the mice livers using a commercial lysis buffer ( intron , usa ) that contained a protease inhibitor cocktail ( roche , usa ) . the protein concentrations were determined with the bio - rad protein assay kit ( bio - rad , usa ) . the western blot analysis was performed with antibodies that were specific to amp - activated protein kinase ( ampk ) , phospho - ampk ( thr 72 ) , acetyl - coa carboxylase ( acc ) , phospho - acc ( ser 79 ) ( cell signaling , usa ) , and -actin ( santa cruz , usa ) . after the sds - page at 10% acrylamide concentrations , the proteins were transferred to the nitrocellulose membrane , blocked with 5% skim milk in a tris - buffered saline / tween buffer ( 10 mm tris - hcl , ph 7.5 , 150 mm nacl , and 0.05% tween 20 ) , and incubated with appropriate antibodies overnight . the blots were washed extensively , incubated with a horseradish - peroxidase - linked anti - rabbit or -mouse ig ( santa cruz , usa ) , and washed again . the detection was carried out with the ecl western blotting kit ( animal genetics , korea ) , after which the blots were exposed to an x - ray film ( agfa , belgium ) . the results of the body weight gain , liver weight , and serum and hepatic biochemistries were expressed as the mean values sem of the eight mice in each group . the results of the semiquantitative rt - pcr and western blot were expressed as the mean values sem of the three independent experiments in which the rna and protein samples from the eight mice were used , respectively . the analysis of the variance ( anova ) and the duncan 's multiple range method were used to compare significant differences among the groups . the level of significance was set at p < 0.05 for all the statistical tests . the nine - week body weight gain of the mice in the ed group was 51% lower than the value for the pair fed nd mice . the animals that were fed the med ( 43% greater ) or the esd ( 41% greater ) exhibited significantly greater body weight gains than the ed mice , whereas the mice that were fed the epd did not recover the weight they lost from ethanol consumption ( table 2 ) . compared to the values for the ed mice , the mice that were fed the med exhibited significantly lower levels of serum ffa ( 17% reduction ) and lower activities of serum alt ( 17% reduction ) and ast ( 22% reduction ) . they also demonstrated a tendency towards decreasing serum triglyceride , total cholesterol , vldl + ldl cholesterol , and atherogenic indices , albeit at statistically insignificant levels than those for the mice that were fed the ed . in contrast , the esd or epd failed to improve both the lipid levels and enzyme activities in the blood of the mice with chronic ethanol consumption ( table 2 ) . histological analysis of the livers with h&e staining revealed prominent lipid accumulation in the livers of the ed fed mice whereas lipid droplets were rare in the livers of the med , esd , and epd - fed mice ( figure 1 ) . ed feeding of the mice for nine weeks resulted in more significant increases in the relative weight of their livers ( by 8% ) and in the hepatic levels of their triglycerides ( 43% increase ) , cholesterol ( 36% increase ) , and ffa ( 52% increase ) than in the nd group ( figure 1 ) . the med or esd significantly improved the ethanol - induced enlargement of the liver ( the relative liver weight was 7% or 6% lower , resp . ) . the med , esd , or epd significantly reversed the ethanol - induced hepatic accumulations of triglycerides ( 18% , 11% , and 18% reductions , resp . ) , cholesterol ( 22% , 12% , and 18% reductions , resp . ) , and ffa ( 29% , 14% , and 22% reductions , resp . ) . the triglyceride and cholesterol levels in the liver were lowest after the med consumption among the diets that were supplemented with different fractions of the c. lanceolata root , but the differences between the diets were not statistically significant . the med group exhibited the most prominent and the most significant improvement in the reduction of the hepatic ffa accumulation , better than did the esd and epd groups ( p < 0.05 ) ( figure 1 ) . med feeding significantly reversed the ethanol - induced upregulation of the a1r and a2br genes in the livers of the mice and restored the ethanol - induced downregulation of the ppar , cpt-1 , mtp , lcad , and mcad genes in the livers of the mice ( figures 2(a ) and 2(b ) ) . the supplementation of the ethanol diet with cme significantly blunted the upregulation of these transcription factors and of their target genes due to ethanol . chronic ethanol ingestion led to more significant decreases in the hepatic expression of the adipor1 and adipor2 genes than in the nd mice , and med feeding significantly restored these ethanol - induced changes in the adiponectin receptor genes . similarly , the med feeding significantly reversed the marked decreases in the mrna levels of the hepatic sirt1 and pgc1 , which were induced by ethanol feeding . in addition , the med efficiently counteracted the ethanol - induced upregulation of the srebp-1c gene ( higher 57% reduction than in the ed mice , p < 0.05 ) ( figure 3 ) . the med significantly blunted the downregulation of the phospho - ampk / total ampk ( 146% increase ) ratio and of the phospho - acc / total acc ratio ( 700% increase ) , which were caused by chronic ethanol ingestion , in the livers of the mice ( figures 3(b ) and 3(c ) ) . the hepatic expression of the tlr mrnas was measured after nine weeks of ethanol exposure . more significant increases in the tlr1 , tlr2 , tlr3 , tlr4 , tlr6 , tlr7 , tlr8 , tlr9 , tlr12 , and tlr13 mrna levels were observed in the livers of the ethanol - fed control mice than of the pair fed nd mice . the med significantly alleviated the ethanol - induced upregulation of the hepatic tlr1 , tlr2 , tlr4 , tlr6 , tlr7 , tlr8 , tlr9 , tlr12 , and tlr13 gene expressions . the hepatic tlr3 , which was upregulated by the ethanol feeding , and the hepatic tlr5 , which was unaffected by the ethanol feeding , did not respond to the med feeding ( figure 4 ) . after endotoxin bound itself to the lps - binding protein ( lbp ) , it associates in the portal blood with the cd14 prior to its binding to tlr4 and all the tlrs that were recruited by myd88 , mal , trif , and traf6 for their proinflammatory signaling . the mrna levels of the lbp and cd14 , as well as of the adaptor molecules of the tlrs ( myd88 , mal , trif , and traf6 ) , all more significantly increased in the livers of the ed mice than of the nd mice , whereas the med feeding led to more significant decreases in the hepatic mrna levels of the lbp , cd14 , myd88 , mal , trif , and traf6 than in the ed mice ( figure 5(a ) ) . among the extremely significant recent discoveries on tlr signaling is that the myd88-dependent pathway also activates some irfs , such as irf1 , 3 , 5 , and 7 . in this study , the mrna levels of the irf1 , 3 , 5 , and 7 genes more significantly increased in the livers of the ed mice than of the nd mice and these ethanol - induced upregulations of the hepatic irfs were significantly reversed by the med feeding ( figure 5(b ) ) . the rt - pcr analysis of the hepatic mrnas of nfb ( p50 and p65 ) and of its target cytokine genes such as tnf , il-6 , ifn , ifn , il-12-p40 , and cxcl2 was performed next . the med feeding significantly downregulated the expressions of the p50 , p65 , tnf , il-6 , ifn , ifn , il-12-p40 , and cxcl2 genes compared to the ed mice ( figure 5(c ) ) . the authors ' previous study showed that the supplementation of the lieber - decarli ethanol diet with c. lanceolata water extract ( 5 mg / l of a liquid diet ) showed a significant improvement in the amount of weight that was gained and in the hepatic lipid levels ( in the press ) . in this study , the effects of three different fractions of c. lanceolata roots , which were prepared using different solvents , on the protection from alcoholic fatty liver were tested . several studies have demonstrated that the protective effects of various plant extracts from alcoholic liver injury in animals , such as of fenugreek seed ( trigonella foenum fraecum ) methanol extract , and hot water extracts of avaram leaves ( cassia auriculata ) and green tea ( camellia sinensis ) were manifested at dosages of between 200 and 500 mg / kg of body weight . based on these studies , c. lanceolata root fractions were added to a liquid diet at 0.091% ( wt / v ) , which is equivalent to a daily intake of 300 mg / kg of body weight , assuming that a mouse weighs 30 g and consumes 10 ml of a liquid diet per day . among the three different fractions that were prepared from the c. lanceolata root , cme exhibited the most remarkable attenuation of alcohol - induced fatty liver in terms of various parameters . for example , cme more significantly reduced the serum alt and ast activities and the hepatic ffa concentration in the mice that were fed ethanol than did ces or cep . besides , alcohol - induced body weight loss and hepatic accumulations of triglycerides and cholesterol more significantly improved in mice that were supplemented with cme than in animals that were supplemented with esd or epd , although the difference was statistically insignificant . therefore , cme appeared to be the most potent , among the fractions that were obtained from the c. lanceolata root , in protecting animals from alcohol - induced hepatic accumulation of lipids . further investigated were the underlying mechanisms of cme against hepatic steatosis induced by chronic ethanol administration . the ethanol - induced elevation of the plasma hdl cholesterol concentration , as observed in the current study , has been suggested as a cardioprotective response in animals chronically loaded with ethanol . the elevated hdl concentration and the reduced ldl concentration , which are characteristic of the lipoprotein pattern in chronic alcoholics , could well explain the reduced risk of coronary heart disease in alcoholics . in this present study , the mrna expression of the hepatic a2br and a1r was markedly elevated by chronic ethanol feeding . furthermore , the expression of ppar and of its target genes , such as cpt-1 , mtp , lcad , and mcad , all significantly decreased , whereas the expression of ppar and of its target genes , such as rxr , c / ebp , lpl , ap2 , and fas , all significantly increased in the livers of the ethanol - fed ed mice than of the nd mice ( figure 6 ) . these results are in accordance with recent findings from experiments with mice that adenosine generated by ethanol metabolism plays an important role in ethanol - induced hepatic steatosis via a1r and a2br , which leads to the upregulation of ppar and the downregulation of ppar , respectively . it was observed that cme supplementation reversed ethanol - induced elevation of the expressions of a2br , a1r , ppar , and their target genes in the liver tissues of mice . these results suggest that cme ameliorates hepatic steatosis in mice , at least partly by modulating adenosine - receptor - mediated signaling molecules that are responsible for fatty acid oxidation and lipogenesis . adiponectin stimulates hepatic ampk , which , in turn , phosphorylates acc on ser-79 and attenuates acc activity . inhibition of acc directly reduces lipid synthesis and indirectly enhances fatty acid oxidation by blocking the production of malonyl - coa , an allosteric inhibitor of cpt-1 . activation of ampk by adiponectin in the liver also leads to decreased mrna and protein expression of srebp-1c [ 3841 ] , which results in decreased hepatic lipid synthesis . adiponectin is also known to stimulate the activities of both pgc1 and ppar , which mainly control the transcription of a panel of genes that encode fatty acid oxidation enzymes [ 42 , 43 ] . as a result , adiponectin - mediated ampk activation favors lipid catabolism and opposes lipid deposition in the liver [ 4449 ] . chronic ethanol administration is known to significantly decrease the plasma adiponectin level in mice . in this study , more significant decreases in the mrna levels of adipor1 and adipor2 were observed in the livers of the ed mice than of the pair fed nd mice . these alcohol - induced reductions in the hepatic adipors expression appear to have been associated with the subsequent decreases in the phosphorylation of ampk and acc as their phosphorylations in the liver more significantly decreased in the ed mice than in the nd mice ( figure 6 ) . besides , the hepatic mrna levels of sirt1 , pgc1 , srebp-1c , ppar , and cpt1 were also more significantly reduced in the ed mice than in the nd mice ( figure 6 ) . sirt1 has been gaining recognition as one of the critical agents in the mediation of adiponectin signaling to ampk , which is the central mechanism in the regulation of lipid metabolism [ 5255 ] . although pgc1 was initially identified as a coactivator of ppar , it has subsequently been shown to serve as a cofactor of several other transcription factors , including ppar [ 56 , 57 ] . treatment with adiponectin restored the ethanol - inhibited pgc1/ppar activity in cultured hepatic cells and in animal livers , which suggests that the stimulation of adiponectin - sirt1 signaling may serve as an effective therapeutic strategy for treating or preventing human alcoholic fatty liver [ 3840 ] . in this study , cme significantly restored the ethanol - induced downregulation of adiponectin - mediated signaling molecules , including adipor1 , adipor2 , pampk / ampk , pacc / acc , sirt1 , and pgc1 , which led to increased fatty acid oxidation and decreased lipogenesis in the livers of the mice . it was recently shown that innate immune cells recognize conserved pathogen - associated molecular patterns through pattern recognition receptors , among which the family of tlrs occupies an important place . lbp , which is present in normal serum , recognizes and binds to lps with high affinity through its lipid moiety [ 58 , 59 ] . lps - lbp complexes then activate cells through the second glycoprotein , the membrane - bound cd14 , to produce inflammatory mediators [ 6062 ] in the presence of only a functional tlr4 . although lps was not detected in the blood , more significant increases in the mrna levels of lbp and cd14 were observed in the livers of the ed mice than of the nd mice ( figure 6 ) . tlrs have a tir domain that initiates the signaling cascade through tir adapters , such as myd88 , mal [ 65 , 66 ] , and trif , which interact with traf6 , the downstream adaptor . traf6 activates the irfs , which leads to nf-b activation and induction of the expressions of proinflammatory cytokines , such as of tnf , il-6 , ifn , and ifn. it was demonstrated that chronic ethanol feeding induces hepatic steatosis and clear upregulation of tlr1 , 2 , 3 , 4 , 6 , 7 , 8 , 9 , 12 , and 13 in the liver . these findings are supported by previous reports that ethanol - fed mice exhibited hepatic inflammation that was dependent on the upregulation of multiple tlrs in the liver . the expressions of the myd88 , mal , trif , traf6 , irf1 , 3 , 5 , and 7 genes , along with the mrna levels of nfb and proinflammatory cytokines , were all more significantly elevated in the livers of the ed mice than of the nd mice ( figure 6 ) . these results are also in accordance with previous observations that myd88 is required for the development of fibrosis , a predisposing condition for hepatocellular cancer development , and that irf7 expression more significantly increased in the livers of alcohol - fed myd88-deficient mice than of pair fed control mice . these results suggest that chronic ethanol intake may activate tlr - mediated proinflammatory signaling cascades . the study data show that these hepatic inductions of tlr - mediated signaling cascade molecules , which involve both myd88-dependent and -independent pathways , along with their target proinflammatory cytokine genes , such as tnf , il-6 , ifn , ifn , il-12-p40 , and cscl2 , due to ethanol feeding were all dramatically reversed with cme supplementation . taken together , these beneficial effects of cme against alcoholic fatty livers in mice appear to have occurred with adenosine- and adiponectin - mediated regulations of hepatic steatosis and tlr - mediated modulation of hepatic proinflammatory responses , since ethanol - induced changes in the expression of the molecules that are involved in these three signaling pathways were all significantly reversed with cme feeding . although alcoholic fatty liver is a major risk factor for advanced liver injuries such as steatohepatitis , fibrosis , and cirrhosis , previous studies have mostly focused on the antilipogenic or -inflammatory activity alone , but not in combination , of a compound and extracts . evaluated the effect of the cassia auriculata leaf extract on lipid metabolism in alcohol - induced hepatic steatosis . resveratrol prevents the development of hepatic steatosis induced by alcohol , by restoring the inhibited hepatic sirt1-ampk signaling system . this study demonstrated that both lipogenesis and inflammation are associated with the development of alcoholic fatty liver and that cme has both antilipogenic and anti - inflammatory effects through coordinated multiple signaling pathways . although much further study is required concerning the exact identities of the chemical constituents of the c. lanceolata methanol extract that are responsible for the findings described herein , the results of this study provide some insights on the basic mechanism that underlies the therapeutic effect of the extract on mice hepatic tissues after chronic ethanol feeding .

this study evaluated the antilipogenic and anti - inflammatory effects of codonopsis lanceolata ( c. lanceolata ) root extract in mice with alcohol - induced fatty liver and elucidated its underlying molecular mechanisms . ethanol was introduced into the liquid diet by mixing it with distilled water at 5% ( wt / v ) , providing 36% of the energy , for nine weeks . among the three different fractions prepared from the c. lanceolata root , the c. lanceolata methanol extract ( cme ) exhibited the most remarkable attenuation of alcohol - induced fatty liver with respect to various parameters such as hepatic free fatty acid concentration , body weight loss , and hepatic accumulations of triglyceride and cholesterol . the hepatic gene and protein expression levels were analysed via rt - pcr and western blotting , respectively . cme feeding significantly restored the ethanol - induced downregulation of the adiponectin receptor ( adipor ) 1 and of adipor2 , along with their downstream molecules . furthermore , the study data showed that cme feeding dramatically reversed ethanol - induced hepatic upregulation of toll - like receptor- ( tlr- ) mediated signaling cascade molecules . these results indicate that the beneficial effects of cme against alcoholic fatty livers of mice appear to be with adenosine- and adiponectin - mediated regulation of hepatic steatosis and tlr - mediated modulation of hepatic proinflammatory responses .

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

more recently , two transcription factors , the peroxisome proliferator - activated receptor ( ppar ) and ppar , have been discovered as new mechanisms that control hepatic fatty acid oxidation and synthesis , respectively [ 3 , 4 ] . in the meantime , dietary supplementation of ethanol - fed animals with a ppar agonist induces the expression of ppar target genes and stimulates the rate of fatty acid -oxidation , which prevents alcoholic fatty liver . ethanol increases the mrna expressions of ppar and lipid synthetic enzymes , atp citrate lyase ( acl ) , and fatty acid synthase ( fas ) in mice liver . reported that ethanol - mediated increases in extracellular adenosine , which act via adenosine a1r and a2br , link the ingestion and metabolism of ethanol to the development of hepatic steatosis . thus , they suggested that targeting adenosine receptors may be effective in the prevention of alcohol - induced fatty liver . alcohol consumption induces a state of a leaky gut that increases plasma and liver endotoxin levels and leads to the production of the tumor necrosis factor ( tnf ) by kupffer cells via the toll - like receptor ( tlr ) 4 , which is known to mediate lipopolysaccharide- ( lps- ) induced signal transduction and to eventually contribute to liver injury . ethanol - fed mice exhibited an oxidative stress that was dependent on the upregulation of multiple tlrs ( tlr1 , 2 , 4 , 6 , 7 , 8 , and 9 ) in the liver and was sensitive to liver inflammation induced by multiple bacterial products recognized by the tlrs . codonopsis lanceolata ( c. lanceolata ) , which belongs to the campanulaceae family , is a perennial herb that grows naturally in moist places in woods , low mountains , and hills . the total methanol extracts of the fresh leaves or roots of c. lanceolata significantly suppress the production of tnf and nitric oxide , the expression of interleukin ( il)-3 and il-6 , and lps - mediated phagocytic uptake in raw 264.7 cells . in a rat model , c. lanceolata water extract significantly improved the hepatic accumulation of triglyceride and cholesterol induced by a high - fat diet . although the lipid - lowering effect of c. lanceolata extract has been demonstrated in a rodent model with diet - induced obesity , the protective effects of this plant against alcoholic fatty liver diseases have not yet been explored . in this study , the potent bioactivities of the c. lanceolata methanol extract that can be used as natural compound or pharmaceutical supplements for the prevention and/or treatment of alcoholic fatty liver were demonstrated . the mechanisms by which the c. lanceolata methanol extract performs its antilipogenic and anti - inflammatory actions were investigated in the hepatic tissues of mice with chronic ethanol consumption . 200 g plant powder was refluxed with 95% methyl alcohol ( meoh ) in a round bottom flask on a water bath for 3 h. crude meoh extract was filtered out and evaporated to dryness for the preparation of the c. lanceolata methanol extract ( cme ) . following their centrifugation at 5,000 g for 30 min at 4c , the supernatant was concentrated in a rotary vacuum evaporator and freeze dried for the preparation of the c. lanceolata ethanol supernatant ( ces ) , and the pellets were air dried and freeze dried for the preparation of the c. lanceolata ethanol precipitate ( cep ) . the yields of these three extracts were 3.84% ( cme ) , 5.05% ( ces ) , and 3.30% ( cep ) of the wet c. lanceolata root , respectively . the animals were then randomly divided into five groups ( n = 8) and fed the normal diet ( nd ) , the ethanol diet ( ed ) , the cme - supplemented ethanol diet ( med ) , the ces - supplemented ethanol diet ( esd ) , or the cep - supplemented ethanol diet ( epd ) for nine weeks . the mice in the ed group consumed a liquid diet wherein ethanol provided 36% of the energy , as described by lieber et al . ethanol was introduced into the diet by gradually mixing it with distilled water , from 0% ( wt / v ) to 5% ( wt / v ) , over a one - week period for adaptation , and was provided at 5% ( wt / v ) for the next eight weeks . the nd mice received the same diet but with isocaloric amounts of dextrin - maltose instead of ethanol , and the med , esd , and epd mice were fed the ethanol diet that contained 0.091% ( wt / v ) cme , ces , and cep , respectively . the hepatic cholesterol , triglyceride , and free fatty acid concentrations were analyzed using the same enzymatic kit that was used in the serum analyses . amplification of the gapdh , a1r , a2ar , a2br , a3r , ppar , carnitine palmitoyltransferase ( cpt-1 ) , microsomal triglyceride transfer protein ( mtp ) , long - chain acyl - coa dehydrogenase ( lcad ) , medium - chain acyl - coa dehydrogenase ( mcad ) , ppar , retinoid x receptor ( rxr ) , ccaat - enhancer - binding protein- ( c / ebp ) , lipoprotein lipase ( lpl ) , adipocyte protein 2 or fatty acid binding protein 4 ( ap2 ) , fas , tlrs , lps binding protein ( lbp ) , cluster of differentiation 14 ( cd14 ) , myeloid differentiation primary response gene 88 ( myd88 ) , myelin and lymphocyte protein ( mal ) , tlr adaptor molecule ( trif ) , tnf - receptor - associated factor 6 ( traf6 ) , interferon regulatory factors ( irfs ) , nuclear factor kappa b - p50 ( p50 ) , p65 , interferon ( ifn ) , ifn , il-12-p40 , chemokine ( c - x - c motif ) ligand 2 ( cxcl2 ) , adiponectin receptor 1 ( adipor1 ) , adipor2 , silent - mating - type information regulation 2 homolog 1 ( sirt1 ) , peroxisome proliferative activated receptor gamma coactivator 1 alpha ( pgc1 ) , and sterol regulatory element binding transcription factor 1 ( srebp-1c ) was initiated via 5 min of denaturation at 94c for 1 cycle , followed by 30 or 35 cycles at 94c for 30 s , 55 or 60c for 30 s , and 72c for 1 min and via 10 min of incubation at 72c . the sequences of the primer pairs and pcr conditions ( annealing temperatures , cycles , and product sizes ) that were used in the rt - pcr experiments are shown in the supplementary material . the results of the body weight gain , liver weight , and serum and hepatic biochemistries were expressed as the mean values sem of the eight mice in each group . the results of the semiquantitative rt - pcr and western blot were expressed as the mean values sem of the three independent experiments in which the rna and protein samples from the eight mice were used , respectively . in contrast , the esd or epd failed to improve both the lipid levels and enzyme activities in the blood of the mice with chronic ethanol consumption ( table 2 ) . ed feeding of the mice for nine weeks resulted in more significant increases in the relative weight of their livers ( by 8% ) and in the hepatic levels of their triglycerides ( 43% increase ) , cholesterol ( 36% increase ) , and ffa ( 52% increase ) than in the nd group ( figure 1 ) . the med or esd significantly improved the ethanol - induced enlargement of the liver ( the relative liver weight was 7% or 6% lower , resp . ) the med , esd , or epd significantly reversed the ethanol - induced hepatic accumulations of triglycerides ( 18% , 11% , and 18% reductions , resp . ) the triglyceride and cholesterol levels in the liver were lowest after the med consumption among the diets that were supplemented with different fractions of the c. lanceolata root , but the differences between the diets were not statistically significant . the med group exhibited the most prominent and the most significant improvement in the reduction of the hepatic ffa accumulation , better than did the esd and epd groups ( p < 0.05 ) ( figure 1 ) . med feeding significantly reversed the ethanol - induced upregulation of the a1r and a2br genes in the livers of the mice and restored the ethanol - induced downregulation of the ppar , cpt-1 , mtp , lcad , and mcad genes in the livers of the mice ( figures 2(a ) and 2(b ) ) . the supplementation of the ethanol diet with cme significantly blunted the upregulation of these transcription factors and of their target genes due to ethanol . chronic ethanol ingestion led to more significant decreases in the hepatic expression of the adipor1 and adipor2 genes than in the nd mice , and med feeding significantly restored these ethanol - induced changes in the adiponectin receptor genes . similarly , the med feeding significantly reversed the marked decreases in the mrna levels of the hepatic sirt1 and pgc1 , which were induced by ethanol feeding . in addition , the med efficiently counteracted the ethanol - induced upregulation of the srebp-1c gene ( higher 57% reduction than in the ed mice , p < 0.05 ) ( figure 3 ) . the med significantly blunted the downregulation of the phospho - ampk / total ampk ( 146% increase ) ratio and of the phospho - acc / total acc ratio ( 700% increase ) , which were caused by chronic ethanol ingestion , in the livers of the mice ( figures 3(b ) and 3(c ) ) . more significant increases in the tlr1 , tlr2 , tlr3 , tlr4 , tlr6 , tlr7 , tlr8 , tlr9 , tlr12 , and tlr13 mrna levels were observed in the livers of the ethanol - fed control mice than of the pair fed nd mice . the med significantly alleviated the ethanol - induced upregulation of the hepatic tlr1 , tlr2 , tlr4 , tlr6 , tlr7 , tlr8 , tlr9 , tlr12 , and tlr13 gene expressions . the mrna levels of the lbp and cd14 , as well as of the adaptor molecules of the tlrs ( myd88 , mal , trif , and traf6 ) , all more significantly increased in the livers of the ed mice than of the nd mice , whereas the med feeding led to more significant decreases in the hepatic mrna levels of the lbp , cd14 , myd88 , mal , trif , and traf6 than in the ed mice ( figure 5(a ) ) . among the extremely significant recent discoveries on tlr signaling is that the myd88-dependent pathway also activates some irfs , such as irf1 , 3 , 5 , and 7 . in this study , the mrna levels of the irf1 , 3 , 5 , and 7 genes more significantly increased in the livers of the ed mice than of the nd mice and these ethanol - induced upregulations of the hepatic irfs were significantly reversed by the med feeding ( figure 5(b ) ) . the rt - pcr analysis of the hepatic mrnas of nfb ( p50 and p65 ) and of its target cytokine genes such as tnf , il-6 , ifn , ifn , il-12-p40 , and cxcl2 was performed next . the authors ' previous study showed that the supplementation of the lieber - decarli ethanol diet with c. lanceolata water extract ( 5 mg / l of a liquid diet ) showed a significant improvement in the amount of weight that was gained and in the hepatic lipid levels ( in the press ) . in this study , the effects of three different fractions of c. lanceolata roots , which were prepared using different solvents , on the protection from alcoholic fatty liver were tested . several studies have demonstrated that the protective effects of various plant extracts from alcoholic liver injury in animals , such as of fenugreek seed ( trigonella foenum fraecum ) methanol extract , and hot water extracts of avaram leaves ( cassia auriculata ) and green tea ( camellia sinensis ) were manifested at dosages of between 200 and 500 mg / kg of body weight . based on these studies , c. lanceolata root fractions were added to a liquid diet at 0.091% ( wt / v ) , which is equivalent to a daily intake of 300 mg / kg of body weight , assuming that a mouse weighs 30 g and consumes 10 ml of a liquid diet per day . among the three different fractions that were prepared from the c. lanceolata root , cme exhibited the most remarkable attenuation of alcohol - induced fatty liver in terms of various parameters . besides , alcohol - induced body weight loss and hepatic accumulations of triglycerides and cholesterol more significantly improved in mice that were supplemented with cme than in animals that were supplemented with esd or epd , although the difference was statistically insignificant . therefore , cme appeared to be the most potent , among the fractions that were obtained from the c. lanceolata root , in protecting animals from alcohol - induced hepatic accumulation of lipids . the ethanol - induced elevation of the plasma hdl cholesterol concentration , as observed in the current study , has been suggested as a cardioprotective response in animals chronically loaded with ethanol . furthermore , the expression of ppar and of its target genes , such as cpt-1 , mtp , lcad , and mcad , all significantly decreased , whereas the expression of ppar and of its target genes , such as rxr , c / ebp , lpl , ap2 , and fas , all significantly increased in the livers of the ethanol - fed ed mice than of the nd mice ( figure 6 ) . these results are in accordance with recent findings from experiments with mice that adenosine generated by ethanol metabolism plays an important role in ethanol - induced hepatic steatosis via a1r and a2br , which leads to the upregulation of ppar and the downregulation of ppar , respectively . it was observed that cme supplementation reversed ethanol - induced elevation of the expressions of a2br , a1r , ppar , and their target genes in the liver tissues of mice . these results suggest that cme ameliorates hepatic steatosis in mice , at least partly by modulating adenosine - receptor - mediated signaling molecules that are responsible for fatty acid oxidation and lipogenesis . these alcohol - induced reductions in the hepatic adipors expression appear to have been associated with the subsequent decreases in the phosphorylation of ampk and acc as their phosphorylations in the liver more significantly decreased in the ed mice than in the nd mice ( figure 6 ) . besides , the hepatic mrna levels of sirt1 , pgc1 , srebp-1c , ppar , and cpt1 were also more significantly reduced in the ed mice than in the nd mice ( figure 6 ) . treatment with adiponectin restored the ethanol - inhibited pgc1/ppar activity in cultured hepatic cells and in animal livers , which suggests that the stimulation of adiponectin - sirt1 signaling may serve as an effective therapeutic strategy for treating or preventing human alcoholic fatty liver [ 3840 ] . in this study , cme significantly restored the ethanol - induced downregulation of adiponectin - mediated signaling molecules , including adipor1 , adipor2 , pampk / ampk , pacc / acc , sirt1 , and pgc1 , which led to increased fatty acid oxidation and decreased lipogenesis in the livers of the mice . tlrs have a tir domain that initiates the signaling cascade through tir adapters , such as myd88 , mal [ 65 , 66 ] , and trif , which interact with traf6 , the downstream adaptor . traf6 activates the irfs , which leads to nf-b activation and induction of the expressions of proinflammatory cytokines , such as of tnf , il-6 , ifn , and ifn. it was demonstrated that chronic ethanol feeding induces hepatic steatosis and clear upregulation of tlr1 , 2 , 3 , 4 , 6 , 7 , 8 , 9 , 12 , and 13 in the liver . the expressions of the myd88 , mal , trif , traf6 , irf1 , 3 , 5 , and 7 genes , along with the mrna levels of nfb and proinflammatory cytokines , were all more significantly elevated in the livers of the ed mice than of the nd mice ( figure 6 ) . these results are also in accordance with previous observations that myd88 is required for the development of fibrosis , a predisposing condition for hepatocellular cancer development , and that irf7 expression more significantly increased in the livers of alcohol - fed myd88-deficient mice than of pair fed control mice . the study data show that these hepatic inductions of tlr - mediated signaling cascade molecules , which involve both myd88-dependent and -independent pathways , along with their target proinflammatory cytokine genes , such as tnf , il-6 , ifn , ifn , il-12-p40 , and cscl2 , due to ethanol feeding were all dramatically reversed with cme supplementation . taken together , these beneficial effects of cme against alcoholic fatty livers in mice appear to have occurred with adenosine- and adiponectin - mediated regulations of hepatic steatosis and tlr - mediated modulation of hepatic proinflammatory responses , since ethanol - induced changes in the expression of the molecules that are involved in these three signaling pathways were all significantly reversed with cme feeding . although alcoholic fatty liver is a major risk factor for advanced liver injuries such as steatohepatitis , fibrosis , and cirrhosis , previous studies have mostly focused on the antilipogenic or -inflammatory activity alone , but not in combination , of a compound and extracts . evaluated the effect of the cassia auriculata leaf extract on lipid metabolism in alcohol - induced hepatic steatosis . this study demonstrated that both lipogenesis and inflammation are associated with the development of alcoholic fatty liver and that cme has both antilipogenic and anti - inflammatory effects through coordinated multiple signaling pathways . although much further study is required concerning the exact identities of the chemical constituents of the c. lanceolata methanol extract that are responsible for the findings described herein , the results of this study provide some insights on the basic mechanism that underlies the therapeutic effect of the extract on mice hepatic tissues after chronic ethanol feeding .

the ant genus mycetagroicus , described in 2001 , is a member of the ant tribe attini , all of which are obligate agriculturalists and fungivores . in establishing the genus , brando and mayh - nunes ( 2001 ) included three species : m. cerradensis , m. triangularis , and m. urbanus . in 2008 , the same authors described a fourth species , m. inflatus , based on two workers collected by r. feitosa and r. rosa da silva in southern par , brazil , in 2005 ( brando and mayh - nunes , 2008 ) . the descriptions of three of the four mycetagroicus species were based solely on workers . for m. triangularis , a single specimen of the gyne was described . until now mycetagroicus occupies a critical position in the phylogeny of the attini ( schultz and brady , 2008 ) . it is the sister group of the so - called higher attini ( a group containing the genera trachymyrmex , sericomyrmex , acromyrmex , and atta ) which cultivate a clade of obligately symbiotic fungal species that share a lower attine fungal ancestor , all of which are highly specialized for symbiosis with their ant hosts ( mueller and gerardo , 2002 ; schultz et al . , 2005 ; mikheyev et al . , 2006 , 2007 ; schultz and brady , 2008 ) . based on worker morphology , brando and mayh - nunes ( 2008 ) suggested that the genus might be a member of the higher attini , but solomon et al . ( 2011 ) , based on a molecular analysis of the cultivar , found that m. cerradensis is in fact a member of the lower attini , i.e. , it is associated with a lower attine cultivar . since the genus is the sister group of the higher attines , study of mycetagroicus species can potentially provide insights regarding the origin of higher attine agriculture , arguably the most ecologically significant transition in the evolutionary history of the attini . during the past few decades , new natural history information has required periodic , sometimes dramatic revisions of our model of the complex co - evolutionary relationships among fungus - growing ants , their fungal cultivars , and other microbial symbionts ( chapela et al . , 1994 ; mueller et al . , 1998 ; currie et al . , 1999 ; mikheyev et al . , 2006 , 2007 ; mehdiabadi and schultz , 2010 ; mueller et al . , 2011a ; mehdiabadi et al . , nest architecture has been studied from an evolutionary perspective in a number of different organisms , especially in birds , termites , and social hymenoptera . in some studies , nest characters were included in a phylogenetic data matrix and analyzed along with other character data to produce a phylogeny ( lanyon , 1986 ; wenzel , 1998 ; zyskowski and prum 1999 ) , whereas in other studies , nest characters were mapped onto pre - existing phylogenies to test whether nest traits were correlated with phylogenies obtained from non - nest - related character information ( packer , 1991 ; rasmussen and camargo , 2008 ) . a study of termites in the genus apicotermes holmgren , 1912 included a key to the species based solely on nest morphology ( schmidt , 1955 ) , concluding that termite nests contain more phylogenetic information than the termites themselves . in ants , studies of underground nests ( tschinkel , 2003 ) and of nest entrance morphology ( schultz et al . , 2002 ) have revealed patterns in structure and morphology that are species - specific . at deeper phylogenetic levels , however , due to convergence and parallelism nest architecture characters are often homoplastic , arising multiple times on the ant tree of life ( tschinkel , 2011 ) . nest architecture in fungus - growing ants is important not only for the ants but also for their fungal cultivars , which require particular environmental conditions ( bollazzi and roces 2002 ; bollazzi et al . nest architecture , therefore , is a significant aspect of attine ant biology , and the importance of documenting attine nest life histories has been recognized by multiple researchers ( weber , 1972 ; solomon et al . , 2004 , 2011 ; moreira et al . , 2004 ; fernndez - marn et al . 2005 ; diehl - fleig and diehl , 2007 ; klingenberg et al . mehdiabadi and schultz , 2010 ; ramos - lacau et al . , 2012 ) . in addition to describing nest architecture , we identify the fungal cultivar of mycetagroicus inflatus . we describe the previously unknown queen , provide the first description of a male for the genus , and compare both to reproductive forms of other , closely related attine genera . field work was conducted from 5 to 10 october 2012 in eastern par state , brazil , at the end of the dry season . during this time period all nests were found at two localities on the west coast of the araguaia river , a southern component of the amazon river basin , across from the town of araguacema , tocantins , in an area characterized as dense alluvial forest ( s 08.75387 , w 49.54544 , elevation 164 m ) , nest entrances occurred approximately 15 m from the river s edge in an area about 7 m above water level , which was at its seasonal low point ; at locality 2 ( s 08.80157 , w 49.57368 , elevation 176 m ) , they occurred approximately 5 m away from the river s edge and about 5 m above the water level . at both localities the beach area is sandy , with occasional low bushes , and banks are covered with gallery forest . based on information from local inhabitants , both locality 1 and locality 2 are entirely submerged during the rainy season ( december march ) . in both localities , nests were located in the morning by following foraging workers returning with bait ( cream of rice cereal ) to their nest entrances . nests were excavated using gerber folding shovels by first digging a trench approximately 1.5 m away from the nest entrance ( rabeling et al . , 2007 ; solomon et al . , 2011 ) . the trench was extended by carefully shaving away soil with a shovel or a knife in the direction of the nest entrance . as the trench grew closer to the nest entrance , digging became increasingly careful in order to avoid missing or destroying chambers and tunnels . since the depths at which m. inflatus constructs its deepest chambers were initially unknown , after locating a chamber excavation continued downward 0.51 m in order to locate possible additional chambers . if a chamber contained a queen , it was assumed that it was the bottom - most chamber and excavation ceased . when a chamber was located , part of the chamber wall near the excavator was removed and the fungus garden , when present , was transferred , using a flame - sterilized forceps , spoon , and/or knife , to a plastic nest box with a plaster bottom pre - saturated with water . chamber dimensions were recorded , including : height ( maximum length along vertical axis ) , width ( maximum length along horizontal axis parallel to the excavation plane ) , depth ( maximum length along horizontal axis perpendicular to the excavation plane ) , and distance from the surface to the chamber roof ( summarized in table 1 ) . nests were maintained in live laboratory culture and were still alive in the lab at the time of this writing ( july 2013 ) . a few days following collection , small portions of fungus gardens were collected from the live lab nests and likewise preserved in 95 % ethanol . the time interval between field collections of the nests and preservation of garden samples allowed the ants to reassemble the gardens , which are usually broken apart during collection , and to remove particles of soil , which are usually introduced during nest excavation . estimated number of individuals for each colony is based on the count of the workers in the laboratory nests ; it may be an underestimate because laboratory colonies appear to have fungus gardens that are smaller than those initially collected in the field.table 1summary of nest datanest idchamber 1chamber 2chamber 3chamber 4jsc121006 - 02 locality 1depth80 cm94 cm103 cm\dimensions6.5 4 5 cm5 4 7 cm7.5 6 7 cmcontentsfn , w , alfn , w , alfn , w , al , qtrs121006 - 07 locality 1depth22 cm68 cm83 cm96 cmdimensions4 cm2 cm7 cm7 8 cmcontentsloose sandw , mw , alfn , w , g , m , 2qtrs121009 - 01 locality 2depth87 cm100 cm230 cm\dimensions8 4 10 cm8 5 9 cm3 3 3.5 cmcontentswfn , 1q?1q?trs121009 - 02 locality 2depth75 cm310 cm\\dimensions9 5 12 cm7 4 7 cmcontentsfn , wfn , w , mdimensions : width height depth fn fungus garden , w workers , q queen , m males , g alate females , al alates ( sex not recorded ) ; for nest trs121009 - 01 , 1q ? indicates that we did not record in which chamber the queen was found dimensions : width height depth fn fungus garden , w workers , q queen , m males , g alate females , al alates ( sex not recorded ) ; for nest trs121009 - 01 , 1q ? indicates that we did not record in which chamber the queen was found in the lab , a few strands of hyphae from 3 different nests ( collection codes jsc121006 - 02 , trs121006 - 07 and trs121007 - 01 ) were separated from the field - preserved garden material with flame - sterilized forceps under a stereomicroscope and dna was extracted from them using the chelex protocol of sen et al . the extracted dna was amplified and sequenced for the nuclear its region following the methods of mueller et al . this dna sequence was incorporated into a large alignment ( > 440 sequences ) of fungal cultivars and free - living leucocoprineae and aligned using mafft ( katoh and standley , 2013 ) using parameter values e - ins - i , 200pam / k = 2 , and gap open penalty = 1.2 . the sequences are deposited in genbank under accession numbers kf562343 , kf562444 , and kf562345 . in the laboratory , specimens were photographed using a jvc ky - f70b video camera with m420 leica stereomicroscope and automontage pro version 5.03.0018 software . five specimens were studied , including three last - instar worker larvae and two worker prepupae ( i.e. , post - feeding last instars ) , all taken from nest collection trs121006 - 07 . morphological terminology and measurement indices follow snodgrass ( 1910 ) , tulloch ( 1935 ) , gauld and bolton ( 1988 ) , hlldobler and wilson ( 1990 ) , bolton ( 1994 ) , schultz and meier ( 1995 ) , mackay et al . ( 2004 ) , klingenberg and brando ( 2009 ) , and sosa - calvo and schultz ( 2010 ) . morphological measurements , index abbreviations , and definitions are as follows : head width ( hw ) : in frontal view , maximum width of the head just above eyes , excluding eyes ; head width in males ( hwm ) : in frontal view , maximum width of the head including eyes ; interocular distance ( iod ) : in the male , maximum width of the head in frontal view measured at the midpoint of the internal margin of eyes ; head length ( hl ) : in frontal view , maximum length of the head from posteriormost margin of the head to median point of anteriormost margin of clypeal apron ; scape length ( sl ) : maximum length of scape excluding the basal condyle ; interfrontal width ( ifw ) : maximum distance between lateral margins of frontal lobes ; mandible length ( ml ) : in frontal view , straight - line length from midpoint of anterior margin of clypeal apron to tip of mandibles when fully closed ; eye length ( el ) : in profile , maximum diameter of eye ; weber length ( wl ) : in lateral view , length of mesosoma from anteriormost point of pronotum , to posteriormost ventral angle of propodeum ; hind femur length ( hfl ) : length of hind femur in lateral view ; petiole length ( pl ) : in lateral view , maximum length of petiole ; postpetiole length ( ppl ) : in lateral view , maximum length of postpetiole ; gaster length ( gl ) : in lateral view , distance from anteriormost point of tergo - sternal gaster suture to the posterior tip ; cephalic index ( ci ) : for females ( hw / hl ) 100 , for males ( iod / hl ) 100 ; frontal lobes index ( fli ) : for females ( ifw / hw ) 100 , for males ( ifw / iod ) 100 ; scape index ( si ) : for females ( sl / hw ) 100 , for males ( sl / iod ) 100 . deposition of material : the specimens examined are deposited in usnm , national museum of natural history , washington , dc , usa ; mzsp , museu de zoologia da universidade de so paulo , so paulo , sp , brazil ; mbc ufu , museu de biodiversidade do cerrado , universidade federal de uberlndia , uberlndia , minas gerais , brazil ; and mcz , museum of comparative zoology , harvard university , cambridge , ma , usa . all nests had a single , simple , very inconspicuous entrance hole approximately 23 mm in diameter . in no case was the nest entrance surrounded by a mound of excavated soil particles , differing in this regard from entrances of the congeneric m. cerradensis ( solomon et al . , 2011 ) . the deepest chamber encountered was 310 cm deep and contained garden and ants , but no queen . the shallowest chamber encountered was 22 cm deep and contained no fungus or ants , only loose sand . the shallowest chamber that contained ants was 68 cm deep , and the shallowest chamber that contained fungus garden was 75 cm deep . tunnels connecting chambers were observed in some cases and were very straight and perpendicular to the surface ( fig . 1 ) . the walls of two nest chambers , from nests trs121009 - 01 and -02 , were punctuated with multiple holes ( fig . colony sizes ranged from approximately 30100 workers.fig . 1 mycetagroicus inflatus nest : a illustration of nest trs121006 - 07 . c photo of chamber 1 of nest trs121009 - 02 showing the many holes punctuating the chamber wall mycetagroicus inflatus nest : a illustration of nest trs121006 - 07 . c photo of chamber 1 of nest trs121009 - 02 showing the many holes punctuating the chamber wall one chamber of a fifth nest was encountered accidentally ( trs121007 - 01 ) while digging , but no data on the position of the nest entrance or other chambers were available for that nest , so it is not treated as a separately excavated nest in table 1 . this isolated chamber was encountered at 80 cm depth , was 6 cm wide , and contained a fungus garden , queen , workers , and brood . its sequences indicate that the fungal cultivars collected from three nests of m. inflatus ( jsc121006 - 02 , trs121006 - 07 , and trs121007 - 01 ) all belong to subclade f of the so - called clade 2 of lower attine ( g3 ) leucocoprineaceous cultivars ( mehdiabadi et al . subclade f is arguably a single fungal species that is also associated with cyphomyrmex faunulus , mycocepurus smithii , myrmicocrypta cf . buenzlii , and other attine species from ecuador , trinidad , guyana , and brazil ( mehdiabadi et al . , 2012 ; unpubl . remarkably , the fungal species cultivated by m. inflatus in par is also grown by its congener mycetagroicus cerradensis ( solomon et al . , 2011 , accession number in genbank hm245775 ) over 1,000 km to the south in minas gerais . while following a m. inflatus forager carrying cream of rice bait back to its nest , a fly , later identified as belonging to the genus pholeomyia bilimek ( milichiidae : diptera ) , was observed following the worker by walking a short distance behind it ( jsc , pers . wilson of a pholeomyia fly following a worker of the fungus - growing ant trachymyrmex septentrionalis mccook in florida and suggested a possible association of certain pholeomyia species with fungus - growing ants . later , waller ( 1980 ) reported finding pholeomyia texensis sabrosky entering the nests of atta texana by riding on the cut leaves , where the fly larvae presumably feed on nest refuse . larvae of some species of milichiids have close associations with hymenoptera ( sabrosky , 1959 ; krombein , 1967 ; moser and neff 1971 ; melo , 1996 ; wild and brake , 2009 ; swann , 2010 ) . . 2 mycetagroicus inflatus gyne : a full - face view , b dorsal view , c lateral profile , d fore- and hindwing , e dorsal view of postpetiole mycetagroicus inflatus gyne : a full - face view , b dorsal view , c lateral profile , d fore- and hindwing , e dorsal view of postpetiole measurements in mm ( range ) : hw 0.600.72 , hl 0.720.76 , scl 0.620.66 , ifw 0.380.39 , ml 0.200.26 , el 0.150.17 , wl 1.01.1 , hfl 0.790.87 , pl 0.180.23 , ppl 0.320.37 , gl 0.891.14 , ci 8494 , fli 5562 , si 91102 ( n = 7 ) . posterolateral corners of head , mesosoma , petiole , and postpetiole light ferruginous brown ; gaster dark ferruginous brown . mandibles , antennae , and anterior borders of frontal lobes distinctly lighter than the rest of the dorsum of the head . integument finely reticulate - punctuate , opaque . erect hairs absent , most of the body covered with short , white , appressed hairs . slightly longer than broad ( ci 8494 ) , posterior corners of cephalic margin rounded , slightly notched medially . mandibles triangular , dorsally striate , masticatory ( inner ) margin of mandible with 5 teeth , the distal - most tooth noticeably prolonged and larger than the rest . margin of anterior clypeal apron convex , with a very broad and shallow but distinct median notch . median clypeal seta ( ~0.15 mm ) arising from border of clypeal apron and clypeal anterior margin , flanked on either side by a pair of additional , long setae as well as 23 shorter setae . distinct frontoclypeal teeth present ( triangular lateral tooth of brando and mayh - nunes , 2008 ) , best seen in lateral view . frontal lobes evenly rounded , separated by a fingerlike extension of the clypeus extending posterad to the level of the eyes . frontal carinae short , diverging toward corners of the head , not extending beyond median ocellus . preocular carina distinct , extending posterad from mandibular insertion , then curving medially above eye level . area laterad of frontal lobes , the ill - defined antennal scrobe , devoid of hairs . eyes convex , 13 ommatidia across largest diameter of dorso - ventral axis and 10 ommatidia across smallest diameter . antennal scape extending only slightly ( ~0.02 mm ) past posterior border of head . scape noticeably curved ventrad in basal one third , best seen in posterior view with scape positioned at 90 from median axis . first funicular segment and segments 810 longer than broad , segments in between almost subquadrate . anterior pronotal tubercle absent ( paired median pronotal teeth of brando and mayh - nunes , 2008 ) . antero - inferior corner of pronotum almost forming a right angle , best seen in lateral view . axillae ( anterior division of scutellum sensu snodgrass , 1910 ; prescutellum sensu tulloch , 1935 ; paraptera sensu brando and mayh - nunes , 2008 ) relatively large , laterally rounded and medially constricted . scutellum mostly flat in profile view , weakly concave and narrowing posteriorly in dorsal view , posterior border medially emarginate , not forming scutellar processes . propodeal spiracle directed posterad , mounted on a tumulus , best seen in dorsal view . propodeal lobe lamellate , connected by a short , longitudinal carina , along bulla of metathoracic gland , to the dorsum of the propodeal spiracle , best seen in dorsolateral view forewing ( length : 3.62 mm ) with 5 closed cells ( terminology follows goulet and huber , 1993 ) : costal ( c ) , radial ( r ) , cubital ( cu ) , first radial 1 ( 1r1 ) , and first radial 2 ( 2r1 ) . hindwing ( length : 2.57 mm ) with reduced venation , just one closed cell , and 6 hamuli . subpetiolar process present anteriorly , pointing forward , sometimes not clearly visible because concealed by propodeal lobes . from ventral view clearly visible as a spine . postpetiole large , > 1.5 the width of the petiole , slightly broader than long in dorsal view . posterior border of postpetiole wider than the rest of petiole , bearing protruding transverse plate that is rounded laterally and slightly emarginated medially ( fig . in dorsal view , gaster elliptical , narrowing posteriorly , lacking ridges or noticeable sculpture , but having finely punctuated microsculpture . rows of long , suberect hairs present on posterior borders of gastral sternites 24 ( a5a7 ) . gastral tergite and sternite i subequal in length , the posteriormost tip of the gaster formed by segment a7 in contrast to the ( presumably derived ) condition in some attini , in which gastral tergite i longer than sternite i so that posteriorly it overlaps the remaining segments , which are shifted anterad . the gyne of m. inflatus is , as in many ant species , quite similar to the worker except for modifications typical for the caste such as the presence of ocelli , the morphology of the mesosoma associated with wings , and the slightly larger size . in addition , in the worker the eyes are smaller , the frontoclypeal teeth are less pronounced , the frontal lobes are narrower , median anterior tubercles are present on the pronotum , and the petiole is shorter . the gyne of m. inflatus can be distinguished from the gynes of other closely related attine genera by characters that are synapomorphic for the genera . sericomyrmex gynes , for instance , are larger in size and covered with dense pilosity , have cordate heads , have gasters that are laterally straight instead of rounded , and have frontal lobes that are more developed than those in mycetagroicus . gynes of trachymyrmex species are in most cases larger in size and have integuments that are moderately to strongly tuberculate , frequently appearing spiny , most noticeably on the posterior lobes of head and on the gaster , which in m. inflatus are smooth . cyphomyrmex gynes can be similar in size to gynes of m. inflatus , but are immediately distinguished by the strongly expanded frontal lobes , often covering a large portion of the head , and in the strigatus and wheeleri groups accompanied by deep antennal scrobes . the gyne of the congeneric m. triangularis can be distinguished from that of m. inflatus by its slightly larger size , the presence of a median longitudinal ridge posterior to the frontal triangle , the presence of a distinct parapsidal furrow , the presence of scutellar processes , and the rugulose microsculpture on the dorsum of first gastral segment ( a4 ) , which also bears two pronounced ridges laterally . 3)fig . 3male of mycetagroicus inflatus : a full - face view , b lateral profile , c dorsal view , d fore- and hindwings male of mycetagroicus inflatus : a full - face view , b lateral profile , c dorsal view , d fore- and hindwings measurements in mm ( range ) : hwm 0.530.61 , iod 0.370.40 , hl 0.450.56 , scl 0.440.62 , ifw 0.15 0.19 , ml 0.140.21 , el 0.130.19 , wl 0.901.16 , hfl 1.051.35 , pl 0.140.20 , ppl 0.210.29 , gl 0.801.07 , ci 7781 , fli 4144 , si 120142 ( n = 7 ) . body color dark brown , integument reticulate - punctuate on head and mesosoma , gaster opaque with reticulate microsculpture . erect hairs absent , short appressed white hairs covering head , dorsal side of mesosoma , gaster , and legs ; only a few hairs present on lateral side of mesosoma . head . mandibles triangular , dorsally striate , basal angle rounded , masticatory margin with three teeth , all arising in the distal half , an unusual arrangement shared with males of trachymyrmex urichi and perhaps other trachymyrmex species . clypeus evenly reticulate , without frontoclypeal carinae or teeth as seen in worker and queen . median clypeal seta ( ~0.12 mm ) arising at the point where posterior margin of clypeal apron and anterior clypeal margin meet flanked by one much shorter seta on each side . frontal lobes small , short , not completely covering the antennal condyle , broadly separated by a fingerlike extension of the clypeus extending posterad . frontal carinae short , extending to the level of the posterior border of the eyes . eyes convex , large , 16 ommatidia across largest diameter of dorso - ventral axis , 15 ommatidia across the smallest diameter of the eye . three similarly sized ocelli present . antenna 12-segmented , a departure from the plesiomorphic number of 13 for ant males ( including attine males ) , the first funicular segment ( pedicel ) as long as second funicular segment , or slightly longer . first funicular segment longer than broad and thicker than the second funicular segment , but not thicker than the most distal segments . anapleural suture present , dividing mesopleuron into katepisternum and anepisternum . a wide , transversely costate groove present on dorsoposterior border of anepisternum below insertion of wing . in some males the anapleural suture scutum with shallow , complete , v - shaped notauli , dividing the scutum into an anteromedian area ( prescutum ) and two lateral areas . scutellum slightly inflated , narrowing posteriorly , posterior border with two short , blunt denticles . forewing ( length : 3.81 mm ) , hindwing ( length : 2.7 mm ) . venation and appearance same as in female , except in male spur of cross vein 1 m - cu is visible protruding from the bottom of 1r1 cell and 7 hamuli instead of 6 on hind wing . integument of tergum 1 of gaster ( i.e. , a4 ) more shining than the other body parts , but still subopaque due to the finely reticulate microsculpture ; bearing sparse , white , appressed hairs . in dorsal view , gaster elliptical , narrowing posteriorly , without any carinae or other macrosculpture . gastral tergite and sternite i subequal in length and gastral tergites 25 ( i.e. , a5a8 ) visible dorsally or posterodorsally . rows of long , suberect hairs present on posterior borders of gastral sternites 25 ( a5a8 ) . males of m. inflatus have 12-segmented antennae , a deviation from the usual 13-segmented antennae present in the males of most attine species , although the 12-segmented condition has arisen independently elsewhere in the tribe in the genus sericomyrmex , in some cyphomyrmex species ( e.g. , c. faunulus and c. auritus ) , in trachymyrmex opulentus , and in a number of social parasites in the genus acromyrmex ( gallardo , 1916 ) . reductions in male antennal segment number are even more dramatic in males of the socially parasitic attine species mycocepurus castrator ( rabeling and bacci , 2010 ) and pseudoatta argentina ( gallardo , 1916 ; schultz et al . , 1998 ) , which have 11-segmented antennae . the male of m. inflatus can be distinguished from males of closely related genera by the following combination of characters : presence of 12 antennal segments , reticulate - punctuate integument ( even on the clypeus ) , dark brown color , transversely costate groove on the mesopleuron , and propodeal teeth very reduced . males of sericomyrmex can be distinguished from males of m. inflatus by the presence of dense appressed pilosity covering the whole body and longer , thicker , decumbent to erect hairs on the head , mesosoma , and dorsum of the gaster . males of trachymyrmex usually also have denser pilosity , are sometimes covered with spiny tubercules on the head and gaster , and usually have sharp propodeal spines and humeral tubercles . males of the closely related species cyphomyrmex costatus and c. wheeleri have 13 antennal segments , strongly developed frontal lobes , antennal scrobes that reach the posterior border of the head , and sharp propodeal spines . prepupae of mycetagroicus inflatus : a lateral profile ( prepupae ) , b head ( prepupae ) , c head , lateral view ( prepupae ) , d mouthparts ( last - instar larvae ) larvae and prepupae of mycetagroicus inflatus : a lateral profile ( prepupae ) , b head ( prepupae ) , c head , lateral view ( prepupae ) , d mouthparts ( last - instar larvae ) specimens examined : three last - instar worker larvae and two worker prepupae ( i.e. , post - feeding last instars ) , all from nest collection trs121006 - 07 . profile attoid sensu wheeler and wheeler 1976 , i.e. , with a moderately curved , ventrally shortened profile . as in all other attini , thoracic - abdominal articulation absent , thoracic intersegmental constrictions superficial , deep lateral depressions associated with abdominal spiracles absent , and leg vestiges present as open slits . as in most other attini , dorsal and lateral body surfaces devoid of setae . differing from other attini in the remarkably small number of head and ventral setae . head devoid of setae except for two setae on each gena , a state previously unknown in attine larvae ; ventral thoracic segments 13 each with a single pair of setae laterally ; and two short setae on abdominal segment 10 ventral to the anus . ventral thoracic segments 1 and 2 medially bearing multidentate spinules , thoracic segment 2 additionally with a low ventromedian boss , in combination with the genal and thoracic setae clearly functioning as a food anchor . as in most neoattini , labrum monolobate , narrow , bulging , a synapomorphy for the attini ; anterior labral setae reduced to papillae . mandibles typically attine : short , fleshy , subconical . a distinct , undivided apical mandibular tooth and no subapical teeth ; spinules sparsely but evenly distributed on all mandibular surfaces . galea reduced , present as two sensilla surmounting a low welt ; a shallow pit distal and immediately adjacent to the galea . one seta on each maxilla between the galea and palp , a second seta present laterad of the palp . as in most attines , all nests had a single , simple , very inconspicuous entrance hole approximately 23 mm in diameter . in no case was the nest entrance surrounded by a mound of excavated soil particles , differing in this regard from entrances of the congeneric m. cerradensis ( solomon et al . , 2011 ) . the deepest chamber encountered was 310 cm deep and contained garden and ants , but no queen . the shallowest chamber encountered was 22 cm deep and contained no fungus or ants , only loose sand . the shallowest chamber that contained ants was 68 cm deep , and the shallowest chamber that contained fungus garden was 75 cm deep . tunnels connecting chambers were observed in some cases and were very straight and perpendicular to the surface ( fig . 1 ) . the walls of two nest chambers , from nests trs121009 - 01 and -02 , were punctuated with multiple holes ( fig . colony sizes ranged from approximately 30100 workers.fig . 1 mycetagroicus inflatus nest : a illustration of nest trs121006 - 07 . c photo of chamber 1 of nest trs121009 - 02 showing the many holes punctuating the chamber wall mycetagroicus inflatus nest : a illustration of nest trs121006 - 07 . c photo of chamber 1 of nest trs121009 - 02 showing the many holes punctuating the chamber wall one chamber of a fifth nest was encountered accidentally ( trs121007 - 01 ) while digging , but no data on the position of the nest entrance or other chambers were available for that nest , so it is not treated as a separately excavated nest in table 1 . this isolated chamber was encountered at 80 cm depth , was 6 cm wide , and contained a fungus garden , queen , workers , and brood . its sequences indicate that the fungal cultivars collected from three nests of m. inflatus ( jsc121006 - 02 , trs121006 - 07 , and trs121007 - 01 ) all belong to subclade f of the so - called clade 2 of lower attine ( g3 ) leucocoprineaceous cultivars ( mehdiabadi et al . , 2012 ; mueller et al . , 1998 ) subclade f is arguably a single fungal species that is also associated with cyphomyrmex faunulus , mycocepurus smithii , myrmicocrypta cf . buenzlii , and other attine species from ecuador , trinidad , guyana , and brazil ( mehdiabadi et al . , 2012 ; unpubl . data ) . remarkably , the fungal species cultivated by m. inflatus in par is also grown by its congener mycetagroicus cerradensis ( solomon et al . , 2011 , accession number in genbank hm245775 ) over 1,000 km to the south in minas gerais . while following a m. inflatus forager carrying cream of rice bait back to its nest , a fly , later identified as belonging to the genus pholeomyia bilimek ( milichiidae : diptera ) , was observed following the worker by walking a short distance behind it ( jsc , pers . wilson of a pholeomyia fly following a worker of the fungus - growing ant trachymyrmex septentrionalis mccook in florida and suggested a possible association of certain pholeomyia species with fungus - growing ants . later , waller ( 1980 ) reported finding pholeomyia texensis sabrosky entering the nests of atta texana by riding on the cut leaves , where the fly larvae presumably feed on nest refuse . larvae of some species of milichiids have close associations with hymenoptera ( sabrosky , 1959 ; krombein , 1967 ; moser and neff 1971 ; melo , 1996 ; wild and brake , 2009 ; swann , 2010 ) . mycetagroicus inflatus , brando & mayh - nunes 2008 gyne ( fig . 2)fig . 2 mycetagroicus inflatus gyne : a full - face view , b dorsal view , c lateral profile , d fore- and hindwing , e dorsal view of postpetiole mycetagroicus inflatus gyne : a full - face view , b dorsal view , c lateral profile , d fore- and hindwing , e dorsal view of postpetiole measurements in mm ( range ) : hw 0.600.72 , hl 0.720.76 , scl 0.620.66 , ifw 0.380.39 , ml 0.200.26 , el 0.150.17 , wl 1.01.1 , hfl 0.790.87 , pl 0.180.23 , ppl 0.320.37 , gl 0.891.14 , ci 8494 , fli 5562 , si 91102 ( n = 7 ) . posterolateral corners of head , mesosoma , petiole , and postpetiole light ferruginous brown ; gaster dark ferruginous brown . mandibles , antennae , and anterior borders of frontal lobes distinctly lighter than the rest of the dorsum of the head . erect hairs absent , most of the body covered with short , white , appressed hairs . slightly longer than broad ( ci 8494 ) , posterior corners of cephalic margin rounded , slightly notched medially . mandibles triangular , dorsally striate , masticatory ( inner ) margin of mandible with 5 teeth , the distal - most tooth noticeably prolonged and larger than the rest . margin of anterior clypeal apron convex , with a very broad and shallow but distinct median notch . median clypeal seta ( ~0.15 mm ) arising from border of clypeal apron and clypeal anterior margin , flanked on either side by a pair of additional , long setae as well as 23 shorter setae . distinct frontoclypeal teeth present ( triangular lateral tooth of brando and mayh - nunes , 2008 ) , best seen in lateral view . frontal lobes evenly rounded , separated by a fingerlike extension of the clypeus extending posterad to the level of the eyes . frontal carinae short , diverging toward corners of the head , not extending beyond median ocellus . preocular carina distinct , extending posterad from mandibular insertion , then curving medially above eye level . area laterad of frontal lobes , the ill - defined antennal scrobe , devoid of hairs . eyes convex , 13 ommatidia across largest diameter of dorso - ventral axis and 10 ommatidia across smallest diameter . antennal scape extending only slightly ( ~0.02 mm ) past posterior border of head . scape noticeably curved ventrad in basal one third , best seen in posterior view with scape positioned at 90 from median axis . first funicular segment and segments 810 longer than broad , segments in between almost subquadrate . anterior pronotal tubercle absent ( paired median pronotal teeth of brando and mayh - nunes , 2008 ) . antero - inferior corner of pronotum almost forming a right angle , best seen in lateral view . axillae ( anterior division of scutellum sensu snodgrass , 1910 ; prescutellum sensu tulloch , 1935 ; paraptera sensu brando and mayh - nunes , 2008 ) relatively large , laterally rounded and medially constricted . scutellum mostly flat in profile view , weakly concave and narrowing posteriorly in dorsal view , posterior border medially emarginate , not forming scutellar processes . propodeal spiracle directed posterad , mounted on a tumulus , best seen in dorsal view . propodeal lobe lamellate , connected by a short , longitudinal carina , along bulla of metathoracic gland , to the dorsum of the propodeal spiracle , best seen in dorsolateral view . forewing ( length : 3.62 mm ) with 5 closed cells ( terminology follows goulet and huber , 1993 ) : costal ( c ) , radial ( r ) , cubital ( cu ) , first radial 1 ( 1r1 ) , and first radial 2 ( 2r1 ) . hindwing ( length : 2.57 mm ) with reduced venation , just one closed cell , and 6 hamuli . subpetiolar process present anteriorly , pointing forward , sometimes not clearly visible because concealed by propodeal lobes . from ventral view clearly visible as a spine . postpetiole large , > 1.5 the width of the petiole , slightly broader than long in dorsal view . posterior border of postpetiole wider than the rest of petiole , bearing protruding transverse plate that is rounded laterally and slightly emarginated medially ( fig . in dorsal view , gaster elliptical , narrowing posteriorly , lacking ridges or noticeable sculpture , but having finely punctuated microsculpture . rows of long , suberect hairs present on posterior borders of gastral sternites 24 ( a5a7 ) . gastral tergite and sternite i subequal in length , the posteriormost tip of the gaster formed by segment a7 in contrast to the ( presumably derived ) condition in some attini , in which gastral tergite i longer than sternite i so that posteriorly it overlaps the remaining segments , which are shifted anterad . the gyne of m. inflatus is , as in many ant species , quite similar to the worker except for modifications typical for the caste such as the presence of ocelli , the morphology of the mesosoma associated with wings , and the slightly larger size . in addition , in the worker the eyes are smaller , the frontoclypeal teeth are less pronounced , the frontal lobes are narrower , median anterior tubercles are present on the pronotum , and the petiole is shorter . the gyne of m. inflatus can be distinguished from the gynes of other closely related attine genera by characters that are synapomorphic for the genera . sericomyrmex gynes , for instance , are larger in size and covered with dense pilosity , have cordate heads , have gasters that are laterally straight instead of rounded , and have frontal lobes that are more developed than those in mycetagroicus . gynes of trachymyrmex species are in most cases larger in size and have integuments that are moderately to strongly tuberculate , frequently appearing spiny , most noticeably on the posterior lobes of head and on the gaster , which in m. inflatus are smooth . cyphomyrmex gynes can be similar in size to gynes of m. inflatus , but are immediately distinguished by the strongly expanded frontal lobes , often covering a large portion of the head , and in the strigatus and wheeleri groups accompanied by deep antennal scrobes . the gyne of the congeneric m. triangularis can be distinguished from that of m. inflatus by its slightly larger size , the presence of a median longitudinal ridge posterior to the frontal triangle , the presence of a distinct parapsidal furrow , the presence of scutellar processes , and the rugulose microsculpture on the dorsum of first gastral segment ( a4 ) , which also bears two pronounced ridges laterally . 3male of mycetagroicus inflatus : a full - face view , b lateral profile , c dorsal view , d fore- and hindwings male of mycetagroicus inflatus : a full - face view , b lateral profile , c dorsal view , d fore- and hindwings measurements in mm ( range ) : hwm 0.530.61 , iod 0.370.40 , hl 0.450.56 , scl 0.440.62 , ifw 0.15 0.19 , ml 0.140.21 , el 0.130.19 , wl 0.901.16 , hfl 1.051.35 , pl 0.140.20 , ppl 0.210.29 , gl 0.801.07 , ci 7781 , fli 4144 , si 120142 ( n = 7 ) . body color dark brown , integument reticulate - punctuate on head and mesosoma , gaster opaque with reticulate microsculpture . erect hairs absent , short appressed white hairs covering head , dorsal side of mesosoma , gaster , and legs ; only a few hairs present on lateral side of mesosoma . head . mandibles triangular , dorsally striate , basal angle rounded , masticatory margin with three teeth , all arising in the distal half , an unusual arrangement shared with males of trachymyrmex urichi and perhaps other trachymyrmex species . clypeus evenly reticulate , without frontoclypeal carinae or teeth as seen in worker and queen . median clypeal seta ( ~0.12 mm ) arising at the point where posterior margin of clypeal apron and anterior clypeal margin meet flanked by one much shorter seta on each side . frontal lobes small , short , not completely covering the antennal condyle , broadly separated by a fingerlike extension of the clypeus extending posterad . frontal carinae short , extending to the level of the posterior border of the eyes . eyes convex , large , 16 ommatidia across largest diameter of dorso - ventral axis , 15 ommatidia across the smallest diameter of the eye . three similarly sized ocelli present . antenna 12-segmented , a departure from the plesiomorphic number of 13 for ant males ( including attine males ) , the first funicular segment ( pedicel ) as long as second funicular segment , or slightly longer . first funicular segment longer than broad and thicker than the second funicular segment , but not thicker than the most distal segments . anapleural suture present , dividing mesopleuron into katepisternum and anepisternum . a wide , transversely costate groove present on dorsoposterior border of anepisternum below insertion of wing . in some males scutum with shallow , complete , v - shaped notauli , dividing the scutum into an anteromedian area ( prescutum ) and two lateral areas . scutellum slightly inflated , narrowing posteriorly , posterior border with two short , blunt denticles . forewing ( length : 3.81 mm ) , hindwing ( length : 2.7 mm ) . venation and appearance same as in female , except in male spur of cross vein 1 m - cu is visible protruding from the bottom of 1r1 cell and 7 hamuli instead of 6 on hind wing . metasoma . integument of tergum 1 of gaster ( i.e. , a4 ) more shining than the other body parts , but still subopaque due to the finely reticulate microsculpture ; bearing sparse , white , appressed hairs . in dorsal view , gaster elliptical , narrowing posteriorly , without any carinae or other macrosculpture . gastral tergite and sternite i subequal in length and gastral tergites 25 ( i.e. , a5a8 ) visible dorsally or posterodorsally . rows of long , suberect hairs present on posterior borders of gastral sternites 25 ( a5a8 ) . males of m. inflatus have 12-segmented antennae , a deviation from the usual 13-segmented antennae present in the males of most attine species , although the 12-segmented condition has arisen independently elsewhere in the tribe in the genus sericomyrmex , in some cyphomyrmex species ( e.g. , c. faunulus and c. auritus ) , in trachymyrmex opulentus , and in a number of social parasites in the genus acromyrmex ( gallardo , 1916 ) . reductions in male antennal segment number are even more dramatic in males of the socially parasitic attine species mycocepurus castrator ( rabeling and bacci , 2010 ) and pseudoatta argentina ( gallardo , 1916 ; schultz et al . , 1998 ) , which have 11-segmented antennae . the male of m. inflatus can be distinguished from males of closely related genera by the following combination of characters : presence of 12 antennal segments , reticulate - punctuate integument ( even on the clypeus ) , dark brown color , transversely costate groove on the mesopleuron , and propodeal teeth very reduced . males of sericomyrmex can be distinguished from males of m. inflatus by the presence of dense appressed pilosity covering the whole body and longer , thicker , decumbent to erect hairs on the head , mesosoma , and dorsum of the gaster . males of trachymyrmex usually also have denser pilosity , are sometimes covered with spiny tubercules on the head and gaster , and usually have sharp propodeal spines and humeral tubercles . males of the closely related species cyphomyrmex costatus and c. wheeleri have 13 antennal segments , strongly developed frontal lobes , antennal scrobes that reach the posterior border of the head , and sharp propodeal spines . prepupae of mycetagroicus inflatus : a lateral profile ( prepupae ) , b head ( prepupae ) , c head , lateral view ( prepupae ) , d mouthparts ( last - instar larvae ) larvae and prepupae of mycetagroicus inflatus : a lateral profile ( prepupae ) , b head ( prepupae ) , c head , lateral view ( prepupae ) , d mouthparts ( last - instar larvae ) specimens examined : three last - instar worker larvae and two worker prepupae ( i.e. , post - feeding last instars ) , all from nest collection trs121006 - 07 . profile attoid sensu wheeler and wheeler 1976 , i.e. , with a moderately curved , ventrally shortened profile . as in all other attini , thoracic - abdominal articulation absent , thoracic intersegmental constrictions superficial , deep lateral depressions associated with abdominal spiracles absent , and leg vestiges present as open slits . as in most other attini , dorsal and lateral body surfaces devoid of setae . differing from other attini in the remarkably small number of head and ventral setae . head devoid of setae except for two setae on each gena , a state previously unknown in attine larvae ; ventral thoracic segments 13 each with a single pair of setae laterally ; and two short setae on abdominal segment 10 ventral to the anus . ventral thoracic segments 1 and 2 medially bearing multidentate spinules , thoracic segment 2 additionally with a low ventromedian boss , in combination with the genal and thoracic setae clearly functioning as a food anchor . as in most neoattini , genal lobes present . labrum monolobate , narrow , bulging , a synapomorphy for the attini ; anterior labral setae reduced to papillae . mandibles typically attine : short , fleshy , subconical . a distinct , undivided apical mandibular tooth and no subapical teeth ; spinules sparsely but evenly distributed on all mandibular surfaces . galea reduced , present as two sensilla surmounting a low welt ; a shallow pit distal and immediately adjacent to the galea . one seta on each maxilla between the galea and palp , a second seta present laterad of the palp . as in most attines , the genus mycetagroicus is the sister group of the higher attini ( sericomyrmex , trachymyrmex , acromyrmex , and atta ) ( schultz and brady , 2008 ) . thus , understanding the biology of mycetagroicus is our best hope for reconstructing the attributes of the most recent common ancestor that it shares with the higher attini , especially when considered in combination with the biology of the most closely related outgroup [ currently thought to be the cyphomyrmex wheeleri clade ( schultz and brady , 2008 ) ] . reconstructing the common ancestor of mycetagroicus + the higher attini is important for reconstructing the evolutionary transitions that occurred on the branch subtending the higher attini , arguably the most ecologically significant period of evolutionary change in all of the attini . these transitions coincide with the origin of the obligate higher attine fungal symbiont , derived from a lower attine fungal species ( schultz et al . , 2005 ; mikheyev et al . , 2006 , 2007 ; mueller et al . , 2010 ; mueller et al . , 2011a this study adds to the scant biological information so far accumulated for the four known mycetagroicus species . it describes the gyne of m. inflatus , increasing to two the numbers of species for which gynes are known , the other being m. triangularis ( brando and mayh - nunes , 2001 ) . the nest architecture of m. inflatus is also described , increasing to two the number of species for which nest architecture is known , the other being m. cerradensis ( solomon et al . , 2011 ) . the walls of one chamber in each of the two nests from locality 2 ( trs121009 - 01 : chamber depth not recorded ; trs121009 - 02 : chamber 75 cm deep ) were punctured by numerous holes of unknown purpose ( fig . it is highly unlikely that all of the holes were the openings of tunnels because no ants were observed entering or exiting and , if they were tunnel openings , they would connect with dozens of horizontal tunnels whereas no such tunnels were encountered ; all of the tunnels observed during excavation were vertically arranged . one possibility is that the holes are somehow associated with flooding , perhaps serving as shelters for the ants during the months that the nests are submerged or serving in some way to capture and hold pockets of air . this conjecture gains support from the observation that similarly punctured chamber walls , as described above , were observed in the deepest chambers of colonies of the sympatric but very distantly related species kalathomyrmex emeryi , colonies of which are certainly flooded during the rainy season . in addition to being punctured by numerous holes , unlike those of m. inflatus the chamber walls of k. emeryi were lined with a layer of brown clay . the nest architecture of mycetagroicus cerradensis , the only other congeneric species for which nesting biology is known , shares some characteristics with that of m. inflatus , including : a single nest entrance , chambers that are more or less vertically arranged below the nest entrance , and an extremely deep lowermost garden chamber ( solomon et al . , 2011 ) suggested that m. cerradensis moves its fungus garden seasonally , using deeper chambers in the dry season and shallower chambers in the wet season . this hypothesis applies equally to m. inflatus because , in both species , nests were excavated in the dry season and the uppermost nest chambers were found to be empty . seasonal repositioning of fungus gardens has been recorded for some other fungus - growing ants , including two species of the genus mycocepurus ( rabeling et al . , 2007 ) and in the north american species atta texana and acromyrmexversicolor ( moser , 1962 , 2006 ; mueller et al . , 2011a , b ) . perhaps the most potentially important result of this study is the identification of the fungal cultivars grown by m. inflatus at the study sites on the araguaia river . the gardens from all three collected nests belong to the same fungal species ( clade 2 , subclade f ; mehdiabadi et al . , 2012 ) , suggesting that m. inflatus may depart from most lower attine species in possessing a high degree of symbiont fidelity . even more surprising , m. inflatus cultivates the same fungal species as its congener m. cerradensis in minas gerais , brazil , over 1000 km to the south ( solomon et al . , 2011 ) . field research during the past two decades has established that attine cultivars are frequently transmitted horizontally between colonies , including colonies of different ant species , and that free - living fungi are frequently imported into the lower attine symbiosis as well ( mueller et al . , 1998 ; vo et al . , 2009 ; green et al . , 2002 ; these observations have led to the current paradigm of diffuse attine ant / fungus coevolution ( mikheyev et al . , 2006 ; mikheyev et al . , 2007 ; 2010 ) and a general expectation of weak symbiont fidelity and frequent reassociations of ants and fungi in attine agriculture . one exception to this rule is the cyphomyrmex wheeleri group , in which it has recently been shown that particular ant and fungal species have been associated for many millions of years ( mehdiabadi et al . , 2012 ) . interestingly , the genus mycetagroicus is the sister group to the higher attini and the c. wheeleri group is thought to be the sister group to the clade containing mycetagroicus and the higher attini , suggesting that an increase in symbiont fidelity may have preceded the origin of the higher attini . although based on only four samples , this pattern is consistent with a scenario of strong symbiont fidelity that spans speciation events . obviously , additional samples are necessary for judging whether this pattern of symbiont fidelity continues to hold or whether the observed association with the same fungal species within and across species of mycetagroicus is due to a rare coincidence . symbiont choice is favored when potential symbionts vary in their abilities to enhance partner fitness . mycetagroicus species live in habitats that may be at least seasonally inhospitable to free - living leucocoprineaceous fungi . three of the four species in the genus mycetagroicus are found in the cerrado ; the fourth , m. inflatus , is so far known from seasonally flooded riverbanks in a region characterized as dense alluvial forest in such habitats , it may be the case that some fungal symbionts are better adapted than others , providing conditions that favor symbiont choice and long - term , close associations of particular ant and fungal species . arguing against this , however , is the observation that most cyphomyrmex wheeleri group ant species live in wet forests where feral leucocoprineaceous cultivars are known to commonly occur ( mueller et al . , 1998 ) and in which nests of different ant species , each containing its particular fungal symbiont species , often co - occur within centimeters of one another ( schultz et al . , 2002 ) . clearly , more data are required for understanding the mechanisms that produced the genetic and morphological modifications encountered today in the spectacularly successful higher attine fungi and ants .

all known fungus - growing ants ( tribe attini ) are obligately symbiotic with their cultivated fungi . the fungal cultivars of lower attine ants are facultative symbionts , capable of living apart from ants , whereas the fungal cultivars of higher attine ants , including leaf - cutting genera atta and acromyrmex , are highly specialized , obligate symbionts . since higher attine ants and fungi are derived from lower attine ants and fungi , understanding the evolutionary transition from lower to higher attine agriculture requires understanding the historical sequence of change in both ants and fungi . the biology of the poorly known ant genus mycetagroicus is of special interest in this regard because it occupies a phylogenetic position intermediate between lower and higher ant agriculture . here , based on the excavations of four nests in par , brazil , we report the first biological data for the recently described species mycetagroicus inflatus , including the first descriptions of mycetagroicus males and larvae . like m. cerradensis , the only other species in the genus for which nesting biology is known , the garden chambers of m.inflatus are unusually deep and the garden is most likely relocated vertically in rainy and dry seasons . due to the proximity of nests to the araguaia river , it is likely that even the uppermost chambers and nest entrances of m. inflatus are submerged during the rainy season . most remarkably , all three examined colonies of m. inflatus cultivate the same fungal species as their congener , m. cerradensis , over 1,000 km away , raising the possibility of long - term symbiont fidelity spanning speciation events within the genus .

Introduction Materials and methods Results Nest architecture Fungal symbiont Arthropod symbiont Systematic treatment Discussion

the ant genus mycetagroicus , described in 2001 , is a member of the ant tribe attini , all of which are obligate agriculturalists and fungivores . in establishing the genus , brando and mayh - nunes ( 2001 ) included three species : m. cerradensis , m. triangularis , and m. urbanus . in 2008 , the same authors described a fourth species , m. inflatus , based on two workers collected by r. feitosa and r. rosa da silva in southern par , brazil , in 2005 ( brando and mayh - nunes , 2008 ) . it is the sister group of the so - called higher attini ( a group containing the genera trachymyrmex , sericomyrmex , acromyrmex , and atta ) which cultivate a clade of obligately symbiotic fungal species that share a lower attine fungal ancestor , all of which are highly specialized for symbiosis with their ant hosts ( mueller and gerardo , 2002 ; schultz et al . ( 2011 ) , based on a molecular analysis of the cultivar , found that m. cerradensis is in fact a member of the lower attini , i.e. since the genus is the sister group of the higher attines , study of mycetagroicus species can potentially provide insights regarding the origin of higher attine agriculture , arguably the most ecologically significant transition in the evolutionary history of the attini . during the past few decades , new natural history information has required periodic , sometimes dramatic revisions of our model of the complex co - evolutionary relationships among fungus - growing ants , their fungal cultivars , and other microbial symbionts ( chapela et al . nest architecture in fungus - growing ants is important not only for the ants but also for their fungal cultivars , which require particular environmental conditions ( bollazzi and roces 2002 ; bollazzi et al . in addition to describing nest architecture , we identify the fungal cultivar of mycetagroicus inflatus . we describe the previously unknown queen , provide the first description of a male for the genus , and compare both to reproductive forms of other , closely related attine genera . field work was conducted from 5 to 10 october 2012 in eastern par state , brazil , at the end of the dry season . during this time period all nests were found at two localities on the west coast of the araguaia river , a southern component of the amazon river basin , across from the town of araguacema , tocantins , in an area characterized as dense alluvial forest ( s 08.75387 , w 49.54544 , elevation 164 m ) , nest entrances occurred approximately 15 m from the river s edge in an area about 7 m above water level , which was at its seasonal low point ; at locality 2 ( s 08.80157 , w 49.57368 , elevation 176 m ) , they occurred approximately 5 m away from the river s edge and about 5 m above the water level . based on information from local inhabitants , both locality 1 and locality 2 are entirely submerged during the rainy season ( december march ) . estimated number of individuals for each colony is based on the count of the workers in the laboratory nests ; it may be an underestimate because laboratory colonies appear to have fungus gardens that are smaller than those initially collected in the field.table 1summary of nest datanest idchamber 1chamber 2chamber 3chamber 4jsc121006 - 02 locality 1depth80 cm94 cm103 cm\dimensions6.5 4 5 cm5 4 7 cm7.5 6 7 cmcontentsfn , w , alfn , w , alfn , w , al , qtrs121006 - 07 locality 1depth22 cm68 cm83 cm96 cmdimensions4 cm2 cm7 cm7 8 cmcontentsloose sandw , mw , alfn , w , g , m , 2qtrs121009 - 01 locality 2depth87 cm100 cm230 cm\dimensions8 4 10 cm8 5 9 cm3 3 3.5 cmcontentswfn , 1q?1q?trs121009 - 02 locality 2depth75 cm310 cm\\dimensions9 5 12 cm7 4 7 cmcontentsfn , wfn , w , mdimensions : width height depth fn fungus garden , w workers , q queen , m males , g alate females , al alates ( sex not recorded ) ; for nest trs121009 - 01 , 1q ? morphological measurements , index abbreviations , and definitions are as follows : head width ( hw ) : in frontal view , maximum width of the head just above eyes , excluding eyes ; head width in males ( hwm ) : in frontal view , maximum width of the head including eyes ; interocular distance ( iod ) : in the male , maximum width of the head in frontal view measured at the midpoint of the internal margin of eyes ; head length ( hl ) : in frontal view , maximum length of the head from posteriormost margin of the head to median point of anteriormost margin of clypeal apron ; scape length ( sl ) : maximum length of scape excluding the basal condyle ; interfrontal width ( ifw ) : maximum distance between lateral margins of frontal lobes ; mandible length ( ml ) : in frontal view , straight - line length from midpoint of anterior margin of clypeal apron to tip of mandibles when fully closed ; eye length ( el ) : in profile , maximum diameter of eye ; weber length ( wl ) : in lateral view , length of mesosoma from anteriormost point of pronotum , to posteriormost ventral angle of propodeum ; hind femur length ( hfl ) : length of hind femur in lateral view ; petiole length ( pl ) : in lateral view , maximum length of petiole ; postpetiole length ( ppl ) : in lateral view , maximum length of postpetiole ; gaster length ( gl ) : in lateral view , distance from anteriormost point of tergo - sternal gaster suture to the posterior tip ; cephalic index ( ci ) : for females ( hw / hl ) 100 , for males ( iod / hl ) 100 ; frontal lobes index ( fli ) : for females ( ifw / hw ) 100 , for males ( ifw / iod ) 100 ; scape index ( si ) : for females ( sl / hw ) 100 , for males ( sl / iod ) 100 . in no case was the nest entrance surrounded by a mound of excavated soil particles , differing in this regard from entrances of the congeneric m. cerradensis ( solomon et al . c photo of chamber 1 of nest trs121009 - 02 showing the many holes punctuating the chamber wall one chamber of a fifth nest was encountered accidentally ( trs121007 - 01 ) while digging , but no data on the position of the nest entrance or other chambers were available for that nest , so it is not treated as a separately excavated nest in table 1 . its sequences indicate that the fungal cultivars collected from three nests of m. inflatus ( jsc121006 - 02 , trs121006 - 07 , and trs121007 - 01 ) all belong to subclade f of the so - called clade 2 of lower attine ( g3 ) leucocoprineaceous cultivars ( mehdiabadi et al . remarkably , the fungal species cultivated by m. inflatus in par is also grown by its congener mycetagroicus cerradensis ( solomon et al . , 2011 , accession number in genbank hm245775 ) over 1,000 km to the south in minas gerais . wilson of a pholeomyia fly following a worker of the fungus - growing ant trachymyrmex septentrionalis mccook in florida and suggested a possible association of certain pholeomyia species with fungus - growing ants . the gyne of m. inflatus is , as in many ant species , quite similar to the worker except for modifications typical for the caste such as the presence of ocelli , the morphology of the mesosoma associated with wings , and the slightly larger size . in addition , in the worker the eyes are smaller , the frontoclypeal teeth are less pronounced , the frontal lobes are narrower , median anterior tubercles are present on the pronotum , and the petiole is shorter . cyphomyrmex gynes can be similar in size to gynes of m. inflatus , but are immediately distinguished by the strongly expanded frontal lobes , often covering a large portion of the head , and in the strigatus and wheeleri groups accompanied by deep antennal scrobes . the gyne of the congeneric m. triangularis can be distinguished from that of m. inflatus by its slightly larger size , the presence of a median longitudinal ridge posterior to the frontal triangle , the presence of a distinct parapsidal furrow , the presence of scutellar processes , and the rugulose microsculpture on the dorsum of first gastral segment ( a4 ) , which also bears two pronounced ridges laterally . males of m. inflatus have 12-segmented antennae , a deviation from the usual 13-segmented antennae present in the males of most attine species , although the 12-segmented condition has arisen independently elsewhere in the tribe in the genus sericomyrmex , in some cyphomyrmex species ( e.g. the male of m. inflatus can be distinguished from males of closely related genera by the following combination of characters : presence of 12 antennal segments , reticulate - punctuate integument ( even on the clypeus ) , dark brown color , transversely costate groove on the mesopleuron , and propodeal teeth very reduced . males of sericomyrmex can be distinguished from males of m. inflatus by the presence of dense appressed pilosity covering the whole body and longer , thicker , decumbent to erect hairs on the head , mesosoma , and dorsum of the gaster . in no case was the nest entrance surrounded by a mound of excavated soil particles , differing in this regard from entrances of the congeneric m. cerradensis ( solomon et al . c photo of chamber 1 of nest trs121009 - 02 showing the many holes punctuating the chamber wall one chamber of a fifth nest was encountered accidentally ( trs121007 - 01 ) while digging , but no data on the position of the nest entrance or other chambers were available for that nest , so it is not treated as a separately excavated nest in table 1 . its sequences indicate that the fungal cultivars collected from three nests of m. inflatus ( jsc121006 - 02 , trs121006 - 07 , and trs121007 - 01 ) all belong to subclade f of the so - called clade 2 of lower attine ( g3 ) leucocoprineaceous cultivars ( mehdiabadi et al . remarkably , the fungal species cultivated by m. inflatus in par is also grown by its congener mycetagroicus cerradensis ( solomon et al . wilson of a pholeomyia fly following a worker of the fungus - growing ant trachymyrmex septentrionalis mccook in florida and suggested a possible association of certain pholeomyia species with fungus - growing ants . gastral tergite and sternite i subequal in length , the posteriormost tip of the gaster formed by segment a7 in contrast to the ( presumably derived ) condition in some attini , in which gastral tergite i longer than sternite i so that posteriorly it overlaps the remaining segments , which are shifted anterad . the gyne of m. inflatus is , as in many ant species , quite similar to the worker except for modifications typical for the caste such as the presence of ocelli , the morphology of the mesosoma associated with wings , and the slightly larger size . in addition , in the worker the eyes are smaller , the frontoclypeal teeth are less pronounced , the frontal lobes are narrower , median anterior tubercles are present on the pronotum , and the petiole is shorter . the gyne of m. inflatus can be distinguished from the gynes of other closely related attine genera by characters that are synapomorphic for the genera . gynes of trachymyrmex species are in most cases larger in size and have integuments that are moderately to strongly tuberculate , frequently appearing spiny , most noticeably on the posterior lobes of head and on the gaster , which in m. inflatus are smooth . cyphomyrmex gynes can be similar in size to gynes of m. inflatus , but are immediately distinguished by the strongly expanded frontal lobes , often covering a large portion of the head , and in the strigatus and wheeleri groups accompanied by deep antennal scrobes . the gyne of the congeneric m. triangularis can be distinguished from that of m. inflatus by its slightly larger size , the presence of a median longitudinal ridge posterior to the frontal triangle , the presence of a distinct parapsidal furrow , the presence of scutellar processes , and the rugulose microsculpture on the dorsum of first gastral segment ( a4 ) , which also bears two pronounced ridges laterally . males of m. inflatus have 12-segmented antennae , a deviation from the usual 13-segmented antennae present in the males of most attine species , although the 12-segmented condition has arisen independently elsewhere in the tribe in the genus sericomyrmex , in some cyphomyrmex species ( e.g. males of sericomyrmex can be distinguished from males of m. inflatus by the presence of dense appressed pilosity covering the whole body and longer , thicker , decumbent to erect hairs on the head , mesosoma , and dorsum of the gaster . as in most attines , the genus mycetagroicus is the sister group of the higher attini ( sericomyrmex , trachymyrmex , acromyrmex , and atta ) ( schultz and brady , 2008 ) . thus , understanding the biology of mycetagroicus is our best hope for reconstructing the attributes of the most recent common ancestor that it shares with the higher attini , especially when considered in combination with the biology of the most closely related outgroup [ currently thought to be the cyphomyrmex wheeleri clade ( schultz and brady , 2008 ) ] . reconstructing the common ancestor of mycetagroicus + the higher attini is important for reconstructing the evolutionary transitions that occurred on the branch subtending the higher attini , arguably the most ecologically significant period of evolutionary change in all of the attini . these transitions coincide with the origin of the obligate higher attine fungal symbiont , derived from a lower attine fungal species ( schultz et al . it describes the gyne of m. inflatus , increasing to two the numbers of species for which gynes are known , the other being m. triangularis ( brando and mayh - nunes , 2001 ) . the nest architecture of m. inflatus is also described , increasing to two the number of species for which nest architecture is known , the other being m. cerradensis ( solomon et al . one possibility is that the holes are somehow associated with flooding , perhaps serving as shelters for the ants during the months that the nests are submerged or serving in some way to capture and hold pockets of air . this conjecture gains support from the observation that similarly punctured chamber walls , as described above , were observed in the deepest chambers of colonies of the sympatric but very distantly related species kalathomyrmex emeryi , colonies of which are certainly flooded during the rainy season . the nest architecture of mycetagroicus cerradensis , the only other congeneric species for which nesting biology is known , shares some characteristics with that of m. inflatus , including : a single nest entrance , chambers that are more or less vertically arranged below the nest entrance , and an extremely deep lowermost garden chamber ( solomon et al . this hypothesis applies equally to m. inflatus because , in both species , nests were excavated in the dry season and the uppermost nest chambers were found to be empty . seasonal repositioning of fungus gardens has been recorded for some other fungus - growing ants , including two species of the genus mycocepurus ( rabeling et al . perhaps the most potentially important result of this study is the identification of the fungal cultivars grown by m. inflatus at the study sites on the araguaia river . the gardens from all three collected nests belong to the same fungal species ( clade 2 , subclade f ; mehdiabadi et al . , 2012 ) , suggesting that m. inflatus may depart from most lower attine species in possessing a high degree of symbiont fidelity . even more surprising , m. inflatus cultivates the same fungal species as its congener m. cerradensis in minas gerais , brazil , over 1000 km to the south ( solomon et al . field research during the past two decades has established that attine cultivars are frequently transmitted horizontally between colonies , including colonies of different ant species , and that free - living fungi are frequently imported into the lower attine symbiosis as well ( mueller et al . , 2007 ; 2010 ) and a general expectation of weak symbiont fidelity and frequent reassociations of ants and fungi in attine agriculture . interestingly , the genus mycetagroicus is the sister group to the higher attini and the c. wheeleri group is thought to be the sister group to the clade containing mycetagroicus and the higher attini , suggesting that an increase in symbiont fidelity may have preceded the origin of the higher attini . obviously , additional samples are necessary for judging whether this pattern of symbiont fidelity continues to hold or whether the observed association with the same fungal species within and across species of mycetagroicus is due to a rare coincidence . three of the four species in the genus mycetagroicus are found in the cerrado ; the fourth , m. inflatus , is so far known from seasonally flooded riverbanks in a region characterized as dense alluvial forest in such habitats , it may be the case that some fungal symbionts are better adapted than others , providing conditions that favor symbiont choice and long - term , close associations of particular ant and fungal species .

the majority of studies on the prehospitalisation phase of cerebral ischaemia ( ischaemic stroke [ is ] and transient ischaemic attack [ tia ] ) have largely been conducted by emergency medical personnel [ 13 ] and have primarily focused on technical considerations , general practitioners ' ( gp ) knowledge of cerebrovascular events , the time lapses before medical care is sought , and the measures needed to shorten these delays . however , the dynamic nature of cerebral ischaemia has been emphasised , and the process in terms of time and space involved in the transition from reversible ischaemia to irreversible infarction is not a uniform one . this may explain the extremely variable outcomes for any given trunk occlusion , including the spectacular shrinking deficit described by kraemer et al . . evidence suggests that management of is patients is not always adapted to individual pathophysiological states and that the natural course of is needs to be assessed with an array of simple and available tests . rapid assessment is all the more crucial given that thrombolytic treatment is highly time dependent ( <3 hours after symptom onset ) . although the management of cerebral ischaemia is considerably helped by neuroimaging techniques such as diffusion - perfusion mr imaging ( dwi - pwi ) , these techniques can not replace clinical findings such as symptom intensity and duration . identifying the initial course and thresholds may constitute a predictive diagnostic tool and lead to better acute management of stroke . indeed placebo group analyses from pivot studies have demonstrated a good reliability between baseline national institutes of health stroke scale ( nihss ) scores on admission and functional outcome or hospital disposition after stroke [ 68 ] . to our knowledge , only one recent study has specifically analysed the natural course of cerebral ischaemia to predict outcome . the aims of this study were firstly to determine the early clinical findings that are predictive of outcome in acute stoke , and secondly , to establish the thresholds for deficit severity and symptom duration that make it possible to stratify outcome . the study had a prospective , observational cohort design . from september 2000 to march 2001 , patients admitted to besanon university hospital within 24 hours of symptom onset with clinical and neuroimaging patterns of is or tia were screened for inclusion in the study ( n = 361 ) . we selected arterial ischaemia and excluded cerebral venous thrombosis and intracerebral haematoma or patients who had previously had a stroke and were dependent ( corresponding to a modified rankin scale ( mrs ) score of 3 ) . patients were excluded as follows : 98 were admitted without consulting a gp , 27 had intraparenchymatous haematoma , 4 had cerebral venous thrombosis , 24 is patients were admitted > 24 hours after symptom onset and two were receiving thrombolytic therapy . among the 206 is patients admitted within < 24 hours , 52 were excluded when the precise course of their symptoms could not be obtained ( n = 32 ) , because the gp could not be contacted or could not establish relevant and detailed clinical patterns ( n = 18 ) , or when the patient showed symptoms of events similar to is ( epilepsy , migraine ) which could cast doubt on their diagnosis ( 2 cases ) . the remaining 154 eligible patients were assessed according to the besanon stroke registry criteria , reported in greater detail elsewhere . tia was defined according to a symptom duration of < 24 hours ( pre-2004 definition ) . for the purposes of this study , posterior and anterior circulation strokes were not differentiated , and side of deficit was not specified . the first , termed the neurological dysfunction score ( nds ) , reflected the patient 's and/or his / her family 's evaluation of the symptoms and their variation prior to assessment on admission ( table 3 ) . for patients who had been referred by a gp , nihss - gp was performed as soon as possible ( up to 12 hours ) after admission by a phone interview with the gp using a standard procedure . upon admission , the in - hospital clinical examination was carried out to establish a new nds ( nds-1 ) and an admission nihss ( nihss-1 ) . in order to monitor stroke course and to estimate the maximal duration of symptoms within 24 hours , a further examination took place every 8 hours during the first day of hospitalisation ( nihss-1a , nihss-1b ) , 24 hours after admission ( nihss-2 ) , and finally at discharge ( nihss-3 ) . the nds was used to assess the variation of the deficit before admission , to identify the degree of variance in the course of is , and to ensure relevance and agreement in score evolution ( nihss and nds ) . this was most frequent for the following items : dysarthria ( 21 patients ) , neglect ( 11 patients ) , visual loss ( 9 patients ) , and ataxia ( 8 patients ) . for these items , the nihss - gp item was considered to have the same value as the corresponding nihss-1 item if the nds was stable during the same period . these relate to initial onset of symptoms ( evaluated by nds ) , intensity of initial deficit ( evaluated by nihss - gp ) , duration of maximal scores ( plateau evaluated by patient 's and/or relatives ' anamnesis and controlled by the nds ) , course of deficit ( evaluated by nihss and nds score changes ) , maximum nihss ( out of nihss - gp , nihss-1 , nihss-1a , nihss-1b , and nihss-2 ) during the first 24 hours ( nihss - max ) , and maximum nds ( nds - max ) . threshold outcomes were established based on the symptom duration plateau corresponding to the average plateau duration estimated by the patient and/or his / her family once the symptoms had regressed , and by nihss - max for the intensity of signs corresponding to the average nihss - max score in the cohort . a worsening of neurological deficit was defined as an increase of 4 + points on the nihss and 2 + on the nds . conversely , a decrease of 4 + points on the nihss and 2 + points on the nds represented a regression . neurological deficit was considered stable for variations below 4 and 2 points on the nihss and the nds respectively . statistical analysis was carried out using spss software , version 13.0 ( spss inc , 2005 ) . the chi - square test , the student 's t - test , linear correlation , variance analysis and roc curves were used to determine outcome thresholds . outcome was considered poor if patients died or were dependent ( i.e. , a score of 4 - 5 on the mrs ) or good if patients were left with slight or no disability ( mrs 0 - 1 ) . the cut - offs corresponded to the point of the roc curve where the tangent to the slope ( s ) was equal to s = ( ( 1 p)/p ) , where p represents the prevalence of the group considered as positive according to the besanon stroke registry data and represents the error overcost due to false positive ( fp ) over that due to false negative ( fn ) , in comparison to real positive ( rp ) and real negative ( rn ) diagnoses . was calculated using the following formula : = ( cost fp - cost rn)/(cost fncost rp ) . ratio was fixed at 2 in order to increase the positive predictive value ( ppv ) ( reliability of the prognosis for the family ) , and when thrombolysis was considered , the risk of haemorrhagic transformation when treating by excess , over the benefit lost when not treating by default . the relative risks used were the same as those from the meta - analysis of results obtained by hacke et al . . ppv and negative predictive value ( npv ) thresholds were calculated using the following formulae : ppv = p se / p se + ( 1 p)(1 sp ) , and npv = ( 1 p ) sp/(1 p ) sp + p(1 se ) where p represents the prevalence of the positive - considered group in the cohort . the model was subsequently evaluated on a new and prospective cohort of 157 patients to validate the sensitivity and specificity of the different thresholds . the study population comprised 154 patients including 88 men and 66 women ( sex ratio 1.33 ) . the mean age was 69.7 years 15.6 ( 2296 years ) . in 36 ( 23% ) patients , the neurological deficit regressed within 24 hours . in 26 ( 17% ) , the clinical presentation suggested an anterior circulatory system tia and in the other 10 ( 6.5% ) a tia in the posterior circulatory system . selected patients were all admitted within 24 hours of symptom onset , <3 hours for 45 ( 29.4% ) patients and < 6 hours for 89 ( 57.5% ) . symptom onset was sudden in 116 ( 75.3% ) patients , progressive in 14 ( 9.2% ) , and unknown in 24 ( 15.5% ) . clinical course during the first 24 hours was monophasic in 134 ( 87% ) patients . this represented a stable initial deficit in 62 ( 40.2% ) , a total regression of symptoms in 36 ( 23.4% ) , and a partial regression in 36 ( 23.4% ) . in 20 ( 13% ) patients , the initial presentation was polyphasic , that is , fluctuating in 10 ( 6.5% ) and progressive in 10 ( 6.5% ) . there was a good correlation between the scores obtained on admission for nds and nihss-1 ( r = 0.87 ) . this was also the case for nihss - gp and nihss-1 scores on admission ( r = 0.79 ) ; mean time between the two physical examinations was 610.7 minutes . of the 36 tia patients , 8 ( 22.2% ) showed changes in their follow - up imaging studies ( 3 mri and 5 ct - scan ) . however , only 12/28 ( 42.8% ) patients with lesion - free tia underwent mri . the severity of neurological deficit , identified by nihss - max or nds - max scores and deficit plateau , was correlated with the type of ischaemia ( lesion - free tia , tia with lesion , stroke ) , but no significant differences were revealed between lesion - free tia and tia with lesion . in addition , initial deficit severity and plateau duration were strongly correlated with outcome after hospitalisation ( table 2 ) . in 150 ( 97.2% ) patients ( = 44.1 ; p < 0.0001 ) , a regression of the initial neurological signs within the first 24 hours was associated with a good prognosis ( mrs 0 - 3 ) . however in 92 ( 59.7% ) patients ( = 49.2 ; p < 0.0001 ) , a stable condition was significantly correlated with a negative prognosis ( mrs 4 - 5 or death ) . nevertheless , there was no significant relationship between the mrs and fluctuating ( = 2.5 ; p < 0.11 ) or progressive courses ( = 1.2 ; p < 0.45 ) within the first 24 hours . an nihss score > 22 with a ppv of 86% and a npv of 88% ( figure 4(a ) , left ) was predictive of functional dependence ( mrs 4 - 5 ) or death . the plateau duration threshold was 1,230 minutes ( ppv = 56% and npv = 89% ) ( figure 4(a ) , right ) . an nihss score < 5 was predictive of good functional outcome with a ppv of 86% and an npv of 73% ( figure 4(b ) , left ) . the plateau duration threshold was 135 minutes ( ppv = 69% and npv = 76% ) ( figure 4(b ) , right ) . good outcome prediction ( mrs 0 - 1 ) showed 91% sensitivity and 88% specificity for nihss < 5 and 100% sensitivity and 51% specificity for a plateau duration of < 135 min . for nihss > 22 , the prediction of poor outcome ( mrs > 3 ) indicated 95% sensitivity and 85% specificity , and 67% sensitivity and 100% specificity for plateau duration > 1,230 min . although clinicians managing acute is benefit from novel therapeutic approaches and better radiological evaluation , there is a lack of quantitative clinical criteria to enable is to be stratified according to the vital and functional risks it generates . this lack of criteria is the result of rigid and uniform management of is patients . the highly unpredictable outcome for any given arterial occlusion argues for a more individualised approach to the dynamic nature of ischaemia . specific clinical criteria and temporal thresholds are more variable than neuroimaging data , even though certain models predicting outcome have recently been reported [ 1316 ] . these models include neuroradiological variables and clinical events after admission , which precludes their use in decision - making on admission . statistical models that predict functional outcome after stroke using 6 simple variables may prove useful in epidemiological studies , but until their impact on patient care and outcome has been evaluated , they should not be applied to clinical management . a study of the course of is in the first 24 hours and identification of the factors predictive of outcome could clarify nontreated patients ' evolution using initial clinical data on admission . previous studies have been based on an a priori model in which specific criteria were used to select patients . in contrast , the homogeneity and reproducibility of our results stem from the fact that our study was based on an a posteriori model involving two consecutive , prospective cohorts who were all admitted in a routine setting . covariables that predict outcome such as age , comorbidity , lesion size , and penumbra were not included for the statistical power of the tests and to ensure a practical application of the thresholds to initial clinical management . despite the good reproducibility of the nihss , a bias may have been introduced by the subjectivity of each examiner ( gps for nihss - gp , neurologist for nihss-1 ) . moreover , even if some gps had undergone initial training in the nihss , most of them did not use it regularly . to limit this bias , we excluded patients when the precise course of symptoms could not be ascertained ( 32 patients ; 20.8% ) or when gps could not provide relevant and detailed clinical data ( 18 patients ; 11.7% ) . however , data for the nihss - gp items were obtained for all selected patients , except for disregarded items ( dysarthria , neglect , visual loss , and ataxia ) . this type of retrospective nihss scoring has already been validated with similar algorithms elsewhere [ 18 , 19 ] with excellent reliability ( resp . r = 0.94 , p < 0.001 and 86% probability of correctly ranking nihss ) . using a similar methodological algorithm to perform nihss - gp , a good correlation between nihss - gp and nihss-1 ( r = 0.79 ) was observed even when the two scores were 610 minutes apart . we feel that reliability is improved by our method in which unknown nihss - gp items were either considered as equal to the corresponding nihss-1 or coded as unknown and not taken into account . furthermore , in order to limit the risk of obtaining incomplete information from the gp , the patient , or his / her relatives , the history was taken as soon as possible after admission . we also performed a double analysis of the nihss and nds scores from the patient 's perspective . this introduced a further possible source of bias , particularly due to the lack of objectivity and precision of the items ( side , evaluation of cognitive disturbance , etc . ) , but it allowed us to evaluate the coherence of the temporal course of the initial symptoms and to ensure relevance and agreement in score evolution . the good correlations between nihss - gp and nihss-1 and between nihss-1 and nds ( r = 0.87 ) both argue for the coherence of the different results and therefore for a limited bias . deficit duration in tia has already been widely discussed in the literature . as far back as 1983 , waxman and toole reported on tia patients with ct scans revealing cerebral infarction . it was later established that evidence of recent infarction on cerebral imaging ( ct scan or mri ) was directly correlated with symptom duration . in 1999 , two definitions of tia were proposed according to whether symptoms lasted less or more than 1 hour . since 2004 , the duration threshold has been reduced from 24 hours to 60 minutes . in our sample of patients with tia , 23.4% had an event as defined before 2004 , which is higher than in other comparable studies in the literature . this is probably due to the way in which medical care is organised in our hospital 's emergency room and also due to the low number of exclusions made possible by our ability to access temporal data with tia . among our tia patients , test imaging study data were modified in 22.2% of cases , despite the low number of mri studies performed ( 42.8% ) . deficit severity and duration were on average higher in lesion - free tia and lower in is . unlike kimura et al . , there was no significant difference in symptom duration between lesion - free and nonlesion - free tia . this is probably due to the lack of statistical power related to the small number of tia patients with lesion . this was true when the mean duration of symptoms exceeded 7 hours ( 442 minutes ) , and the mean duration of lesion - free tia deficit ( 75.2 minutes ) was close to the 1 hour found by kimura et al . . the severity of neurological deficit , expressed by the initial nihss , predicted the functional prognosis at the end of hospitalisation : average initial nihss of 5.5 for nondependent , 17 for dependent , and 24.1 for deceased patients . correlations between the severity of initial clinical presentation and a poor prognosis have been reported in only a few studies [ 2428 ] . in these studies , progressive stroke also has a poor prognosis , but with a progression time definition subject to high variations ( from < 6 hours to 8 days ) . moreover , degraba et al . studied the criteria predictive of negative prognosis and worsening of the initial deficit in is . according to these authors , an nihss score > 7 represents a negative threshold in terms of worsening and functional outcome using a qualitative variable . in our study the duration of neurological deficit was also a predictive criterion of functional outcome at discharge . on average , the plateau duration in our sample was > 6 hours ( 399.1 minutes ) for nondependent patients , > 15 hours ( 931.5 minutes ) for dependent patients , and close to 24 hours ( 1,230 minutes ) for patients who died . although these results appear evident in clinical practice , there is a dearth of precise data relating to the duration of the initial neurological deficit in the literature . the symptom thresholds that we defined met the selection requirements of patients for whom a therapeutic decision was likely to be considered . here the first corresponds to all patients who are likely to be dependent or dead at discharge ; the prognosis threshold has a therapeutic implication ( serious therapeutic decision ) and implications for the family ( negative prognosis announcement , application for nursing home care ) . however , the temporal criteria threshold obtained was 1,230 minutes , with a ppv of 56% and an npv of 89% . this criterion does not appear to accurately predict poor outcome since it was too long ( close to 21 hours ) , and it was negative in nearly 50% of cases . moreover , a regression of symptoms before this maximum threshold may also be predictive of an absence of functional dependence or death ( npv in 89% of cases ) . in the same way , an initial nihss score > 22 was a better predictor of poor prognosis with a ppv of 86% and an npv of 88% . this threshold would seem to more accurately define those patients who are more likely to have a poor outcome , even if it fails in about 10% of cases . secondly , although recent studies indicate that patients with mild but disabling symptoms could be treated with tpa regardless of their baseline nihss score [ 29 , 30 ] , the risk of haemorrhagic transformation secondary to thrombolysis may be greater than the expected benefits for patients whose natural course appears favourable ( mrs 0 - 1 ) . a maximum ppv is therefore required for this threshold since an aggressive therapy may result . in our study where the initial nihss was < 5 or when the initial deficit regressed within the first 135 minutes , the expected benefit of thrombolysis would have been minimal or nil in 87% and 69% of cases respectively . for initial nihss < 5 , since the gains and risks of thrombolysis are both low , it is difficult to differentiate between the positive effect of thrombolysis and patients ' natural outcome . in our study , for these two threshold values ( initial nihss was < 5 or initial deficit regressed within the first 135 minutes ) , the npv was acceptable ( 73% and 76% , respectively ) . in other words , an initial nihss score > 5 represented a good prognosis ( mrs 0 - 1 ) in only 27% of cases , and a neurological deficit that was stable for > 135 minutes was also a sign of good prognosis in only 24% of cases . the nihss thresholds as defined in our study are very similar to those recommended for selecting candidates for thrombolysis . the duration threshold would not be applicable though as it is too close to the cut - off onset - to - needle time of 180 minutes . furthermore , if symptoms start to regress at 135 minutes , thrombolysis may not be advisable , because in 69% of cases , recovery is likely to be excellent . in the stars study where patients who were admitted earliest were the last to receive treatment . this raises an important question over the possible benefits of waiting as long as possible before commencing thrombolysis in order to be certain that symptoms are not going to regress , even if national and international guidelines recommend that it be administered as early as possible [ 31 , 33 ] . conversely , a less intense set of symptoms lasting for > 24 hours does not seem to have a negative prognosis since this type of course is frequently encountered in minor infarctions . thus , the extent of ischaemic lesions could be approached using a combination of clinical and temporal data and thereby define a gradient such as nihss per minute . in conclusion , predicting natural course and stroke outcome at the acute phase seems possible . low ( < 5 ) and high ( > 22 ) nihss cut - off points are effective positive predictive values for good ( mrs 0 - 1 ) and poor ( mrs 4 - 5 or death ) outcomes . results are less conclusive for intermediate initial nihss or for thresholds for symptom duration . in order to stratify decision making , anatomophysiological data resulting from the use of functional mri techniques ( dwi - pwi ) must be associated with clinicotemporal data in order to establish precise predictive is criteria for each individual patient . indeed some authors have highlighted a higher probability of infarction growth and early neurological deterioration when a mismatch between clinical data and dwi is observed [ 34 , 35 ] . however , it seems that these data alone can not identify independent predictors of outcome at 3 months [ 36 , 37 ] . in order to stratify decision making , clinical and temporal variables should be integrated into the equation alongside neuroimaging data in order to determine natural outcome , and thus the best course of treatment .

background . few studies have analysed the natural course of cerebral ischaemia for predicting outcome . we aimed to determine the early clinical findings and the thresholds for deficit severity and symptom duration that make it possible to stratify outcome . methods . we included 154 patients with transient ischaemic attack or ischaemic stroke . stroke profiles and neurological status were assessed from onset to 24 hrs , on admission , at 48 hrs , and at discharge . outcomes were evaluated using the modified rankin scale . positive and negative predictive values were calculated for the different thresholds . the model was subsequently evaluated on a new prospective cohort of 157 patients . results . initial national institute of health stroke scale ( nihss ) score < 5 and symptoms regressing within 135 min were predictive of good outcome . initial nihss score > 22 and symptom stability after 1,230 min were predictive of physical dependency or death . conclusions . low and high nihss cut - off points are effective positive predictive values for good and poor outcomes . thresholds for symptom duration are less conclusive .

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

the majority of studies on the prehospitalisation phase of cerebral ischaemia ( ischaemic stroke [ is ] and transient ischaemic attack [ tia ] ) have largely been conducted by emergency medical personnel [ 13 ] and have primarily focused on technical considerations , general practitioners ' ( gp ) knowledge of cerebrovascular events , the time lapses before medical care is sought , and the measures needed to shorten these delays . however , the dynamic nature of cerebral ischaemia has been emphasised , and the process in terms of time and space involved in the transition from reversible ischaemia to irreversible infarction is not a uniform one . this may explain the extremely variable outcomes for any given trunk occlusion , including the spectacular shrinking deficit described by kraemer et al . . evidence suggests that management of is patients is not always adapted to individual pathophysiological states and that the natural course of is needs to be assessed with an array of simple and available tests . rapid assessment is all the more crucial given that thrombolytic treatment is highly time dependent ( <3 hours after symptom onset ) . although the management of cerebral ischaemia is considerably helped by neuroimaging techniques such as diffusion - perfusion mr imaging ( dwi - pwi ) , these techniques can not replace clinical findings such as symptom intensity and duration . indeed placebo group analyses from pivot studies have demonstrated a good reliability between baseline national institutes of health stroke scale ( nihss ) scores on admission and functional outcome or hospital disposition after stroke [ 68 ] . to our knowledge , only one recent study has specifically analysed the natural course of cerebral ischaemia to predict outcome . the aims of this study were firstly to determine the early clinical findings that are predictive of outcome in acute stoke , and secondly , to establish the thresholds for deficit severity and symptom duration that make it possible to stratify outcome . from september 2000 to march 2001 , patients admitted to besanon university hospital within 24 hours of symptom onset with clinical and neuroimaging patterns of is or tia were screened for inclusion in the study ( n = 361 ) . we selected arterial ischaemia and excluded cerebral venous thrombosis and intracerebral haematoma or patients who had previously had a stroke and were dependent ( corresponding to a modified rankin scale ( mrs ) score of 3 ) . among the 206 is patients admitted within < 24 hours , 52 were excluded when the precise course of their symptoms could not be obtained ( n = 32 ) , because the gp could not be contacted or could not establish relevant and detailed clinical patterns ( n = 18 ) , or when the patient showed symptoms of events similar to is ( epilepsy , migraine ) which could cast doubt on their diagnosis ( 2 cases ) . the remaining 154 eligible patients were assessed according to the besanon stroke registry criteria , reported in greater detail elsewhere . tia was defined according to a symptom duration of < 24 hours ( pre-2004 definition ) . for the purposes of this study , posterior and anterior circulation strokes were not differentiated , and side of deficit was not specified . the first , termed the neurological dysfunction score ( nds ) , reflected the patient 's and/or his / her family 's evaluation of the symptoms and their variation prior to assessment on admission ( table 3 ) . for patients who had been referred by a gp , nihss - gp was performed as soon as possible ( up to 12 hours ) after admission by a phone interview with the gp using a standard procedure . upon admission , the in - hospital clinical examination was carried out to establish a new nds ( nds-1 ) and an admission nihss ( nihss-1 ) . in order to monitor stroke course and to estimate the maximal duration of symptoms within 24 hours , a further examination took place every 8 hours during the first day of hospitalisation ( nihss-1a , nihss-1b ) , 24 hours after admission ( nihss-2 ) , and finally at discharge ( nihss-3 ) . the nds was used to assess the variation of the deficit before admission , to identify the degree of variance in the course of is , and to ensure relevance and agreement in score evolution ( nihss and nds ) . this was most frequent for the following items : dysarthria ( 21 patients ) , neglect ( 11 patients ) , visual loss ( 9 patients ) , and ataxia ( 8 patients ) . for these items , the nihss - gp item was considered to have the same value as the corresponding nihss-1 item if the nds was stable during the same period . these relate to initial onset of symptoms ( evaluated by nds ) , intensity of initial deficit ( evaluated by nihss - gp ) , duration of maximal scores ( plateau evaluated by patient 's and/or relatives ' anamnesis and controlled by the nds ) , course of deficit ( evaluated by nihss and nds score changes ) , maximum nihss ( out of nihss - gp , nihss-1 , nihss-1a , nihss-1b , and nihss-2 ) during the first 24 hours ( nihss - max ) , and maximum nds ( nds - max ) . threshold outcomes were established based on the symptom duration plateau corresponding to the average plateau duration estimated by the patient and/or his / her family once the symptoms had regressed , and by nihss - max for the intensity of signs corresponding to the average nihss - max score in the cohort . a worsening of neurological deficit was defined as an increase of 4 + points on the nihss and 2 + on the nds . neurological deficit was considered stable for variations below 4 and 2 points on the nihss and the nds respectively . the chi - square test , the student 's t - test , linear correlation , variance analysis and roc curves were used to determine outcome thresholds . the cut - offs corresponded to the point of the roc curve where the tangent to the slope ( s ) was equal to s = ( ( 1 p)/p ) , where p represents the prevalence of the group considered as positive according to the besanon stroke registry data and represents the error overcost due to false positive ( fp ) over that due to false negative ( fn ) , in comparison to real positive ( rp ) and real negative ( rn ) diagnoses . was calculated using the following formula : = ( cost fp - cost rn)/(cost fncost rp ) . ratio was fixed at 2 in order to increase the positive predictive value ( ppv ) ( reliability of the prognosis for the family ) , and when thrombolysis was considered , the risk of haemorrhagic transformation when treating by excess , over the benefit lost when not treating by default . the relative risks used were the same as those from the meta - analysis of results obtained by hacke et al . ppv and negative predictive value ( npv ) thresholds were calculated using the following formulae : ppv = p se / p se + ( 1 p)(1 sp ) , and npv = ( 1 p ) sp/(1 p ) sp + p(1 se ) where p represents the prevalence of the positive - considered group in the cohort . the model was subsequently evaluated on a new and prospective cohort of 157 patients to validate the sensitivity and specificity of the different thresholds . the study population comprised 154 patients including 88 men and 66 women ( sex ratio 1.33 ) . the mean age was 69.7 years 15.6 ( 2296 years ) . in 26 ( 17% ) , the clinical presentation suggested an anterior circulatory system tia and in the other 10 ( 6.5% ) a tia in the posterior circulatory system . selected patients were all admitted within 24 hours of symptom onset , <3 hours for 45 ( 29.4% ) patients and < 6 hours for 89 ( 57.5% ) . symptom onset was sudden in 116 ( 75.3% ) patients , progressive in 14 ( 9.2% ) , and unknown in 24 ( 15.5% ) . this represented a stable initial deficit in 62 ( 40.2% ) , a total regression of symptoms in 36 ( 23.4% ) , and a partial regression in 36 ( 23.4% ) . in 20 ( 13% ) patients , the initial presentation was polyphasic , that is , fluctuating in 10 ( 6.5% ) and progressive in 10 ( 6.5% ) . there was a good correlation between the scores obtained on admission for nds and nihss-1 ( r = 0.87 ) . this was also the case for nihss - gp and nihss-1 scores on admission ( r = 0.79 ) ; mean time between the two physical examinations was 610.7 minutes . however , only 12/28 ( 42.8% ) patients with lesion - free tia underwent mri . the severity of neurological deficit , identified by nihss - max or nds - max scores and deficit plateau , was correlated with the type of ischaemia ( lesion - free tia , tia with lesion , stroke ) , but no significant differences were revealed between lesion - free tia and tia with lesion . in addition , initial deficit severity and plateau duration were strongly correlated with outcome after hospitalisation ( table 2 ) . however in 92 ( 59.7% ) patients ( = 49.2 ; p < 0.0001 ) , a stable condition was significantly correlated with a negative prognosis ( mrs 4 - 5 or death ) . nevertheless , there was no significant relationship between the mrs and fluctuating ( = 2.5 ; p < 0.11 ) or progressive courses ( = 1.2 ; p < 0.45 ) within the first 24 hours . an nihss score > 22 with a ppv of 86% and a npv of 88% ( figure 4(a ) , left ) was predictive of functional dependence ( mrs 4 - 5 ) or death . the plateau duration threshold was 1,230 minutes ( ppv = 56% and npv = 89% ) ( figure 4(a ) , right ) . an nihss score < 5 was predictive of good functional outcome with a ppv of 86% and an npv of 73% ( figure 4(b ) , left ) . good outcome prediction ( mrs 0 - 1 ) showed 91% sensitivity and 88% specificity for nihss < 5 and 100% sensitivity and 51% specificity for a plateau duration of < 135 min . for nihss > 22 , the prediction of poor outcome ( mrs > 3 ) indicated 95% sensitivity and 85% specificity , and 67% sensitivity and 100% specificity for plateau duration > 1,230 min . the highly unpredictable outcome for any given arterial occlusion argues for a more individualised approach to the dynamic nature of ischaemia . specific clinical criteria and temporal thresholds are more variable than neuroimaging data , even though certain models predicting outcome have recently been reported [ 1316 ] . these models include neuroradiological variables and clinical events after admission , which precludes their use in decision - making on admission . a study of the course of is in the first 24 hours and identification of the factors predictive of outcome could clarify nontreated patients ' evolution using initial clinical data on admission . previous studies have been based on an a priori model in which specific criteria were used to select patients . covariables that predict outcome such as age , comorbidity , lesion size , and penumbra were not included for the statistical power of the tests and to ensure a practical application of the thresholds to initial clinical management . despite the good reproducibility of the nihss , a bias may have been introduced by the subjectivity of each examiner ( gps for nihss - gp , neurologist for nihss-1 ) . moreover , even if some gps had undergone initial training in the nihss , most of them did not use it regularly . to limit this bias , we excluded patients when the precise course of symptoms could not be ascertained ( 32 patients ; 20.8% ) or when gps could not provide relevant and detailed clinical data ( 18 patients ; 11.7% ) . however , data for the nihss - gp items were obtained for all selected patients , except for disregarded items ( dysarthria , neglect , visual loss , and ataxia ) . r = 0.94 , p < 0.001 and 86% probability of correctly ranking nihss ) . using a similar methodological algorithm to perform nihss - gp , a good correlation between nihss - gp and nihss-1 ( r = 0.79 ) was observed even when the two scores were 610 minutes apart . we feel that reliability is improved by our method in which unknown nihss - gp items were either considered as equal to the corresponding nihss-1 or coded as unknown and not taken into account . furthermore , in order to limit the risk of obtaining incomplete information from the gp , the patient , or his / her relatives , the history was taken as soon as possible after admission . this introduced a further possible source of bias , particularly due to the lack of objectivity and precision of the items ( side , evaluation of cognitive disturbance , etc . ) , but it allowed us to evaluate the coherence of the temporal course of the initial symptoms and to ensure relevance and agreement in score evolution . the good correlations between nihss - gp and nihss-1 and between nihss-1 and nds ( r = 0.87 ) both argue for the coherence of the different results and therefore for a limited bias . as far back as 1983 , waxman and toole reported on tia patients with ct scans revealing cerebral infarction . it was later established that evidence of recent infarction on cerebral imaging ( ct scan or mri ) was directly correlated with symptom duration . in 1999 , two definitions of tia were proposed according to whether symptoms lasted less or more than 1 hour . in our sample of patients with tia , 23.4% had an event as defined before 2004 , which is higher than in other comparable studies in the literature . deficit severity and duration were on average higher in lesion - free tia and lower in is . , there was no significant difference in symptom duration between lesion - free and nonlesion - free tia . this is probably due to the lack of statistical power related to the small number of tia patients with lesion . this was true when the mean duration of symptoms exceeded 7 hours ( 442 minutes ) , and the mean duration of lesion - free tia deficit ( 75.2 minutes ) was close to the 1 hour found by kimura et al . . the severity of neurological deficit , expressed by the initial nihss , predicted the functional prognosis at the end of hospitalisation : average initial nihss of 5.5 for nondependent , 17 for dependent , and 24.1 for deceased patients . correlations between the severity of initial clinical presentation and a poor prognosis have been reported in only a few studies [ 2428 ] . in these studies , progressive stroke also has a poor prognosis , but with a progression time definition subject to high variations ( from < 6 hours to 8 days ) . studied the criteria predictive of negative prognosis and worsening of the initial deficit in is . according to these authors , an nihss score > 7 represents a negative threshold in terms of worsening and functional outcome using a qualitative variable . in our study the duration of neurological deficit was also a predictive criterion of functional outcome at discharge . on average , the plateau duration in our sample was > 6 hours ( 399.1 minutes ) for nondependent patients , > 15 hours ( 931.5 minutes ) for dependent patients , and close to 24 hours ( 1,230 minutes ) for patients who died . here the first corresponds to all patients who are likely to be dependent or dead at discharge ; the prognosis threshold has a therapeutic implication ( serious therapeutic decision ) and implications for the family ( negative prognosis announcement , application for nursing home care ) . this criterion does not appear to accurately predict poor outcome since it was too long ( close to 21 hours ) , and it was negative in nearly 50% of cases . moreover , a regression of symptoms before this maximum threshold may also be predictive of an absence of functional dependence or death ( npv in 89% of cases ) . in the same way , an initial nihss score > 22 was a better predictor of poor prognosis with a ppv of 86% and an npv of 88% . secondly , although recent studies indicate that patients with mild but disabling symptoms could be treated with tpa regardless of their baseline nihss score [ 29 , 30 ] , the risk of haemorrhagic transformation secondary to thrombolysis may be greater than the expected benefits for patients whose natural course appears favourable ( mrs 0 - 1 ) . in our study where the initial nihss was < 5 or when the initial deficit regressed within the first 135 minutes , the expected benefit of thrombolysis would have been minimal or nil in 87% and 69% of cases respectively . for initial nihss < 5 , since the gains and risks of thrombolysis are both low , it is difficult to differentiate between the positive effect of thrombolysis and patients ' natural outcome . in our study , for these two threshold values ( initial nihss was < 5 or initial deficit regressed within the first 135 minutes ) , the npv was acceptable ( 73% and 76% , respectively ) . in other words , an initial nihss score > 5 represented a good prognosis ( mrs 0 - 1 ) in only 27% of cases , and a neurological deficit that was stable for > 135 minutes was also a sign of good prognosis in only 24% of cases . the duration threshold would not be applicable though as it is too close to the cut - off onset - to - needle time of 180 minutes . furthermore , if symptoms start to regress at 135 minutes , thrombolysis may not be advisable , because in 69% of cases , recovery is likely to be excellent . in the stars study where patients who were admitted earliest were the last to receive treatment . this raises an important question over the possible benefits of waiting as long as possible before commencing thrombolysis in order to be certain that symptoms are not going to regress , even if national and international guidelines recommend that it be administered as early as possible [ 31 , 33 ] . in conclusion , predicting natural course and stroke outcome at the acute phase seems possible . low ( < 5 ) and high ( > 22 ) nihss cut - off points are effective positive predictive values for good ( mrs 0 - 1 ) and poor ( mrs 4 - 5 or death ) outcomes . results are less conclusive for intermediate initial nihss or for thresholds for symptom duration . in order to stratify decision making , anatomophysiological data resulting from the use of functional mri techniques ( dwi - pwi ) must be associated with clinicotemporal data in order to establish precise predictive is criteria for each individual patient . however , it seems that these data alone can not identify independent predictors of outcome at 3 months [ 36 , 37 ] . in order to stratify decision making , clinical and temporal variables should be integrated into the equation alongside neuroimaging data in order to determine natural outcome , and thus the best course of treatment .

in 2009 , glaxosmithkline began test marketing an affordable brand of horlicks , its ubiquitous malt - based drink , to be sold in 2.5 rupee sachets in villages across andhra pradesh in south india . in marketing the product , which it called asha ( hope , in hindi ) , glaxosmithkline claimed it would provide poor rural consumers with an alternative to local cereal mixes of what they call , pepsico india launched a new salty biscuit product called lehar iron chusti , which it rolled out across andhra pradesh in 2 rupee packets , alongside a major education campaign on iron deficiency for women and teenagers . at the same time , in neighbouring orissa , coca - cola was launching vitingo , a new sachet - based orange - flavoured drink fortified with 12 vitamins and minerals that it promoted as helping to combat blindness , anemia and other common diseases , in collaboration with a local ngo and self - help group . these are all examples of what industry and marketing groups refer to as nutraceuticals : nutritionally fortified or engineered foods , beverages or supplements that are marketed for their health - giving properties . in recent years , indian economists and market research agencies have championed the indian nutraceuticals market as a potential engine of growth . with current growth rates exceeding 18% and market forecasts of a fivefold increase by 2020 , nutraceuticals are celebrated as the most successful sector of the food and pharmaceuticals market ( ikon marketing consultants , 2013 ; indian business news agency , 2012 ) . in 2010 , revenues from the indian nutraceutical industry were estimated at us $ 2 billion ( frost & sullivan , 2011 , p. 5 ) . by 2017 , they are expected to reach us $ 4.2 billion ( techsi research , 2013 ) . the potential for expansion is deemed to be vast , with an envisaged latent market of 148 million customers ( ernst & young , 2012 , p. 25 ) . until recently , the nutraceutical industry had concentrated on responding to the rise in obesity and lifestyle diseases in india by marketing more expensive , higher margin products to india s aspirant middle class . marketing consultants celebrate this population as having a growing awareness of health and lifestyle diseases , rising disposable incomes and , increasingly , a self - care ethos that is associated with a demand for preventative healthcare products . to market analysts , food and beverage companies appear to be dividing their urban portfolio into complementary segments of health and wellness on the one hand and indulgence on the other ( gupta , 2009 ) . horlicks is now the second - biggest selling beverage in india after bottled water and has double the sales figures of coca - cola and pepsi combined . but with intense competition in the urban sector , the focus of both domestic and multinational companies is now moving to the design of affordable nutraceutical goods for consumers at the bottom of the pyramid ; indians who live on less than $ 2 a day ( prahalad , 2009 ) . this is a largely rural population whose anticipated purchasing power is based on an economy of speed and scale rather than the size of individual incomes . where the penetration rate of nutraceuticals in urban areas is 22.51% , in rural areas it is currently only 6.32% . for the growing nutraceutical industry and those seeking to make new acquisitions in this area , this presents a huge latent market that could ultimately account for a third of the market scope ( ikon marketing consultants , 2013 ) . the migration of the nutraceuticals industry into rural markets has seen the industry shift away from the claim that their products can prevent non - communicable diseases associated with urban working lifestyles , such as diabetes , cardiovascular disorders and cancers , and towards the claim that products specifically designed for the rural poor can tackle child malnutrition and save vulnerable lives . such claims are couched in a language of humanitarianism and focused on a target that the food industry increasingly shares with the international development sector : micronutrient deficiency , its long - term health implications and its national and global economic cost . today , double burden of obesity and malnutrition is a double opportunity . with the rise of micronutrient deficiency as a development problem , an international consensus has emerged around the corporate laboratory and marketing agency as the solution ( kimura , 2013 ) . where government food programs in india have failed to address rural malnutrition , it is anticipated that big food , with its scientific , marketing and distribution capacities can succeed . selling nutraceuticals to the rural poor , according to governments ; the food industry , a global community of market consultancy agencies and accountancy firms ; and the global development community , is a clear case of doing well by doing good. this commentary sketches out the politics of the expansion of affordable , fast - moving nutraceutical products into rural india , with a focus on fortified foods and beverages . it examines the relationships between industry , government and humanitarian organisations that are being forged alongside the development of markets for nutraceuticals ; the production of evidence and the harnessing of science to support nutraceutical companies claims ; the ways in which nutraceuticals are being marketed and distributed in rural areas ; and the concepts of health and wellbeing that are being promulgated through those marketing campaigns . lastly , it asks what kinds of impact fast - moving nutraceuticals are likely to have on the lives of india s rural poor . it concludes by questioning how smooth a transition to nutraceutical consumption big food marketing strategies can really achieve and how readily low - income families seeking to feed their families and safeguard health will actually adopt concepts of wellness and internalise micro - nutrient associated risks . the us physician , dr stephen de felice , claims to have coined the term nutraceuticals in 1989 as follows : a food or part of a food that provides medical or health benefits , including the prevention and/or treatment of a disease. for de felice , this category includes any food that we already eat , but which molecular and clinical research establishes as having a particular health benefit . in other words , according to this definition , a food can become a nutraceutical simply through the production of new scientific knowledge . as food science has developed in relation to advances in molecular biology , the term has more commonly come to refer to foods that have been transformed and enhanced through technological processes to have particular health - giving properties . such processes include the fortification of foods with vitamins and minerals , the biofortification of food through plant breeding for nutrient - rich crops and the manufacture of dietary supplements . despite the wide circulation of the term and its inclusion in the oxford english dictionary , a review of the literature shows that nutraceuticals do not yet constitute a legal entity or specific object of regulation in any country . instead , the term has largely been used and promoted by representatives from the global food industry , for whom the nutraceutical s association with scientific evidence and medicine generates additional economic value . indeed , nutraceuticals might be defined as food products that have been created solely for the capacity to make health claims about them ( katan & roos , 2004 ; schneider , n.d . ) . how did the privately manufactured and marketed nutraceutical product also become a cornerstone of the global development agenda ? by the late 1990s , international development organisations such as unicef or the world bank , had shifted from talking about hunger as a function of poverty and famine to talking about hidden hunger. the focus was now on the quality as well as the quantity of food , and the qualities that foods have were monitored at a molecular level . hidden hunger was defined as a lack of micronutrients vitamins and minerals such as iron , zinc , vitamin a and iodine . as the food sociologist kimura notes , over the course of the 1990s , the hidden hunger concept slowly accrued global authority through discussion at a series of international conferences on nutrition , global food security and child health , including the 1990 world summit for children , the 1991 montreal meeting ending hidden hunger , the 1992 international conference on nutrition and the 1996 world food summit ( kimura , 2008 ) . the global consensus that micronutrient deficiency is a high priority area is today indicated by its incorporation into the programs of bi - lateral and multi - lateral development agencies such as dfid and ausaid and its inclusion in the first millennium development goal . the global distribution of micronutrient deficiency is now monitored , measured and mapped by a growing number of international organisations and academics . by all these measures , fifty - six percent of ever - married women and 70% of children under the age of five are anemic ( international institute for population science ( iips ) and macro international , 2007 ) and at least 15% are at risk of iodine deficiency ( chakravarty & sinha , 2002 ) . in this context , the affordable nutraceutical that is designed for the rural poor is celebrated as a humanitarian technology , capable of relieving suffering and ameliorating the loss of life in contexts of crisis ( cross , 2013 ; redfield , 2012 ) . the notion of hidden hunger has also been picked up by the international economic community , who draw on medical research about the effects of micronutrient deficiency on long - term health and mental development to make links between hidden hunger and human capital . general improvements in health to the specified mental and physical benefits of micronutrients that has been significant for economists at institutions like asian development bank ( adb , 2004 ) and world bank ( 2006 ) : well - nourished people , it is claimed , are able to work harder and be potentially more innovative ( bekefi , 2006 , p. 8) . the link between micronutrient deficiency and economic development is rendered concrete and measured by disability - adjusted life years ( dalys ) , with a recent study suggesting that india lost 1000 dalys to micronutrient deficiencies per 100,000 population ( muthayya et al . based partly on daly estimates , the copenhagen consensus of 2004 convened by a panel of distinguished economists determined that after the control of hiv / aids , providing micronutrients had the best economic benefit - to - cost ratio of alleviating poverty in the developing world ( copenhagen consensus center , 2004 ) . governments have been providing vitamin and iron supplements , for example , to pregnant women since the 1970s . however , the growing prominence of the concept of micronutrients as a scientific category and the concept of hidden hunger as a major cause of ill - health and poverty in the development industry has coincided with , on the one hand , developments in food science that have enabled the isolation and transformation of individual bioactive components in foods ( kim , 2012 , p. 7 ) and on the other hand , the growing consensus that food fortification is the best solution to micronutrient deficiency . like its counterpart , obesity , micronutrient deficiency has been artifactually constructed through epidemiological measures and economic algorithms in ways that shape its problematisation and subsequent interventions ( guthman , 2013 ) . since the 1990s , we have seen the demise of alternative solutions to micronutrient deficiency , such as supplementation , kitchen gardens or dietary education , in favour of the notion that the most effective and efficient point of transformation for established diets is the corporate laboratory ( morris , cogill , & uauy , 2008 ; victora , 2009 , p. 1124 ) . as a world bank report describes food fortification : probably no other technology available today offers as large an opportunity to improve lives and accelerate development at such low cost and in such a short time ( the world bank , 1994 ) . affordable fortified foods have emerged as the site of opportunity in which market growth , healthy economies and humanitarian ethics coincide . with the shift from poverty and hunger to hidden hunger and micronutrient deficiency , government responses to malnutrition have expanded from direct humanitarian action through food aid to the creation of an enabling environment , in which affordable fortified food markets can flourish . in india , the integrated child development scheme began in 1975 as a subsidised food program that distributes basic foods such as wheat and grain to poor families in anganwadi centres , village - level centres for government - funded social welfare programmes . yet , 30 years after the establishment of the icds , the most recent national family health survey reported that 48% of the country s children under 5 are stunted , 20% are wasted and 43% are underweight ( international institute for population science ( iips ) and macro international , 2007 ) . the state - sponsored food program has been widely criticised for its inefficiency and corruption . more recently , the influential development economists abhijit banerjee and esther duflo have argued that food subsidies , which are designed to ameliorate poverty - related hunger , are an inadequate response to micronutrient deficiency because they fail to address the fact that the poor can usually afford to buy enough food but prioritise better - tasting food : developing ways to pack foods that people like to eat with additional nutrients , and coming up with new strains of nutritious and tasty crops that can be grown in a wider range of environments , need to become priorities for food technology , on an equal footing with raising productivity . ( banerjee & duflo , 2011 , 40 ) developing ways to pack foods that people like to eat with additional nutrients , and coming up with new strains of nutritious and tasty crops that can be grown in a wider range of environments , need to become priorities for food technology , on an equal footing with raising productivity . ( banerjee & duflo , 2011 , 40 ) packing better - tasting food with micronutrients is where the private sector is seen to have skills and resources that are not always available to governments . while the state is still thought to have a role in regulation and facilitation , the private sector is increasingly considered to have the intellectual property , manufacturing capacities and marketing skills needed to take food fortification the last mile ( kimura , 2013 ) . private partnership is now the favoured model for improving nutrition in developing countries for the world bank , the who and the millennium project taskforce on hunger . in 2002 , the un established the global alliance for improved nutrition ( gain ) as a collaborative mechanism for business , civil society and governmental organisations to address micronutrient challenges worldwide . gain received funding from the bill and melinda gates foundation , the canadian development agency , usaid and administrative support from the undp . private national fortification programs , for research to remove technical challenges in food fortification and to support market mechanisms for securing access to fortified foods . to obtain a gain grant , the organisation requires that each country establish a national fortification alliance , made up of government and corporate membership , to ensure that the appropriate foods are fortified and that they are made available through market mechanisms . gain aims to provide corporations with technical assistance to scale up access to their products through facilitating partnerships such as school feeding programs , joint awareness - raising and educational activities with organisations such as unicef and the flour fortification initiative , assistance in social marketing and helping to promote food fortification through publications and reports . in 2008 , gain opened an india office to assist ngos and businesses to create and distribute with funding from gain and the world bank institute , the business alliance for food fortification was launched in beijing 2005 to promote relationships between the food industry and the international development sector . the baff s purpose , as stated on their website , is to identify new financial mechanisms and new business models , expand scientific knowledge and expertise in fortification , and catalyse joint action between companies , development partners , and government. the private sector , it states , is crucial in providing the products , technology and marketing for the creation of market - viable and sustainable food fortification. for multinational companies like coca - cola or unilever , who are co - chairs of the baff , or danone , who joined the gain board in 2006 , these organisations create opportunities for collaboration and networking with representatives from government and development agencies , and access to public and philanthropic funding and political support . all these companies are seeking to expand their fortified foods portfolios in india . today , coca - cola , pepsico and glaxosmithkline each distribute and market their bottom - of - the - pyramid nutraceutical products in partnership with local ngos and microenterprise organisations . in many cases , these ngos have received funding from global organisations like unicef ; in some cases , mediated by gain , to provide educational programs on micronutrients in rural areas . educational campaigns by ngos and development agencies that are run in concert with the rollout of new fortified food products prime a market for corporate access . such partnerships are indications of the creeping privatisation of health education ( powell , 2014 ) . for their nutristar drink fortified with patented triple - fortification technology for iron , vitamin a and iodine , for example , proctor & gamble partnered with unicef , who provided health education about micronutrients , at the same time as proctor & gamble distributed the product . these partnerships have also been crucial to last - mile distribution strategies . in orissa , coca - cola partnered with the ngo biswa to distribute vitingo through self help groups ; in andhra pradesh , glaxosmithkline developed a partnership with sks microfinance , a microcredit organisation , to distribute asha. the distribution of nutraceutical products in rural areas is being tied into direct - selling employment opportunities , with rural distributors being both personal consumers of the product and social conduits through whom companies can access wider social networks ( cross & street , 2009 ) . when glaxosmithkline launched asha , it added 4000 sub - distributors to its existing 500 distributors in small towns ( kashyap & raut , 2008 , p. 552 ) . to its champions , this partnership model for affordable nutraceuticals is celebrated as a mechanism for enabling access to vital health products and an engine of entrepreneurship and rural employment ( dolan , johnstone - louis , & scott , 2012 ) . yet , its critics have highlighted the ways in which gain s support for multinational companies crowd out local businesses , leading to the loss of local industry and employment opportunities . meanwhile , nutritionists have raised questions about the nutritional quality of fortified foods that are also laced with sugar and salt i.e. where better tasting also corresponds with being damaging to health ( see nestle , 2013 ) and social activists have queried the potentially dangerous marketing activities of gain partners such as nestle , in relation to infant feeding and complementary foods for young children . in a decision that appears to suggest such concerns are taken seriously , in january 2013 , the who decided to postpone formal relations with gain on the basis of its links to the food industry and the need for further information on its partners compliance with who nutritional policy ( who , 2013 ) . the acquisition of nutraceutical portfolios has become increasingly attractive to corporations already invested in pharmaceuticals , such as glaxosmithkline . according to market analysts , pharmaceuticals have increasingly narrow profit margins , especially in developing world contexts where generics are providing greater competition ( e.g. frost & sullivan , 2011 ) . over the past three years , following consultant advice , glaxosmithkline has begun to shed its pharmaceutical portfolio and expand its nutraceutical portfolio in the bric economies . nutraceuticals have lower profit margins than pharmaceuticals but require a fraction of the outlay on research and development ( ikon marketing consultants , 2013 ) . at the same time , nutraceuticals are seen as an opportunity for india s food industry and fast - moving consumer goods industry to increase revenues . making claims about the health - giving qualities of particular products enables multi - nationals to sell more expensive products in a highly competitive , often locally dominated , marketplace ( bourne partners , 2013 ) . in a widely circulated newsletter , the accountancy firm price waterhouse coopers ( pwc ) recently identified increased collaboration between the fast - moving consumer goods(fmcg ) sector and the pharmaceutical industry as the primary opportunity for innovation in the nutraceuticals sector ( pwc , 2013 ) . a clear sign of the pharmaceuticalisation of food is the establishment of scientific foundations and institutes by food and fmcg companies in india to generate the scientific evidence needed to market their products as nutraceuticals . for example , coca - cola established the beverage institute for health & wellness in 2004 ; brittania established the brittania nutritional foundation in 2009 ; and glaxosmithkline launched the horlicks nutrition academy in 2011 . these institutes conduct research to establish micronutrient deficiency in target populations and on the products developed to meet that deficiency . one effect of corporate - funded scientific research into micronutrient deficiency is that homemade food and established diets are considered inadequate and in need of fortification with commercial foods that can be better quantified and monitored in terms of their micronutrient content . as kimura argues , nutritionism the reduction of food to its nutritional qualities has shifted authority over food production and consumption from a domestic to scientific domain ( kimura , 2013 ) . the horlicks nutrition academy website , for example , features photographs of scientists in white coats and emphasises their expertise in nutritional science : our panel of scientists , nutritionists and researchers are committed to provide you with the expertise and nutritional solutions to help you lead a healthier life. indeed , kimura argues that fortified baby food is attractive to policy - makers precisely because of its potential to bypass mothers as the gatekeeper of babies health ( kimura , 2008 , p. 234 ) . yet , as critics have pointed out , most of the scientific evidence for the efficacy of nutraceuticals is generated by the corporations themselves . indeed , there is almost no regulation of the effectiveness and quality of nutraceuticals in india , precisely because they do not occupy a distinct legal category from food and beverages . it is therefore much easier to make health claims about fortified foods in india than drugs . in making scientific claims , the nutraceutical industry has borrowed from the pharmaceutical industry its dominant product testing mechanism : the randomised controlled trial . the results of randomised controlled trials are converted into dalys , which enable corporations to scale up the cost - effectiveness of nutraceutical products to a national level . as critics have pointed out , however , randomised controlled trials take place in highly controlled environments and tell us little about the impact of nutraceuticals in places where compliance may not be high : the types of delivery systems used , country characteristics , year in which the study was done , program characteristics , and costing methodologies contribute to the variability in cost estimates found in the literature . it is not clear whether these cost estimates include expenses necessary to attain such high compliance rates . ( berry , mukherjee , & shastry , 2012 , p. 6 ) the types of delivery systems used , country characteristics , year in which the study was done , program characteristics , and costing methodologies contribute to the variability in cost estimates found in the literature . it is not clear whether these cost estimates include expenses necessary to attain such high compliance rates . ) what corporate - funded nutritional academies research and how , what is left out of those studies ( e.g. the effects of poor nutritional content , such as sugar and salt , or the local availability of nutritional foods ) and how evidence from those studies is subsequently drawn on to market corporate products or garner political support demand enquiry from social scientists equivalent to recent research on the politics of evidence in the pharmaceutical industry ( mcgoey , 2012 ; will & moreira , 2010 ) . how do you make a nutraceutical market for the rural poor ? as the rising costs of health care put it beyond reach of much of this population , investment analysts anticipate that they will become increasingly susceptible to notions of wellness and its associated products . and yet the cornerstone of wellness , the micronutrient , is not a familiar concept for much of the indian population : the indian consumer s awareness about conventional nutraceutical ingredients such as omega-3 fatty acids or lutein is severely limited , and nutraceutical manufacturers need to take up the cause and spread awareness about their products to the indian masses ( frost & sullivan , 2011 , p. 5 ) . the major challenge in this area is considered to be that of translating the established acceptance of alternative and herbal remedies and supplements in indian ayurveda traditions into concepts of wellness and micro - nutrient deficiency. the problem is how to make people think about their food and their bodies in terms of micronutrient content and the long - term health risks associated with micronutrient deficiency . one of the reasons why horlicks , and the fortified beverage market in general , does so well in india may be because the commodification of ayurveda tonics is already well established . companies like himalayan , for example , repackage ayurveda herbal preparations in the symbolic forms of biomedical , english , medicine brightly coloured capsules , plastic bottles , english labels and [ distribute ] it by prescription through biomedical physicians ( cohen , 2000 , p. 133 ) . indeed , companies like horlicks achieve many of their sales through the prescription of their products by family physicians . as one marketing analyst report put it : the existence of alternative medicine in india , and the indian consumer s belief in them , could provide a platform for the nutraceutical industry to capitalize on ( frost & sullivan , 2011 , p. 5 ) . recent research on nutraceuticals in medical anthropology and sociology has concentrated on the ways in which the transformation of food into a micronutrient health product transforms people s sense of self and their relationship to their body in ways that afford new techniques of health governance ( herrick , 2011 ; powell , 2014 ) . it is suggested that the introduction of nutraceuticals is associated with a growing concern with the risks of living ( kim , 2012 ) and the fostering of individual responsibility for managing the risk of chronic disease . food becomes another everyday space in which we monitor our health status and a molecular understanding of foods expands into the social realm ( ibid . the majority of this research , however , has been carried out in urban , middle class environments , often in middle- or high - income countries . as a consequence , there remains little understanding of the concepts of wellness that may be emerging in the context of nutraceutical products designed as humanitarian technologies for the rural poor . in rural india , corporations recognise the need to market their affordable products through associations with different aspirational teleologies from those they use to market products to urban consumers . where the standard horlicks is marketed on the basis of people s aspirations for their children s educational achievement and the prevention of future lifestyle diseases , for example , affordable horlicks was rebranded as poor associations , but it also suggests that marketing agencies believe consumption in rural families , where poor health and hunger may already be the norm , is not oriented towards the prevention of ill - health , but rather a future where the health and prospects of their children may improve . the product sells the possibility for survival amidst the struggle to sustain life in the present . established supplementary food practices in rural india are seen by the nutraceutical industry as an opportunity to sell fortified foods . but how easily can one set of foods and practices be metaphorically extended to incorporate another ? as education and marketing campaigns are rolled out alongside nutraceutical products , will women in rural andhra pradesh and orissa internalise a notion of the molecular self ? will they see commercially produced nutraceuticals as better , more scientifically valid versions of the everyday foods that they give their children to promote vitality and growth ? or will nutraceuticals be received as overly expensive foods ? the possibility that food marketing experiments , such as those of micropackaging or direct selling , that are explicitly designed to overcome obstacles associated with frontier markets in culturally unfamiliar and logistically difficult environments may be less successful than anticipated is suggested by the disappearance of asha from the glaxosmithkline website and the absence of any publicly available information on the product since its test marketing stage in 2010 . the hegemonic power exerted by multinational food companies through integrated marketing techniques used to promote unhealthy foods is well documented ( e.g. jackson , harrison , swinburn , & lawrence , 2014 ) . how successful rural marketing techniques such as multilevel marketing are in promoting new concepts of health and wellness is , by comparison , relatively underexplored . this is where future in - depth , ethnographic research capable of detailing the rich worlds of food and health practices into which multinational companies seek entry for their nutraceutical products will be crucial . on the one hand , new partnerships between international development organisations and the food industry and new strategies for accessing nutraceutical markets at the bottom of the pyramid need to be approached critically . the increasingly dominant message that commercially fortified foods are the solution to hidden hunger obviates the importance of living conditions and structural impediments that may prevent families from creating a balanced diet . indeed , we should be asking why fortified foods have replaced a balanced diet as the basis for nutritional health and what is at stake in such transitions when they are made in the name of humanitarianism . as the nutritionist marion nestle pointed out in 2002 , the complexity of food composition means that no single nutrient is likely to work nearly so well as a diet rich in the fruits and vegetables from which that nutrient was isolated ( nestle , 2013 , part five ) . on the other hand , it is important to bear in mind the logistical , economic and cultural limitations that multinational food companies face when they seek to make new markets for nutraceutical products . smooth segue of corporate capitalism into the world of humanitarian food ( errington , fujikura , & gewertz , 2012 ) . but the amalgamation of the values of profit and humanitarianism in a single nutraceutical product is an achievement that must be actively and repeatedly brokered in the conference halls of the international development sector , the reports of marketing consultants and analysts , the pot - holed roads of india s rural districts and the homes of rural distributors and indian consumers . as research on analogous products , such as the selling of soap ( cross & street , 2009 ) or solar lanterns ( cross , 2013 ) as public health goods has indicated , multinationals often encounter rougher social and physical terrain in rural markets than they anticipate . just how far the nutraceutical can travel remains an important question for capitalists , development specialists , consumers and social scientists alike .

this commentary sketches out the politics of the expansion of affordable , fast - moving nutraceutical products into rural india , with a focus on fortified foods and beverages . it examines the relationships between industry , government and humanitarian organisations that are being forged alongside the development of markets for nutraceuticals ; the production of evidence and the harnessing of science to support nutraceutical companies claims ; the ways in which nutraceuticals are being marketed and distributed in rural areas ; and the concepts of health and well - being that are being promulgated through those marketing campaigns . lastly , it asks what kinds of impact fast - moving nutraceuticals are likely to have on the lives of india s rural poor . it concludes by questioning how smooth a transition to nutraceutical consumption big food marketing strategies can really facilitate and how readily low - income families seeking to feed their families and safeguard health will actually adopt concepts of wellness and internalise micro - nutrient associated risks .

Introduction What are nutraceuticals? Public-private partnership Harnessing science Selling wellness Conclusion

at the same time , in neighbouring orissa , coca - cola was launching vitingo , a new sachet - based orange - flavoured drink fortified with 12 vitamins and minerals that it promoted as helping to combat blindness , anemia and other common diseases , in collaboration with a local ngo and self - help group . these are all examples of what industry and marketing groups refer to as nutraceuticals : nutritionally fortified or engineered foods , beverages or supplements that are marketed for their health - giving properties . with current growth rates exceeding 18% and market forecasts of a fivefold increase by 2020 , nutraceuticals are celebrated as the most successful sector of the food and pharmaceuticals market ( ikon marketing consultants , 2013 ; indian business news agency , 2012 ) . the potential for expansion is deemed to be vast , with an envisaged latent market of 148 million customers ( ernst & young , 2012 , p. 25 ) . until recently , the nutraceutical industry had concentrated on responding to the rise in obesity and lifestyle diseases in india by marketing more expensive , higher margin products to india s aspirant middle class . marketing consultants celebrate this population as having a growing awareness of health and lifestyle diseases , rising disposable incomes and , increasingly , a self - care ethos that is associated with a demand for preventative healthcare products . to market analysts , food and beverage companies appear to be dividing their urban portfolio into complementary segments of health and wellness on the one hand and indulgence on the other ( gupta , 2009 ) . but with intense competition in the urban sector , the focus of both domestic and multinational companies is now moving to the design of affordable nutraceutical goods for consumers at the bottom of the pyramid ; indians who live on less than $ 2 a day ( prahalad , 2009 ) . where the penetration rate of nutraceuticals in urban areas is 22.51% , in rural areas it is currently only 6.32% . for the growing nutraceutical industry and those seeking to make new acquisitions in this area , this presents a huge latent market that could ultimately account for a third of the market scope ( ikon marketing consultants , 2013 ) . the migration of the nutraceuticals industry into rural markets has seen the industry shift away from the claim that their products can prevent non - communicable diseases associated with urban working lifestyles , such as diabetes , cardiovascular disorders and cancers , and towards the claim that products specifically designed for the rural poor can tackle child malnutrition and save vulnerable lives . where government food programs in india have failed to address rural malnutrition , it is anticipated that big food , with its scientific , marketing and distribution capacities can succeed . selling nutraceuticals to the rural poor , according to governments ; the food industry , a global community of market consultancy agencies and accountancy firms ; and the global development community , is a clear case of doing well by doing good. this commentary sketches out the politics of the expansion of affordable , fast - moving nutraceutical products into rural india , with a focus on fortified foods and beverages . it examines the relationships between industry , government and humanitarian organisations that are being forged alongside the development of markets for nutraceuticals ; the production of evidence and the harnessing of science to support nutraceutical companies claims ; the ways in which nutraceuticals are being marketed and distributed in rural areas ; and the concepts of health and wellbeing that are being promulgated through those marketing campaigns . lastly , it asks what kinds of impact fast - moving nutraceuticals are likely to have on the lives of india s rural poor . it concludes by questioning how smooth a transition to nutraceutical consumption big food marketing strategies can really achieve and how readily low - income families seeking to feed their families and safeguard health will actually adopt concepts of wellness and internalise micro - nutrient associated risks . the us physician , dr stephen de felice , claims to have coined the term nutraceuticals in 1989 as follows : a food or part of a food that provides medical or health benefits , including the prevention and/or treatment of a disease. in other words , according to this definition , a food can become a nutraceutical simply through the production of new scientific knowledge . as food science has developed in relation to advances in molecular biology , the term has more commonly come to refer to foods that have been transformed and enhanced through technological processes to have particular health - giving properties . such processes include the fortification of foods with vitamins and minerals , the biofortification of food through plant breeding for nutrient - rich crops and the manufacture of dietary supplements . despite the wide circulation of the term and its inclusion in the oxford english dictionary , a review of the literature shows that nutraceuticals do not yet constitute a legal entity or specific object of regulation in any country . instead , the term has largely been used and promoted by representatives from the global food industry , for whom the nutraceutical s association with scientific evidence and medicine generates additional economic value . how did the privately manufactured and marketed nutraceutical product also become a cornerstone of the global development agenda ? the focus was now on the quality as well as the quantity of food , and the qualities that foods have were monitored at a molecular level . as the food sociologist kimura notes , over the course of the 1990s , the hidden hunger concept slowly accrued global authority through discussion at a series of international conferences on nutrition , global food security and child health , including the 1990 world summit for children , the 1991 montreal meeting ending hidden hunger , the 1992 international conference on nutrition and the 1996 world food summit ( kimura , 2008 ) . in this context , the affordable nutraceutical that is designed for the rural poor is celebrated as a humanitarian technology , capable of relieving suffering and ameliorating the loss of life in contexts of crisis ( cross , 2013 ; redfield , 2012 ) . the notion of hidden hunger has also been picked up by the international economic community , who draw on medical research about the effects of micronutrient deficiency on long - term health and mental development to make links between hidden hunger and human capital . general improvements in health to the specified mental and physical benefits of micronutrients that has been significant for economists at institutions like asian development bank ( adb , 2004 ) and world bank ( 2006 ) : well - nourished people , it is claimed , are able to work harder and be potentially more innovative ( bekefi , 2006 , p. 8) . the link between micronutrient deficiency and economic development is rendered concrete and measured by disability - adjusted life years ( dalys ) , with a recent study suggesting that india lost 1000 dalys to micronutrient deficiencies per 100,000 population ( muthayya et al . however , the growing prominence of the concept of micronutrients as a scientific category and the concept of hidden hunger as a major cause of ill - health and poverty in the development industry has coincided with , on the one hand , developments in food science that have enabled the isolation and transformation of individual bioactive components in foods ( kim , 2012 , p. 7 ) and on the other hand , the growing consensus that food fortification is the best solution to micronutrient deficiency . since the 1990s , we have seen the demise of alternative solutions to micronutrient deficiency , such as supplementation , kitchen gardens or dietary education , in favour of the notion that the most effective and efficient point of transformation for established diets is the corporate laboratory ( morris , cogill , & uauy , 2008 ; victora , 2009 , p. 1124 ) . affordable fortified foods have emerged as the site of opportunity in which market growth , healthy economies and humanitarian ethics coincide . with the shift from poverty and hunger to hidden hunger and micronutrient deficiency , government responses to malnutrition have expanded from direct humanitarian action through food aid to the creation of an enabling environment , in which affordable fortified food markets can flourish . in india , the integrated child development scheme began in 1975 as a subsidised food program that distributes basic foods such as wheat and grain to poor families in anganwadi centres , village - level centres for government - funded social welfare programmes . yet , 30 years after the establishment of the icds , the most recent national family health survey reported that 48% of the country s children under 5 are stunted , 20% are wasted and 43% are underweight ( international institute for population science ( iips ) and macro international , 2007 ) . ( banerjee & duflo , 2011 , 40 ) packing better - tasting food with micronutrients is where the private sector is seen to have skills and resources that are not always available to governments . private partnership is now the favoured model for improving nutrition in developing countries for the world bank , the who and the millennium project taskforce on hunger . private national fortification programs , for research to remove technical challenges in food fortification and to support market mechanisms for securing access to fortified foods . to obtain a gain grant , the organisation requires that each country establish a national fortification alliance , made up of government and corporate membership , to ensure that the appropriate foods are fortified and that they are made available through market mechanisms . in 2008 , gain opened an india office to assist ngos and businesses to create and distribute with funding from gain and the world bank institute , the business alliance for food fortification was launched in beijing 2005 to promote relationships between the food industry and the international development sector . the private sector , it states , is crucial in providing the products , technology and marketing for the creation of market - viable and sustainable food fortification. for multinational companies like coca - cola or unilever , who are co - chairs of the baff , or danone , who joined the gain board in 2006 , these organisations create opportunities for collaboration and networking with representatives from government and development agencies , and access to public and philanthropic funding and political support . all these companies are seeking to expand their fortified foods portfolios in india . today , coca - cola , pepsico and glaxosmithkline each distribute and market their bottom - of - the - pyramid nutraceutical products in partnership with local ngos and microenterprise organisations . in many cases , these ngos have received funding from global organisations like unicef ; in some cases , mediated by gain , to provide educational programs on micronutrients in rural areas . educational campaigns by ngos and development agencies that are run in concert with the rollout of new fortified food products prime a market for corporate access . such partnerships are indications of the creeping privatisation of health education ( powell , 2014 ) . the distribution of nutraceutical products in rural areas is being tied into direct - selling employment opportunities , with rural distributors being both personal consumers of the product and social conduits through whom companies can access wider social networks ( cross & street , 2009 ) . when glaxosmithkline launched asha , it added 4000 sub - distributors to its existing 500 distributors in small towns ( kashyap & raut , 2008 , p. 552 ) . yet , its critics have highlighted the ways in which gain s support for multinational companies crowd out local businesses , leading to the loss of local industry and employment opportunities . meanwhile , nutritionists have raised questions about the nutritional quality of fortified foods that are also laced with sugar and salt i.e. in a decision that appears to suggest such concerns are taken seriously , in january 2013 , the who decided to postpone formal relations with gain on the basis of its links to the food industry and the need for further information on its partners compliance with who nutritional policy ( who , 2013 ) . nutraceuticals have lower profit margins than pharmaceuticals but require a fraction of the outlay on research and development ( ikon marketing consultants , 2013 ) . at the same time , nutraceuticals are seen as an opportunity for india s food industry and fast - moving consumer goods industry to increase revenues . in a widely circulated newsletter , the accountancy firm price waterhouse coopers ( pwc ) recently identified increased collaboration between the fast - moving consumer goods(fmcg ) sector and the pharmaceutical industry as the primary opportunity for innovation in the nutraceuticals sector ( pwc , 2013 ) . a clear sign of the pharmaceuticalisation of food is the establishment of scientific foundations and institutes by food and fmcg companies in india to generate the scientific evidence needed to market their products as nutraceuticals . these institutes conduct research to establish micronutrient deficiency in target populations and on the products developed to meet that deficiency . yet , as critics have pointed out , most of the scientific evidence for the efficacy of nutraceuticals is generated by the corporations themselves . indeed , there is almost no regulation of the effectiveness and quality of nutraceuticals in india , precisely because they do not occupy a distinct legal category from food and beverages . it is therefore much easier to make health claims about fortified foods in india than drugs . the results of randomised controlled trials are converted into dalys , which enable corporations to scale up the cost - effectiveness of nutraceutical products to a national level . as critics have pointed out , however , randomised controlled trials take place in highly controlled environments and tell us little about the impact of nutraceuticals in places where compliance may not be high : the types of delivery systems used , country characteristics , year in which the study was done , program characteristics , and costing methodologies contribute to the variability in cost estimates found in the literature . ( berry , mukherjee , & shastry , 2012 , p. 6 ) the types of delivery systems used , country characteristics , year in which the study was done , program characteristics , and costing methodologies contribute to the variability in cost estimates found in the literature . what corporate - funded nutritional academies research and how , what is left out of those studies ( e.g. the effects of poor nutritional content , such as sugar and salt , or the local availability of nutritional foods ) and how evidence from those studies is subsequently drawn on to market corporate products or garner political support demand enquiry from social scientists equivalent to recent research on the politics of evidence in the pharmaceutical industry ( mcgoey , 2012 ; will & moreira , 2010 ) . as the rising costs of health care put it beyond reach of much of this population , investment analysts anticipate that they will become increasingly susceptible to notions of wellness and its associated products . and yet the cornerstone of wellness , the micronutrient , is not a familiar concept for much of the indian population : the indian consumer s awareness about conventional nutraceutical ingredients such as omega-3 fatty acids or lutein is severely limited , and nutraceutical manufacturers need to take up the cause and spread awareness about their products to the indian masses ( frost & sullivan , 2011 , p. 5 ) . the major challenge in this area is considered to be that of translating the established acceptance of alternative and herbal remedies and supplements in indian ayurveda traditions into concepts of wellness and micro - nutrient deficiency. the problem is how to make people think about their food and their bodies in terms of micronutrient content and the long - term health risks associated with micronutrient deficiency . one of the reasons why horlicks , and the fortified beverage market in general , does so well in india may be because the commodification of ayurveda tonics is already well established . as one marketing analyst report put it : the existence of alternative medicine in india , and the indian consumer s belief in them , could provide a platform for the nutraceutical industry to capitalize on ( frost & sullivan , 2011 , p. 5 ) . recent research on nutraceuticals in medical anthropology and sociology has concentrated on the ways in which the transformation of food into a micronutrient health product transforms people s sense of self and their relationship to their body in ways that afford new techniques of health governance ( herrick , 2011 ; powell , 2014 ) . it is suggested that the introduction of nutraceuticals is associated with a growing concern with the risks of living ( kim , 2012 ) and the fostering of individual responsibility for managing the risk of chronic disease . food becomes another everyday space in which we monitor our health status and a molecular understanding of foods expands into the social realm ( ibid . the majority of this research , however , has been carried out in urban , middle class environments , often in middle- or high - income countries . as a consequence , there remains little understanding of the concepts of wellness that may be emerging in the context of nutraceutical products designed as humanitarian technologies for the rural poor . in rural india , corporations recognise the need to market their affordable products through associations with different aspirational teleologies from those they use to market products to urban consumers . where the standard horlicks is marketed on the basis of people s aspirations for their children s educational achievement and the prevention of future lifestyle diseases , for example , affordable horlicks was rebranded as poor associations , but it also suggests that marketing agencies believe consumption in rural families , where poor health and hunger may already be the norm , is not oriented towards the prevention of ill - health , but rather a future where the health and prospects of their children may improve . established supplementary food practices in rural india are seen by the nutraceutical industry as an opportunity to sell fortified foods . but how easily can one set of foods and practices be metaphorically extended to incorporate another ? as education and marketing campaigns are rolled out alongside nutraceutical products , will women in rural andhra pradesh and orissa internalise a notion of the molecular self ? the possibility that food marketing experiments , such as those of micropackaging or direct selling , that are explicitly designed to overcome obstacles associated with frontier markets in culturally unfamiliar and logistically difficult environments may be less successful than anticipated is suggested by the disappearance of asha from the glaxosmithkline website and the absence of any publicly available information on the product since its test marketing stage in 2010 . how successful rural marketing techniques such as multilevel marketing are in promoting new concepts of health and wellness is , by comparison , relatively underexplored . this is where future in - depth , ethnographic research capable of detailing the rich worlds of food and health practices into which multinational companies seek entry for their nutraceutical products will be crucial . on the one hand , new partnerships between international development organisations and the food industry and new strategies for accessing nutraceutical markets at the bottom of the pyramid need to be approached critically . the increasingly dominant message that commercially fortified foods are the solution to hidden hunger obviates the importance of living conditions and structural impediments that may prevent families from creating a balanced diet . indeed , we should be asking why fortified foods have replaced a balanced diet as the basis for nutritional health and what is at stake in such transitions when they are made in the name of humanitarianism . as the nutritionist marion nestle pointed out in 2002 , the complexity of food composition means that no single nutrient is likely to work nearly so well as a diet rich in the fruits and vegetables from which that nutrient was isolated ( nestle , 2013 , part five ) . on the other hand , it is important to bear in mind the logistical , economic and cultural limitations that multinational food companies face when they seek to make new markets for nutraceutical products . but the amalgamation of the values of profit and humanitarianism in a single nutraceutical product is an achievement that must be actively and repeatedly brokered in the conference halls of the international development sector , the reports of marketing consultants and analysts , the pot - holed roads of india s rural districts and the homes of rural distributors and indian consumers .

amyloid peptides are derived from the amyloid precursor protein ( app ) by sequential proteolytic cleavages by - and -secretases [ 1 , 2 ] . amyloid peptides , particularly the longer a42 species , aggregate into various and different chemical or physical entities , which are now considered the primary pathological agents in ad . controversial experimental data leave nevertheless many questions open with regards to the exact composition and size of the amyloid oligomers in vivo [ 35 ] . moreover , the precise molecular actions of the amyloid peptides and their aggregates remain unknown and enigmatic , largely because their molecular targets , or their specific receptors remain undefined . this issue raises more fundamental questions . for one , while the proteolytic enzymes and their mechanisms responsible for the generation of the amyloid peptides become well - known and understood at the molecular level , the eventual physiological function of the peptides is still questioned and remains questionable . the original pathological importance ascribed to amyloid plaques was weakened , if not eliminated based on information gathered in transgenic models expressing mutant app . we have advocated this hypothesis since we discovered early defects in cognition and ltp in app.v717i mice [ 611 ] . matters are , however , complicated further by recent data originating from several clinical trials that identified a large fraction of individuals in the control groups with considerable brain amyloid load using pet - imaging [ 1214 ] . obviously , this means that high - amyloid concentration in the brain is not per se incompatible with normal cognitive functioning in old age . this calls for other factors to be at least coresponsible for the cognitive demise in ad - forwarding protein tau as the most obvious candidate . primary tauopathy is recognized in a vast and still increasing number of cns - disorders . the pathology is visualized postmortem as intraneuronal aggregates of protein tau , known as neuropil treads and neurofibrillary tangles . these are similar if not identical to those observed in ad brain and mostly present without other associated pathological hallmarks , except inflammation markers . the large clinical variability among the primary tauopathies stems from the presence of the tau pathology in different types of neurons and in different brain - regions [ 1518 ] . biochemically , all tauopathies consist of smaller and larger aggregates and fibrils that become intertwined to form larger tangles in the soma as well as in axons and dendrites of affected neurons . the aggregates consist almost exclusively of protein tau , albeit as different isoforms in different diseases , that is , either tau.3r or tau.4r or as a variable admixture . their widely different grading of phosphorylation is usually referred to as hyperphosphorylated tau , denoted here as hp - tau . importantly , no accurate definition based on either the level or the specific phosphorylated residues can typify any tauopathy exactly , because of the large variability with each primary disease . . rare familial cases of frontotemporal dementia ( ftd ) are linked to exonic and intronic mutations in the mapt gene coding for protein tau ( chromosome 17 ) . although rare , their identification marked a major breakthrough and boosted interest in tauopathy , because of the evident implication that tauopathy itself is sufficient to cause cognitive decline and dementia [ 15 , 16 , 1821 ] . both intronic and exonic mutations have important mechanistical implications : ( i ) most expressed mutations are in the microtubule binding domain of tau ; ( ii ) intronic mutations promote or hinder splicing of exon 10 coding for the second repeat domain in the microtubule binding domain , skewing expression of isoforms to either tau3r or tau4r , but both with normal sequences and consequently normal binding - affinity for microtubules . the pathology resulting from intronic mutations in the mapt gene can only be explained by effects on rna splicing , eventually resulting in changes in absolute or relative levels of tau isoforms that otherwise have normal wild - type protein sequences . the overexpression or overrepresentation of one or the other tau isoform can then be extrapolated to the primary tauopathies that are sporadic , as well as to sporadic ad as the most prominent secondary tauopathy involving normal , wild - type tau - isoforms . the major unresolved problem in ad is the mechanistic relation of amyloid and tau pathology . amyloid oligomers are proposed to precede , and eventually trigger the intracellular tauopathy , likely by increasing the phosphorylation of protein tau [ 11 , 15 , 17 , 22 ] . while this hypothesis remains at this moment in time impossible to prove in sporadic ad cases , indeed , even in the most early onset familial ad that are caused by mutant presenilin or mutant app , and therefore by definition are amyloid - triggered , tauopathy is always evident and codiagnostic . experimentally , double and triple transgenic mouse models with combined amyloid and tau - pathology prove the same intimate relation [ 2326 ] . the genetic data imply that deranged tau - microtubule interactions , caused either directly by the mutation , by phosphorylation , by increased absolute or by disturbed relative concentrations of tau , all can contribute or even be sufficient to cause neurodegeneration in primary tauopathies , that is , in the absence of amyloid pathology . importantly , experimental decrease of the level of tau in amyloid transgenic mice can actually ameliorate several clinical symptoms and defects . the combined data further corroborate the hypothesis that protein tau is essentially contributing to amyloid - induced cognitive defects . not resolved are the identity and exact nature of the receptors and associated signaling pathways that lead from amyloid to tau . these pathways obligatory involve kinases such as gsk3 [ 25 , 26 ] that contribute , directly or indirectly to the phosphorylation of tau at residues that affect its binding to microtubuli . this is thought , but not proven , to instigate its eventual aggregation into fibrils that are deposited locally as neuropil treads or transported to the soma and form the neurofibrillary tangles , and both types of aggregates occur in the same neurons . nevertheless , tauopathy does not per se cause immediate or even delayed neuronal death , as tangled and tau - loaded neurons are observed in humans as well as in experimental models , that is , transgenic mice , zebra - fish , and flies [ 26 , 28 , 29 ] . the pertinent question to be answered then is : what chemical or physical form of protein tau causes synapto- and neurodegeneration ? invaluable insight in the problems that we are concerned with , have resulted from analyzing different transgenic mouse models . our research - group generated , characterized , and validated many different transgenic mouse strains and their bigenic combinations as preclinical models over the last two decades . so far none of the models recapitulates robustly the evident neurodegeneration observed in the brain of ad patients . we explored and reported on additional models to increase our understanding of this aspect of the pathogenesis of ad [ 11 , 30 ] . here , we review data and insights obtained with adeno - associated viral vectors that were engineered to express wild - type or mutant app or protein tau in pyramidal neurons of the hippocampus of wild - type mice . thereby , we aimed to recapitulate aspects of ad pathology and related features that are not attainable in transgenic mice , particularly neurodegeneration . among the different viral vectors available , we selected aav because these have been demonstrated as excellent and safe tools for gene delivery into the cns . recent technical advances have improved aav - based vectors as tools in neuronal research [ 31 , 32 ] . the expression of the human transgenes used in our studies was controlled by the human synapsin1 gene promoter , with constructs packed in chimeric capsids aav1/2 , expressing efficiently in pyramidal neurons of the ca1/2 subfield of the hippocampus ( figure 1 ) . control aav vectors expressed egfp in the designated neurons and areas that persisted for months without negative signs or symptoms ( figure 1 ) and [ 31 , 32 ] . we first constructed aav vectors to recapitulate the amyloid pathology in the limbic region of wild - type mice . the constructs contained app695 as the most abundant neuronal isoform , either as wild type sequence or as the engineered triple mutant ( denoted app.sla ) . the incorporated swedish , london and austrian mutations are each associated with familial forms of ad [ 3335 ] . following intracerebral injection into wild - type mice , the app.sla mutant produced a gradual accumulation of app and amyloid peptides in pyramidal neurons in the hippocampal ca1/2-region , as well as in the deeper layers of the neighboring cortex . similar expression of wild - type human app695 failed to produce any deposition of amyloid or of the related effects observed with mutant app.sla . initially , from 3 weeks to 3 months postinjection ( p.i . ) , amyloid immunoreactivity appeared as intracellular inclusions or vacuolar bodies inside pyramidal neurons . at 6 months , amyloid plaques developed as immunoreactive and thioflavins positive deposits in the hippocampus and cortex ( figure 2 ) . at this late time - point , we also observed increased phosphorylation of endogenous mouse protein tau , particularly at residues t181 and t231 , both known to be substrate for gsk3 . these phosphoepitopes , defined by antibodies at270 and at180 were differentially expressed , that is , in hippocampal pyramidal neurons ( t231 ) and in dystrophic neurites around amyloid plaques ( t181 ) . in contrast , intracerebral injection of aav-app.sla not in wild - type but in tau.p301l mice led to the formation of neurofibrillary tangles ( nft ) , demonstrating that the viral assault did not prevent protein tau from forming tangles ( figure 2 ) . moreover , the combination demonstrated that besides the primary hit , also the receiving genetic background is of utmost importance . most interesting for the interpretation of the data obtained with protein tau , discussed in the next section , was the observation that at 6 months p.i . the phosphorylation of endogenous mouse tau was increased and coincided with a minor but significant reduction in the number of neurons in the ca1 region of aav-app.sla injected mice . in second instance , we similarly expressed either wild - type tau.4r or mutant tau.p301l by aav - vectors in wild - type mouse brain . in sharp contrast to app - sla , aav - mediated expression of protein tau resulted already at 3 weeks p.i . these findings were rather unexpected and we analyzed them in different directions , yielding novel and important insight into the mechanisms causing neuronal death by protein tau . most remarkably , we could not detect any major form of aggregates of protein tau in pyramidal neurons that expressed either wild - type or mutant protein tau . extensive analysis by histochemistry and immunohistochemistry with a panel of indicator compounds and monoclonal antibodies to protein tau , did not reveal any appreciable signs of formation of intraneuronal tau - aggregates , either before or during or after the pyramidal neurons in ca1/2 succumbed . the actual levels in hippocampal extracts were near - physiological and only about twice those of endogenous mouse tau4r . note that also in the aav-app.sla model described in the previous section , the hippocampal levels of human app.sla were only about twice those of endogenous murine app . in addition , expression of egfp by the same aav - type vectors proved harmless to pyramidal neurons over long time - periods ( figure 1 ) and [ 30 , 31 ] . intracerebral injection of 3- and 10-fold less aav - tau , with consequently less expression of human protein tau , produced a graded , lesser loss of hippocampal neurons . the aav - tau - induced neurodegeneration is thereby further demonstrated to be directly caused by and proportional to the near - physiological levels of protein tau . consequently , the observed pyramidal cell - death is qualified as specific for human protein tau . this conclusion holds up for wild - type tau.4r and for mutant tau.p301l , making the model at least conform the observations in familial ftd . indeed , as discussed above , intronic and exonic mutations yield very similar clinical outcome , despite the expression of mutant or wild - type tau4r , respectively . the actual contribution of the mutation to the mechanism underlying the pathogenesis requires a rational explanation in human ftd patients , as well as in the aav - models . extensive analysis to define the mechanisms that underlie the observed tau - mediated pyramidal neurodegeneration are described , summarizing reported data and preliminary data of work in progress . similar aav - mediated expression of a truncated version of protein tau.4r , lacking the microtubule binding c - terminal domain , proved completely harmless and did not cause any neurodegeneration over similar time - periods . the inherent conclusion must be that microtubule binding of protein tau is essentially involved in the neurotoxic degenerative mechanism . consequently , we must orient further analysis to mechanisms that involve axonal and dendritic transport over microtubuli , which is of paramount importance for all cells but especially for neurons with their intricate branching . of note , microgliosis was observed to be intense , and spatially and temporally closely associated with aav - tau induced degenerating neurons . this was most recently also reported in a similar model based on aav vectors but in rats and for a different pathology in a different brain - region . previously , we have observed a similar relation of microgliosis to neurodegeneration in an unrelated model for hippocampal sclerosis , caused by conditional expression of p25 , the truncated activator of cdk5 . also , in that model , intense neurodegeneration was not marked by aggregation of protein tau , further strengthening our conclusion that cdk5 is not a major tau - kinase in vivo [ 3941 ] . the data originating from the aav - models led us to conclude that not large aggregates of protein tau cause neurodegeneration . this confirms our observations in transgenic mice that show aggregation of tau leading to tauopathy in somata and neuropil , but not accompanied by marked neurodegeneration [ 25 , 26 , 36 ] . we proposed that neurons affected by tauopathy can either engage in aggregation of tau and thereby try to decrease the toxic species and hope to survive , or to enter the path to death by failing to aggregate protein tau . clearly , the viral models comply with the transgenic models that aggregation of tau and neurodegeneration are not closely linked . thereby , questions are raised that are addressed in more detail in the next sections . the classic concept that tau - fibrils or tangles are neurotoxic in tauopathies can be abandoned . we advocate that neurofibrillary tangles , like amyloid plaques are the final pathological hallmarks , but not the neurotoxic agents . the outcome fits independent observations that the extent of neuronal loss exceeds the number of nft in patients with ad [ 42 , 43 ] . in inducible tg4510 mice , memory decline and neuronal loss are dissociated from tangle formation in time and brain region [ 44 , 45 ] . in the aav - tau injected mice , phosphorylation of tau was evident at many pathological epitopes , including those defined by antibodies at8 , at100 , at180 , and at270 . despite the increased phosphorylation no aggregates of protein tau were deposited in degenerating pyramidal neurons , analyzed histochemically or immunohistochemically . biochemical analysis indicated the formation of low molecular weight aggregates which need and deserve further analysis . intriguingly , experiments whereby aav-app.sla was injected intracerebrally not in wild - type mice but in transgenic tau.p301l mice , produced not only amyloid plaques but also intracellular tau aggregates . nevertheless , no substantial neuronal loss was evident , substantiating our previous data of absence of marked neurodegeneration in tau.p301l mice and tau.p301lxgsk3b bigenic mice ( bigt ) , despite extensive or even dramatic tauopathy [ 26 , 36 ] . the combined results from our experimental models and from a drosophila model of tauopathy consolidates the thesis that neurotoxicity is not exerted by large aggregates of protein tau but rather by tau - species that are intermediate between normally phosphorylated protein tau and the hyper - phosphorylated fibrils . the identity of the toxic tau species , which we have termed tau - p * [ 11 , 30 ] remains to be defined a challenge equaling that of the identification of the amyloid receptor . it must be remembered that tau is subject to other posttranslational modifications , besides phosphorylation . indeed , tau can be ubiquitinated , truncated , glycosylated , glycated , oxidized , and , moreover , undergoes isomerization at proline residues . at any given moment protein tau is present in various molecular forms , which most likely differ subtly in properties of binding to microtubuli , interaction with other proteins , binding to membranes , being transported for normal duty or for degradation . the decision whether neurons will enter the cell death path or protect itself by forming tauopathy , must depend on the actual levels of protein tau and on its modifications . the rapid accumulation of tau - p * will lead to cell death , while more gradual accumulation of tau - p * would result in formation of aggregates . both pathways will be affected by various external factors , for example , inflammation , amyloid , stress , and so forth . in this concept , evidence from patients , whereby ca1 hippocampal neurons survive for decades despite neurofibrillary tangles and experimental animals show tangle bearing neurons survive , even with lost membrane integrity . two major pathways are responsible for removal of damaged , unfolded , or aggregated cellular proteins : the ubiquitin / proteasome system and the autophagy / lysosomal system that also can remove aged or damaged organelles . malfunction of autophagy can result in accumulation of protein aggregates that may contribute to the disease pathology in ad and other neurodegenerative disorders [ 4952 ] . stimulating autophagy pharmacologically , for example , by inhibition of mtor , ameliorates cognitive deficits and reduces amyloid and tau pathology in the 3xtg mouse model for ad . autophagosomes form in a stepwise process that requires transport over the microtubular network , which is known to be affected by protein tau , a major microtubule - associated protein . in our mouse models , degenerating neurons in the aav - tau.p301l injected mice displayed as dark neurons with extensive vacuolization that could be taken to indicate a defective autophagic process . a similar facet of neurons we observed in an unrelated mouse model , that is , the p25 inducible mice that recapitulate hippocampal sclerosis . the combined data do lend more support to the hypothesis that vacuolization is a correlate or consequence of the degenerative process , as an intrinsic mechanism needed to clean up defective organelles , protein aggregates or even entire dead or dying neurons . consistent with this view is our observation in the aav - tau.p301l mice that vacuoles do not form early in the disease process but only after the neurons are already degenerating [ 11 , 30 ] . finally , we searched but did not find biochemical evidence for the conversion of lc3-i to lc3-ii , which is generally accepted as the necessary step in and read - out of the increased formation of autophagosomes . after differentiation from neuronal precursors , neurons enter their post - mitotic state , while attempts to reenter the cell cycle is considered pathological and eventually result in cell death . in ad and other tauopathies , cell - cycle related events we tested this hypothesis in the aav - tau model by examining cell cycle - related markers . various markers were increased , that is , cyclind2 , phosphorylated retinoblastoma protein , proliferating cell nuclear antigen ( pcna ) , cyclin b1 . however , intracerebral injection of aav - tau in cyclind2 deficient mice showed no extra effect on neurodegeneration , suggesting that cell cycle reentry is not a pre - requisite for neuronal death . microtubules ensure cell shape and constitute roads of transport , a feature prominent in neurons with the most complex architecture of all cells . microtubule dependent transport is ensured by families of motor proteins dyneins and kinesins , respectively for retrograde transport from distal processes towards soma and as plus - end directed motor for anterograde transport . first , mutant tau and hp - tau can cause it to detach from the microtubules and decrease its ability to control microtubule dynamics . on the other hand , increased levels of protein tau can saturate microtubules and hinder the foot - stepping of the motor proteins needed for axonal and dendritic transport . both aspects of tau - related transport deficits have been observed and both can fit into a model leading to starving synapses that eventually culminates in neuronal death . in this hypothesis , tau - mediated neurodegeneration is likely to start in distal neuronal processes , that is , axons and apical dendrites , which degenerate progressively towards the cell soma . synaptic pathology could be an early defect in neurodegenerative tauopathies as proper functioning of distal synapses is totally dependent on adequate transport of essential cargos and uninterrupted provision of energy . impaired oxidative phosphorylation and mitochondrial dysfunction has been observed in mouse models expressing mutant tau , which was potentiated by mutant app and presenilin1 [ 56 , 57 ] . in tau.4r transgenic mice , we previously documented axonopathy and motor deficits that were rescued by coexpression of constitutively active gsk3b [ 37 , 58 ] . the data explain how excess tau in first instance blocks transport by covering microtubuli , while gsk3 acting as major tau - kinase i phosphorylates tau to detach it from the microtubules and relieves the transport blockade . interestingly , treatment of tau.4r mice with lithium salts significantly increased the axonopathy , an effect that can be ascribed to its inhibition of gsk3b . as discussed above , aav - mediated expression of truncated tau , lacking the microtubule binding domain did not cause neurodegeneration , in marked contrast to full length protein tau ( figures 3 and 4 ) and . the combined data from various approaches allow us to conclude that tau mediated pathology is , at least in part , mediated by its microtubule binding properties . chronic inflammation is evident in brain of ad patients and proposed to contribute essentially to the vicious circle leading eventually to neuronal death , brain atrophy and severe dementia . cns - related features of the immune system remain hardly understood , and supposed to be janus - faced , that is , providing protection as well as causing damage , steered by complex mostly unknown control mechanisms [ 60 , 61 ] . protection could be impaired in ad , fueled by and fueling debates on possible prevention or therapeutic benefits of antioxidants , radical scavengers , nonsteroidal anti - inflammatory drugs ( nsaid ) , and so forth , although clinical trials do not substantiate the claims . amyloid deposits attract microglia in a phagosomal attempt of their elimination , but activated microglia release proinflammatory cytokines , chemokines , reactive oxygen species , prostaglandins , and other mediators that harm neurons . much attention has been paid to neurotoxicity mediated by free radicals , both reactive oxygen and nitrogen species , released by microglia . astrocytes are the most common cell - type in the brain , involved in many functions and increasingly appreciated also in synaptic signaling and integrity . astrocytes co - localize with microglia at damage - sites , but remain to be explored in detail in ad . strictly speaking , neurons must also be considered when discussing neuroinflammation because they ( can ) produce active factors , that is , complement , interleukins , tumor necrosis factor , acute - phase proteins . we observed and discussed that degenerating neurons were temporally and spatially closely associated with activated microglia in two independent models , that is , the aav - tau viral model and the p25 transgenic model [ 30 , 39 ] . too many unknowns prevent us from answering the question how tau - mediated damage can lead to neurodegeneration by involving activation of microglia . we maintain the most straightforward hypothesis , that is , that the expression of protein tau , either wild - type or mutant by the viral vectors , is imposing stress on the pyramidal neurons that can not adapt rapidly enough to produce tau - aggregates as a means of temporarily detoxification . the impact on various neuronal functions is evident , including disturbances in post - mitotic state and cell - cycle control , as well as disturbing microtubule - mediated transport . the latter is most destructive in axons and larger dendrites , compromising the major synapses that become dys- or nonfunctional . secreted neuronal proteins then activate nearby microglia that counter - react by secreting proinflammatory and potentially neurotoxic factors , fueling the viscous cycle that eventually leads to neuronal death . based on the combination of transgenic and viral models , and taking into account pathological and clinical data from human patients , we defend the thesis that neurodegeneration is not caused by a single defect but by at least dual actions ( figure 4 ) . the first is proposed to be intrinsically neuronal , for example , damage by accumulation of endogenous proteins in various molecular forms . in the case discussed here , protein tau is the cause in primary tauopathies . in ad , the upstream triggers are the amyloid peptides that accumulate because of mutations or other imposed defects in the proteolytic machinery that controls their formation and turnover . the second factor is also essential , and can be neuronal or microglial in origin , related to any type of stress the brain can experience or has to endure , that is , partial hypoxia , glucose overload or shortage , problems with lipid or cholesterol , or other essential metabolites . protein - aggregates , that is , plaques and tangles , are not the most essential in causing cognitive defects and dementia . the challenge to define the neurotoxic tau - species and its mode of action does nevertheless become not less complicated . the combination of transgenic and viral models illustrates eloquently the power of the approach as well as its weaknesses . we still have more modeling to do , taking into account the data and indications that are provided by ongoing clinical studies , because only the human patient can eventually confirm any hypothesis based on experimental models .

patients suffering from alzheimer 's disease ( ad ) are typified and diagnosed postmortem by the combined accumulations of extracellular amyloid plaques and of intracellular tauopathy , consisting of neuropil treads and neurofibrillary tangles in the somata . both hallmarks are inseparable and remain diagnostic as described by alois alzheimer more than a century ago . nevertheless , these pathological features are largely abandoned as being the actual pathogenic or neurotoxic factors . the previous , almost exclusive experimental attention on amyloid has shifted over the last 10 years in two directions . firstly , from the concrete deposits of amyloid plaques to less well - defined soluble or pseudosoluble oligomers of the amyloid peptides , ranging from dimers to dodecamers and even larger aggregates . a second shift in research focus is from amyloid to tauopathy , and to their mutual relation . the role of tau in the pathogenesis and disease progression is appreciated as leading to synaptic and neuronal loss , causing cognitive deficits and dementia . both trends are incorporated in a modified amyloid cascade hypothesis , briefly discussed in this paper that is mainly concerned with the second aspect , that is , protein tau and its associated fundamental questions .

1. Background 2. Transgenic and Adeno-Associated Virus Models 3. Mechanisms Underlying Tau-Mediated Pyramidal Neurodegeneration 4. Conclusions

amyloid peptides are derived from the amyloid precursor protein ( app ) by sequential proteolytic cleavages by - and -secretases [ 1 , 2 ] . amyloid peptides , particularly the longer a42 species , aggregate into various and different chemical or physical entities , which are now considered the primary pathological agents in ad . controversial experimental data leave nevertheless many questions open with regards to the exact composition and size of the amyloid oligomers in vivo [ 35 ] . moreover , the precise molecular actions of the amyloid peptides and their aggregates remain unknown and enigmatic , largely because their molecular targets , or their specific receptors remain undefined . for one , while the proteolytic enzymes and their mechanisms responsible for the generation of the amyloid peptides become well - known and understood at the molecular level , the eventual physiological function of the peptides is still questioned and remains questionable . obviously , this means that high - amyloid concentration in the brain is not per se incompatible with normal cognitive functioning in old age . this calls for other factors to be at least coresponsible for the cognitive demise in ad - forwarding protein tau as the most obvious candidate . primary tauopathy is recognized in a vast and still increasing number of cns - disorders . the pathology is visualized postmortem as intraneuronal aggregates of protein tau , known as neuropil treads and neurofibrillary tangles . the large clinical variability among the primary tauopathies stems from the presence of the tau pathology in different types of neurons and in different brain - regions [ 1518 ] . biochemically , all tauopathies consist of smaller and larger aggregates and fibrils that become intertwined to form larger tangles in the soma as well as in axons and dendrites of affected neurons . the aggregates consist almost exclusively of protein tau , albeit as different isoforms in different diseases , that is , either tau.3r or tau.4r or as a variable admixture . importantly , no accurate definition based on either the level or the specific phosphorylated residues can typify any tauopathy exactly , because of the large variability with each primary disease . rare familial cases of frontotemporal dementia ( ftd ) are linked to exonic and intronic mutations in the mapt gene coding for protein tau ( chromosome 17 ) . although rare , their identification marked a major breakthrough and boosted interest in tauopathy , because of the evident implication that tauopathy itself is sufficient to cause cognitive decline and dementia [ 15 , 16 , 1821 ] . both intronic and exonic mutations have important mechanistical implications : ( i ) most expressed mutations are in the microtubule binding domain of tau ; ( ii ) intronic mutations promote or hinder splicing of exon 10 coding for the second repeat domain in the microtubule binding domain , skewing expression of isoforms to either tau3r or tau4r , but both with normal sequences and consequently normal binding - affinity for microtubules . the pathology resulting from intronic mutations in the mapt gene can only be explained by effects on rna splicing , eventually resulting in changes in absolute or relative levels of tau isoforms that otherwise have normal wild - type protein sequences . the major unresolved problem in ad is the mechanistic relation of amyloid and tau pathology . amyloid oligomers are proposed to precede , and eventually trigger the intracellular tauopathy , likely by increasing the phosphorylation of protein tau [ 11 , 15 , 17 , 22 ] . while this hypothesis remains at this moment in time impossible to prove in sporadic ad cases , indeed , even in the most early onset familial ad that are caused by mutant presenilin or mutant app , and therefore by definition are amyloid - triggered , tauopathy is always evident and codiagnostic . the genetic data imply that deranged tau - microtubule interactions , caused either directly by the mutation , by phosphorylation , by increased absolute or by disturbed relative concentrations of tau , all can contribute or even be sufficient to cause neurodegeneration in primary tauopathies , that is , in the absence of amyloid pathology . importantly , experimental decrease of the level of tau in amyloid transgenic mice can actually ameliorate several clinical symptoms and defects . the combined data further corroborate the hypothesis that protein tau is essentially contributing to amyloid - induced cognitive defects . not resolved are the identity and exact nature of the receptors and associated signaling pathways that lead from amyloid to tau . these pathways obligatory involve kinases such as gsk3 [ 25 , 26 ] that contribute , directly or indirectly to the phosphorylation of tau at residues that affect its binding to microtubuli . this is thought , but not proven , to instigate its eventual aggregation into fibrils that are deposited locally as neuropil treads or transported to the soma and form the neurofibrillary tangles , and both types of aggregates occur in the same neurons . nevertheless , tauopathy does not per se cause immediate or even delayed neuronal death , as tangled and tau - loaded neurons are observed in humans as well as in experimental models , that is , transgenic mice , zebra - fish , and flies [ 26 , 28 , 29 ] . the pertinent question to be answered then is : what chemical or physical form of protein tau causes synapto- and neurodegeneration ? invaluable insight in the problems that we are concerned with , have resulted from analyzing different transgenic mouse models . our research - group generated , characterized , and validated many different transgenic mouse strains and their bigenic combinations as preclinical models over the last two decades . so far none of the models recapitulates robustly the evident neurodegeneration observed in the brain of ad patients . we explored and reported on additional models to increase our understanding of this aspect of the pathogenesis of ad [ 11 , 30 ] . here , we review data and insights obtained with adeno - associated viral vectors that were engineered to express wild - type or mutant app or protein tau in pyramidal neurons of the hippocampus of wild - type mice . the expression of the human transgenes used in our studies was controlled by the human synapsin1 gene promoter , with constructs packed in chimeric capsids aav1/2 , expressing efficiently in pyramidal neurons of the ca1/2 subfield of the hippocampus ( figure 1 ) . we first constructed aav vectors to recapitulate the amyloid pathology in the limbic region of wild - type mice . following intracerebral injection into wild - type mice , the app.sla mutant produced a gradual accumulation of app and amyloid peptides in pyramidal neurons in the hippocampal ca1/2-region , as well as in the deeper layers of the neighboring cortex . similar expression of wild - type human app695 failed to produce any deposition of amyloid or of the related effects observed with mutant app.sla . initially , from 3 weeks to 3 months postinjection ( p.i . ) at 6 months , amyloid plaques developed as immunoreactive and thioflavins positive deposits in the hippocampus and cortex ( figure 2 ) . these phosphoepitopes , defined by antibodies at270 and at180 were differentially expressed , that is , in hippocampal pyramidal neurons ( t231 ) and in dystrophic neurites around amyloid plaques ( t181 ) . in contrast , intracerebral injection of aav-app.sla not in wild - type but in tau.p301l mice led to the formation of neurofibrillary tangles ( nft ) , demonstrating that the viral assault did not prevent protein tau from forming tangles ( figure 2 ) . most interesting for the interpretation of the data obtained with protein tau , discussed in the next section , was the observation that at 6 months p.i . the phosphorylation of endogenous mouse tau was increased and coincided with a minor but significant reduction in the number of neurons in the ca1 region of aav-app.sla injected mice . in sharp contrast to app - sla , aav - mediated expression of protein tau resulted already at 3 weeks p.i . most remarkably , we could not detect any major form of aggregates of protein tau in pyramidal neurons that expressed either wild - type or mutant protein tau . extensive analysis by histochemistry and immunohistochemistry with a panel of indicator compounds and monoclonal antibodies to protein tau , did not reveal any appreciable signs of formation of intraneuronal tau - aggregates , either before or during or after the pyramidal neurons in ca1/2 succumbed . the actual levels in hippocampal extracts were near - physiological and only about twice those of endogenous mouse tau4r . note that also in the aav-app.sla model described in the previous section , the hippocampal levels of human app.sla were only about twice those of endogenous murine app . intracerebral injection of 3- and 10-fold less aav - tau , with consequently less expression of human protein tau , produced a graded , lesser loss of hippocampal neurons . the aav - tau - induced neurodegeneration is thereby further demonstrated to be directly caused by and proportional to the near - physiological levels of protein tau . consequently , the observed pyramidal cell - death is qualified as specific for human protein tau . the actual contribution of the mutation to the mechanism underlying the pathogenesis requires a rational explanation in human ftd patients , as well as in the aav - models . similar aav - mediated expression of a truncated version of protein tau.4r , lacking the microtubule binding c - terminal domain , proved completely harmless and did not cause any neurodegeneration over similar time - periods . the inherent conclusion must be that microtubule binding of protein tau is essentially involved in the neurotoxic degenerative mechanism . of note , microgliosis was observed to be intense , and spatially and temporally closely associated with aav - tau induced degenerating neurons . this was most recently also reported in a similar model based on aav vectors but in rats and for a different pathology in a different brain - region . also , in that model , intense neurodegeneration was not marked by aggregation of protein tau , further strengthening our conclusion that cdk5 is not a major tau - kinase in vivo [ 3941 ] . the data originating from the aav - models led us to conclude that not large aggregates of protein tau cause neurodegeneration . this confirms our observations in transgenic mice that show aggregation of tau leading to tauopathy in somata and neuropil , but not accompanied by marked neurodegeneration [ 25 , 26 , 36 ] . we proposed that neurons affected by tauopathy can either engage in aggregation of tau and thereby try to decrease the toxic species and hope to survive , or to enter the path to death by failing to aggregate protein tau . clearly , the viral models comply with the transgenic models that aggregation of tau and neurodegeneration are not closely linked . we advocate that neurofibrillary tangles , like amyloid plaques are the final pathological hallmarks , but not the neurotoxic agents . the outcome fits independent observations that the extent of neuronal loss exceeds the number of nft in patients with ad [ 42 , 43 ] . in inducible tg4510 mice , memory decline and neuronal loss are dissociated from tangle formation in time and brain region [ 44 , 45 ] . in the aav - tau injected mice , phosphorylation of tau was evident at many pathological epitopes , including those defined by antibodies at8 , at100 , at180 , and at270 . despite the increased phosphorylation no aggregates of protein tau were deposited in degenerating pyramidal neurons , analyzed histochemically or immunohistochemically . nevertheless , no substantial neuronal loss was evident , substantiating our previous data of absence of marked neurodegeneration in tau.p301l mice and tau.p301lxgsk3b bigenic mice ( bigt ) , despite extensive or even dramatic tauopathy [ 26 , 36 ] . the combined results from our experimental models and from a drosophila model of tauopathy consolidates the thesis that neurotoxicity is not exerted by large aggregates of protein tau but rather by tau - species that are intermediate between normally phosphorylated protein tau and the hyper - phosphorylated fibrils . the identity of the toxic tau species , which we have termed tau - p * [ 11 , 30 ] remains to be defined a challenge equaling that of the identification of the amyloid receptor . indeed , tau can be ubiquitinated , truncated , glycosylated , glycated , oxidized , and , moreover , undergoes isomerization at proline residues . at any given moment protein tau is present in various molecular forms , which most likely differ subtly in properties of binding to microtubuli , interaction with other proteins , binding to membranes , being transported for normal duty or for degradation . the decision whether neurons will enter the cell death path or protect itself by forming tauopathy , must depend on the actual levels of protein tau and on its modifications . both pathways will be affected by various external factors , for example , inflammation , amyloid , stress , and so forth . in this concept , evidence from patients , whereby ca1 hippocampal neurons survive for decades despite neurofibrillary tangles and experimental animals show tangle bearing neurons survive , even with lost membrane integrity . stimulating autophagy pharmacologically , for example , by inhibition of mtor , ameliorates cognitive deficits and reduces amyloid and tau pathology in the 3xtg mouse model for ad . autophagosomes form in a stepwise process that requires transport over the microtubular network , which is known to be affected by protein tau , a major microtubule - associated protein . in our mouse models , degenerating neurons in the aav - tau.p301l injected mice displayed as dark neurons with extensive vacuolization that could be taken to indicate a defective autophagic process . a similar facet of neurons we observed in an unrelated mouse model , that is , the p25 inducible mice that recapitulate hippocampal sclerosis . the combined data do lend more support to the hypothesis that vacuolization is a correlate or consequence of the degenerative process , as an intrinsic mechanism needed to clean up defective organelles , protein aggregates or even entire dead or dying neurons . consistent with this view is our observation in the aav - tau.p301l mice that vacuoles do not form early in the disease process but only after the neurons are already degenerating [ 11 , 30 ] . finally , we searched but did not find biochemical evidence for the conversion of lc3-i to lc3-ii , which is generally accepted as the necessary step in and read - out of the increased formation of autophagosomes . various markers were increased , that is , cyclind2 , phosphorylated retinoblastoma protein , proliferating cell nuclear antigen ( pcna ) , cyclin b1 . first , mutant tau and hp - tau can cause it to detach from the microtubules and decrease its ability to control microtubule dynamics . on the other hand , increased levels of protein tau can saturate microtubules and hinder the foot - stepping of the motor proteins needed for axonal and dendritic transport . both aspects of tau - related transport deficits have been observed and both can fit into a model leading to starving synapses that eventually culminates in neuronal death . in this hypothesis , tau - mediated neurodegeneration is likely to start in distal neuronal processes , that is , axons and apical dendrites , which degenerate progressively towards the cell soma . the data explain how excess tau in first instance blocks transport by covering microtubuli , while gsk3 acting as major tau - kinase i phosphorylates tau to detach it from the microtubules and relieves the transport blockade . interestingly , treatment of tau.4r mice with lithium salts significantly increased the axonopathy , an effect that can be ascribed to its inhibition of gsk3b . as discussed above , aav - mediated expression of truncated tau , lacking the microtubule binding domain did not cause neurodegeneration , in marked contrast to full length protein tau ( figures 3 and 4 ) and . the combined data from various approaches allow us to conclude that tau mediated pathology is , at least in part , mediated by its microtubule binding properties . cns - related features of the immune system remain hardly understood , and supposed to be janus - faced , that is , providing protection as well as causing damage , steered by complex mostly unknown control mechanisms [ 60 , 61 ] . protection could be impaired in ad , fueled by and fueling debates on possible prevention or therapeutic benefits of antioxidants , radical scavengers , nonsteroidal anti - inflammatory drugs ( nsaid ) , and so forth , although clinical trials do not substantiate the claims . amyloid deposits attract microglia in a phagosomal attempt of their elimination , but activated microglia release proinflammatory cytokines , chemokines , reactive oxygen species , prostaglandins , and other mediators that harm neurons . astrocytes are the most common cell - type in the brain , involved in many functions and increasingly appreciated also in synaptic signaling and integrity . strictly speaking , neurons must also be considered when discussing neuroinflammation because they ( can ) produce active factors , that is , complement , interleukins , tumor necrosis factor , acute - phase proteins . we observed and discussed that degenerating neurons were temporally and spatially closely associated with activated microglia in two independent models , that is , the aav - tau viral model and the p25 transgenic model [ 30 , 39 ] . we maintain the most straightforward hypothesis , that is , that the expression of protein tau , either wild - type or mutant by the viral vectors , is imposing stress on the pyramidal neurons that can not adapt rapidly enough to produce tau - aggregates as a means of temporarily detoxification . secreted neuronal proteins then activate nearby microglia that counter - react by secreting proinflammatory and potentially neurotoxic factors , fueling the viscous cycle that eventually leads to neuronal death . in the case discussed here , protein tau is the cause in primary tauopathies . in ad , the upstream triggers are the amyloid peptides that accumulate because of mutations or other imposed defects in the proteolytic machinery that controls their formation and turnover . the second factor is also essential , and can be neuronal or microglial in origin , related to any type of stress the brain can experience or has to endure , that is , partial hypoxia , glucose overload or shortage , problems with lipid or cholesterol , or other essential metabolites . protein - aggregates , that is , plaques and tangles , are not the most essential in causing cognitive defects and dementia . the challenge to define the neurotoxic tau - species and its mode of action does nevertheless become not less complicated . the combination of transgenic and viral models illustrates eloquently the power of the approach as well as its weaknesses .

the impact of body mass index ( bmi ; weight ( kg)/height ( m ) ) and waist circumference ( wc ) on mortality in the growing elderly population is still controversial . there is concern about the potential increase in mortality related to excess weight,1 whereas mortality in underweight individuals has received less attention . as reviewed by heiat et al,2 zamboni et al,3 and janssen and mark,4 the recommendations of ideal weight for adults5 seem to be too restrictive for elderly individuals and being moderately overweight appears to entail limited risk . previous studies on body weight and mortality in elderly individuals have often focused on obesity and mainly assessed total mortality . several reports have found increased mortality in underweight individuals , but this association in elderly people has not been fully explained . thus , there is a need for additional studies with special attention to the lower weight categories . we explored the associations between bmi , wc and mortality in elderly men and women based on two large population - based norwegian studies . the fourth troms study was carried out in 19941995 and the second north - trndelag health study ( hunt ) in 19951997 . troms is a medium - sized town in northern norway and north - trndelag is a mostly rural county located in central norway . a total of 19 515 men and women ( 15 250 from north - trndelag and 4265 from troms ) aged 65 years and older participated and the overall participation rate was 70.0% . we excluded participants with a follow - up time under 1 year ( 425 participants ) and those missing information about cause - specific mortality ( 5 participants ) or questionnaire data concerning smoking , marital status or level of education ( 2374 participants ) . both surveys have been previously described in detail6 7 and they used the same core variables and questions . the regional boards of research ethics approved the surveys and each participating subject provided written informed consent . bmi was calculated as weight ( kg ) divided by height ( m ) squared . wc was measured horizontally to the nearest centimetre at the height of the umbilicus using a steel tape . information about educational level , smoking habits , alcohol intake , physical activity and chronic diseases was obtained from self - reported questionnaires . alcohol intake was categorised into three levels ( < once a month , 13 times / month and 4 times / month ) and physical activity into three levels ( low , medium and high ) . heart disease and lung disease were defined as myocardial infarction or angina pectoris and asthma or chronic bronchitis , respectively . data on cancer were obtained from the norwegian cancer registry and data on marital status were obtained from the population register of norway . in the mortality analysis , we first divided the participants into nine categories based on bmi values ( < 18.5 , 18.519.9 , 20.022.4 , 22.524.9 , 25.027.4 , 27.529.9 , 30.032.4 , 32.534.9 and 35 ) ; thereby including the standard definitions of overweight ( bmi 25.029.9 ) and obesity ( bmi30 ) ( who , 2000).5 in the analyses of cause - specific mortality , we merged all bmi categories below 20 and above 30 due to the limited number of deaths for some of the disease categories . the bmi category with the most participants and the most deaths ( 25.027.4 ) was chosen as the reference . wc was available for all the hunt participants and for all troms participants aged 6574 years as well as for a random sample of participants aged 7584 years . to compare the impacts of wc and bmi on mortality , we followed a procedure similar to that described by flegal et al8 and created wc categories that identified similar proportions of participants as each of the nine bmi categories . the cut - off for each wc category was set to the closest shift in wc value ; consequently , the numbers of participants in the wc categories were not exactly identical to the numbers of participants in the corresponding bmi categories . the wc category equivalent to the 25.027.4 bmi category was used as the reference . in a separate set of analyses , we modelled bmi as a continuous variable within the < 25.0 ( lower bmi ) and 25.0 ( higher bmi ) ranges yielding hrs for each 2.5 kg / m difference in bmi . for ease of understanding , the hrs for the lower bmi range indicate increased risk with decreasing bmi , whereas the hrs in the higher bmi range indicate increased risk with increasing bmi . both total and cause - specific mortality a similar method of modelling bmi was applied in a recent large study of bmi and mortality.9 we linked each participant to data from the norwegian causes of death registry using a personal identification number to identify vital status . cause of death is in norway based on the international classification of diseases ( icd ) system . icd-9 was used for deaths occurring up to 1996 and icd-10 was used for deaths occurring in 1996 and thereafter . we identified10 three main categories of causes of death : cvd ( icd-9 : 390459 ; icd-10 : i00i99 ) , respiratory disease ( icd-9 : 460519 ; icd-10 : j00j98 ) and cancer ( icd-9 : 140208 ; icd-10 : c00c97 ) . all subjects were followed for emigration or mortality from date of study enrolment until 31 december 2007 . to reduce the impact of weight loss due to pre - existing serious diseases , we excluded deaths occurring during the first year of follow - up . the differences in baseline characteristics between surviving and non - surviving participants were assessed by the test or t - test . hrs for both bmi categories and bmi modelled as a continuous variable were determined using a cox proportional hazards regression model controlling for potential confounders . we assessed the proportional hazards assumptions by inspecting the log(log ) survival curves for the various bmi categories . adjustments were performed for initial age , smoking , marital status , study site and educational level . in table 1 however , because there was no difference between adjusted models with a six - level and a three - level ( never , previous or current ) smoking variable , the three - level variable was used for adjustment in the mortality analyses . baseline characteristics of participating elderly men and women according to mortality status during follow - up . participants from the troms ( 19941995 ) and hunt ( 19951997 ) studies p value for differences between groups using the t test or test . the fourth troms study was carried out in 19941995 and the second north - trndelag health study ( hunt ) in 19951997 . troms is a medium - sized town in northern norway and north - trndelag is a mostly rural county located in central norway . a total of 19 515 men and women ( 15 250 from north - trndelag and 4265 from troms ) aged 65 years and older participated and the overall participation rate was 70.0% . we excluded participants with a follow - up time under 1 year ( 425 participants ) and those missing information about cause - specific mortality ( 5 participants ) or questionnaire data concerning smoking , marital status or level of education ( 2374 participants ) . both surveys have been previously described in detail6 7 and they used the same core variables and questions . the regional boards of research ethics approved the surveys and each participating subject provided written informed consent . bmi was calculated as weight ( kg ) divided by height ( m ) squared . wc was measured horizontally to the nearest centimetre at the height of the umbilicus using a steel tape . information about educational level , smoking habits , alcohol intake , physical activity and chronic diseases was obtained from self - reported questionnaires . alcohol intake was categorised into three levels ( < once a month , 13 times / month and 4 times / month ) and physical activity into three levels ( low , medium and high ) . heart disease and lung disease were defined as myocardial infarction or angina pectoris and asthma or chronic bronchitis , respectively . data on cancer were obtained from the norwegian cancer registry and data on marital status were obtained from the population register of norway . in the mortality analysis , we first divided the participants into nine categories based on bmi values ( < 18.5 , 18.519.9 , 20.022.4 , 22.524.9 , 25.027.4 , 27.529.9 , 30.032.4 , 32.534.9 and 35 ) ; thereby including the standard definitions of overweight ( bmi 25.029.9 ) and obesity ( bmi30 ) ( who , 2000).5 in the analyses of cause - specific mortality , we merged all bmi categories below 20 and above 30 due to the limited number of deaths for some of the disease categories . the bmi category with the most participants and the most deaths ( 25.027.4 ) was chosen as the reference . wc was available for all the hunt participants and for all troms participants aged 6574 years as well as for a random sample of participants aged 7584 years . to compare the impacts of wc and bmi on mortality , we followed a procedure similar to that described by flegal et al8 and created wc categories that identified similar proportions of participants as each of the nine bmi categories . the cut - off for each wc category was set to the closest shift in wc value ; consequently , the numbers of participants in the wc categories were not exactly identical to the numbers of participants in the corresponding bmi categories . the wc category equivalent to the 25.027.4 bmi category was used as the reference . in a separate set of analyses , we modelled bmi as a continuous variable within the < 25.0 ( lower bmi ) and 25.0 ( higher bmi ) ranges yielding hrs for each 2.5 kg / m difference in bmi . for ease of understanding , the hrs for the lower bmi range indicate increased risk with decreasing bmi , whereas the hrs in the higher bmi range indicate increased risk with increasing bmi . both total and cause - specific mortality a similar method of modelling bmi was applied in a recent large study of bmi and mortality.9 we linked each participant to data from the norwegian causes of death registry using a personal identification number to identify vital status . cause of death is in norway based on the international classification of diseases ( icd ) system . icd-9 was used for deaths occurring up to 1996 and icd-10 was used for deaths occurring in 1996 and thereafter . we identified10 three main categories of causes of death : cvd ( icd-9 : 390459 ; icd-10 : i00i99 ) , respiratory disease ( icd-9 : 460519 ; icd-10 : j00j98 ) and cancer ( icd-9 : 140208 ; icd-10 : c00c97 ) . all subjects were followed for emigration or mortality from date of study enrolment until 31 december 2007 . to reduce the impact of weight loss due to pre - existing serious diseases the differences in baseline characteristics between surviving and non - surviving participants were assessed by the test or t - test . hrs for both bmi categories and bmi modelled as a continuous variable were determined using a cox proportional hazards regression model controlling for potential confounders . we assessed the proportional hazards assumptions by inspecting the log(log ) survival curves for the various bmi categories . adjustments were performed for initial age , smoking , marital status , study site and educational level . in table 1 however , because there was no difference between adjusted models with a six - level and a three - level ( never , previous or current ) smoking variable , the three - level variable was used for adjustment in the mortality analyses . baseline characteristics of participating elderly men and women according to mortality status during follow - up . participants from the troms ( 19941995 ) and hunt ( 19951997 ) studies p value for differences between groups using the t test or test . during a mean 9.3 years of follow - up , 51.5% of the 7604 men and 39.1% of the 9107 women aged 65 years died . there were 7474 deaths in total ( 643 deaths from respiratory diseases , 3419 deaths from cvd , 1794 deaths from cancer and 1618 deaths from other diseases / conditions ) . the crude total mortality rates per 1000 person - years were 57.1 and 39.4 in men and women , respectively . we compared baseline characteristics of the participants according to whether they died during follow - up ( table 1 ) . men and women who died tended to be older , not to be married and have low education . the mean bmi was also slightly lower in individuals who died and the proportion of subjects with bmi below 25.0 was substantially higher among these individuals than among the surviving group . in both men and women , individuals in the 2529.9 bmi category had the lowest mortality ( table 2 and figure 1 ) . the same relatively low mortality was observed in moderately obese women . in all bmi categories below 25.0 , adjustment for smoking status , educational level and marital status had only a minor impact on the risk estimates . adjusted hrs for total mortality by category of body mass index ( bmi ) and waist circumference ( wc ) in elderly men and women . participants from the troms ( 19941995 ) and hunt ( 19951997 ) studies adjusted for initial age and study site . adjusted for initial age , study site , smoking , educational level and marital status . adjusted hrs ( 95% ci ) * for total mortality rate by body mass index ( bmi ; kg / m ) category in elderly men and women . adjusted for smoking status , age , marital status , educational level and study site . wc categories , mortality was increased only in the lowest category . in the upper categories , the hrs were statistically significantly increased in the three highest categories in men and the highest category in women . when modelling bmi as a continuous variable ( figure 2 ) , we found a 20% increase in mortality per 2.5 kg / m decrease in bmi in the lower bmi range ( < 25 ) . in the upper bmi range ( 25 ) , we found a 79% increase in mortality per 2.5 kg / m increase in bmi . adjusted hrs * for total mortality for every 2.5 kg / m difference in body mass index ( bmi ) in elderly men and women . in the lower range , hr indicates risk of mortality with decreasing bmi , whereas in the higher range , hr indicates risk of mortality with increasing bmi . adjusted for smoking status ( all participants only ) , age , marital status , educational level and study site . information about physical activity and alcohol intake was missing for 6353 participants and 1464 participants , respectively . we repeated the analyses of total mortality and cause - specific mortality with adjustment for frequency of alcohol intake and physical activity , respectively , in the subpopulations with information concerning these two variables . however , these additional adjustments had only a minimal impact on the hrs for the relation between bmi and mortality ( results not shown ) . approximately 20% of the participants were current smokers , 36% were previous smokers and 43% had never smoked . the interaction between bmi and smoking status in the analysis of total mortality was significant only for women in the higher bmi range ( p=0.047 ) ; it was non - significant for men in the higher bmi range and for both men and women in the lower bmi range ( figure 2 ) . about 1 in 10 deaths was from respiratory diseases and the corresponding mortality was three to four times higher in individuals with a bmi below 20 compared to the reference category ( table 3 and figure 3 ) . when bmi was modelled as a continuous variable ( figure 4 ) , a 5474% increase in mortality from respiratory diseases was observed per 2.5 kg / m lower bmi below 25 . in women , no interaction between smoking and bmi was observed . for men in the lower bmi range who had never smoked , there was no relation between bmi and respiratory diseases mortality . however , the corresponding ci was wide due to the small number of deaths . adjusted hrs * for cause - specific mortality by body mass index ( bmi ) category in elderly men and women . participants from the troms ( 19941995 ) and hunt ( 19951997 ) studies adjusted for smoking status , age , marital status , educational level and study site . adjusted hrs * for cause - specific mortality by body mass index ( bmi ; kg / m ) category in elderly men and women . adjusted for smoking status , age , marital status , educational level and study site . adjusted hrs * for cause - specific mortality for every 2.5 kg / m difference in body mass index ( bmi ) in elderly men and women . in the lower range , hr indicates risk of mortality with decreasing bmi , whereas in the higher range , hr indicates risk of mortality with increasing bmi . adjusted for smoking status , age , marital status , educational level and study site . to further explore mortality from respiratory diseases , we analysed the associations between bmi and respiratory disease mortality in participants without lung disease at baseline . the contribution of deaths from respiratory diseases was also explored by analysing total mortality excluding deaths from respiratory diseases . in the lower bmi range , we found that the hr for a 2.5 kg / m decline in bmi below 25 decreased from 1.21 to 1.13 in men . in women , for the categorical analyses ( table 2 ) , the overall pattern of the bmi however , the hrs for mortality in the lower bmi categories were somewhat reduced , especially in men . increased cvd mortality was found in the higher bmi range ; the effect was most pronounced in men with a bmi 30 ( table 3 ; figure 3 ) . cvd mortality was also increased in women with a bmi < 22.5 kg / m . cancer accounted for one - quarter of all deaths and the relationships between bmi and cancer mortality followed the u - shaped pattern found for total mortality ( table 3 ) . prior to inclusion in the study , a history of heart disease was reported by 21% , lung disease by 13% and 9% of the participants had a history of cancer . exclusion of these subjects from the analyses revealed similar relations as described for the main cohort both for total and cause - specific mortality ( results not shown ) . we compared baseline characteristics of the participants according to whether they died during follow - up ( table 1 ) . men and women who died tended to be older , not to be married and have low education . the mean bmi was also slightly lower in individuals who died and the proportion of subjects with bmi below 25.0 was substantially higher among these individuals than among the surviving group . in both men and women , individuals in the 2529.9 bmi category had the lowest mortality ( table 2 and figure 1 ) . the same relatively low mortality was observed in moderately obese women . in all bmi categories below 25.0 , adjustment for smoking status , educational level and marital status had only a minor impact on the risk estimates . adjusted hrs for total mortality by category of body mass index ( bmi ) and waist circumference ( wc ) in elderly men and women . participants from the troms ( 19941995 ) and hunt ( 19951997 ) studies adjusted for initial age and study site . adjusted for initial age , study site , smoking , educational level and marital status . adjusted hrs ( 95% ci ) * for total mortality rate by body mass index ( bmi ; kg / m ) category in elderly men and women . adjusted for smoking status , age , marital status , educational level and study site . table 2 also shows total mortality in the various wc categories . in the lower wc categories , the hrs were statistically significantly increased in the three highest categories in men and the highest category in women . when modelling bmi as a continuous variable ( figure 2 ) , we found a 20% increase in mortality per 2.5 kg / m decrease in bmi in the lower bmi range ( < 25 ) . in the upper bmi range ( 25 ) , we found a 79% increase in mortality per 2.5 kg / m increase in bmi . adjusted hrs * for total mortality for every 2.5 kg / m difference in body mass index ( bmi ) in elderly men and women . in the lower range , hr indicates risk of mortality with decreasing bmi , whereas in the higher range , hr indicates risk of mortality with increasing bmi . adjusted for smoking status ( all participants only ) , age , marital status , educational level and study site . information about physical activity and alcohol intake was missing for 6353 participants and 1464 participants , respectively . we repeated the analyses of total mortality and cause - specific mortality with adjustment for frequency of alcohol intake and physical activity , respectively , in the subpopulations with information concerning these two variables . however , these additional adjustments had only a minimal impact on the hrs for the relation between bmi and mortality ( results not shown ) . approximately 20% of the participants were current smokers , 36% were previous smokers and 43% had never smoked . the interaction between bmi and smoking status in the analysis of total mortality was significant only for women in the higher bmi range ( p=0.047 ) ; it was non - significant for men in the higher bmi range and for both men and women in the lower bmi range ( figure 2 ) . about 1 in 10 deaths was from respiratory diseases and the corresponding mortality was three to four times higher in individuals with a bmi below 20 compared to the reference category ( table 3 and figure 3 ) . when bmi was modelled as a continuous variable ( figure 4 ) , a 5474% increase in mortality from respiratory diseases was observed per 2.5 kg / m lower bmi below 25 . in women , no interaction between smoking and bmi was observed . for men in the lower bmi range who had never smoked , . adjusted hrs * for cause - specific mortality by body mass index ( bmi ) category in elderly men and women . participants from the troms ( 19941995 ) and hunt ( 19951997 ) studies adjusted for smoking status , age , marital status , educational level and study site . adjusted hrs * for cause - specific mortality by body mass index ( bmi ; kg / m ) category in elderly men and women . * adjusted for smoking status , age , marital status , educational level and study site . adjusted hrs * for cause - specific mortality for every 2.5 kg / m difference in body mass index ( bmi ) in elderly men and women . in the lower range , hr indicates risk of mortality with decreasing bmi , whereas in the higher range , hr indicates risk of mortality with increasing bmi . adjusted for smoking status , age , marital status , educational level and study site . to further explore mortality from respiratory diseases , we analysed the associations between bmi and respiratory disease mortality in participants without lung disease at baseline . the contribution of deaths from respiratory diseases was also explored by analysing total mortality excluding deaths from respiratory diseases . in the lower bmi range , we found that the hr for a 2.5 kg / m decline in bmi below 25 decreased from 1.21 to 1.13 in men . in women , for the categorical analyses ( table 2 ) , the overall pattern of the bmi mortality relationship was retained when deaths from respiratory diseases were excluded . however , the hrs for mortality in the lower bmi categories were somewhat reduced , especially in men . increased cvd mortality was found in the higher bmi range ; the effect was most pronounced in men with a bmi 30 ( table 3 ; figure 3 ) . cvd mortality was also increased in women with a bmi < 22.5 kg / m . cancer accounted for one - quarter of all deaths and the relationships between bmi and cancer mortality followed the u - shaped pattern found for total mortality ( table 3 ) . prior to inclusion in the study , a history of heart disease was reported by 21% , lung disease by 13% and 9% of the participants had a history of cancer . exclusion of these subjects from the analyses revealed similar relations as described for the main cohort both for total and cause - specific mortality ( results not shown ) . in this population - based study of elderly men and women , we found increased total mortality in elderly individuals with a bmi below 25 . no excess mortality was found in overweight individuals ( bmi 2529.9 ) and only a moderate increase in mortality was observed with increasing bmi in obese individuals ( bmi30 ) . in the lower bmi range , these results were not explained by the presence of cancer or lung disease at baseline . the crude mortality rates per 1000 person - years ( 57 in men and 39 in women ) observed in the study population were lower than the expected mortality rates ( 73 in men and 55 in women ) if the mortality rates of the entire elderly population of norway11 were applied to the study population . this is probably explained by a better general health in the participants than in non - participating elderly individuals . the impact of a decrease in bmi in the lower bmi range was twice the impact of the corresponding increase in bmi in the higher bmi range ( figure 2 ) . however , the most striking finding was the relative increase in mortality found in all normal weight individuals in the bmi categories below 25 . mortality from respiratory diseases explained about 40% of the increased total mortality found in men in the lower bmi range . previous studies of elderly individuals have demonstrated increased mortality from respiratory diseases in selected groups with a bmi below 2022.7.12 13 we also found substantially increased mortality from respiratory diseases in the 2022.4 and 22.524.9 bmi categories , which are generally regarded as healthy weight . asthma and chronic bronchitis are prevalent conditions associated with the lower bmi categories in elderly men and women.14 when we analysed subjects without these conditions , the impact of bmi on mortality in the lower range persisted . smoking is a major risk factor for both premature death15 and malnutrition16 in elderly individuals , and we adjusted all analyses for smoking status . our results suggest that the impact of bmi 25 on mortality was greater in current smoking women than in women who had never smoked . a number of studies have reported increased mortality among individuals in the lowest bmi categories.24 9 17 mortality tends to increase when bmi falls below the range of 1923 . very few studies of elderly men and women , with information about smoking habits included , have been able to demonstrate a significant increase in mortality in the normal bmi range of 22.524.9 compared to moderately overweight subjects . for some of the studies , this may be explained by a focus on obesity , the use of wide reference categories ( bmi 18.525 ) or a limited number of participants . being underweight is associated with loss of both peripheral and respiratory muscles,18 and this association may partly explain the increases in both total mortality and mortality from respiratory diseases observed in this study among underweight participants . furthermore , a low bmi may increase vulnerability to acute diseases . an elevated in - hospital case - fatality rate among underweight patients has been found for several conditions.19 it has been shown that the immune response is decreased in elderly malnourished individuals,20 which may increase the relative mortality rate among underweight elderly subjects during intercurrent diseases . reverse causation that is , the possibility that pre - existing illnesses or conditions associated with increased mortality lead to loss of body weight is a concern in studies of lower bmi and mortality.21 22 to overcome this problem in our analyses , we used several methods . second , we repeated the analyses of mortality excluding participants with a history of cancer at baseline as these patients might have reduced weight because of the disease . however , the relation between low bmi and mortality was not influenced by this exclusion . finally , to explore the effect of underweight - associated pulmonary disease , we analysed both total mortality and mortality from respiratory diseases in individuals not reporting asthma / chronic bronchitis at baseline . in addition , we stratified the analyses for smoking habits ( figure 2 ) and found that the increased total mortality in subjects with low bmi was not due to smoking . nevertheless , some unknown bias from pre - existing diseases or conditions might have influenced the bmi the prevalence of obesity is rising in elderly individuals and the proportion of obese individuals in the present study ( 20.7% ) was almost at the level found in us populations in the same period.23 we found moderately increased mortality in obese individuals more so in men than in women . most previous studies have found increased mortality in obese elderly individuals , but the increase was somewhat less pronounced than in middle - aged adults.4 using bmi alone for assessment of obesity may be a limitation because it provides only an indirect estimate of abdominal adiposity . in the present study , wc as a measure of abdominal obesity however , overall , wc was not a stronger risk factor for total mortality than bmi in the higher weight categories . this finding is in line with the results of a recent study by flegal et al.8 in the lower weight categories , bmi was a stronger risk factor than wc . several explanations have been proposed for the limited impact of overweight or obesity on mortality in this age group.3 possible explanations include a healthy survival effect in obese individuals meaning that those most vulnerable to the effects of obesity have already died before reaching older age . obesity - related consequences develop slowly and before these consequences appear , individuals who become obese later in life may die from other diseases.24 we found the optimal weight with the lowest mortality to be in the overweight categories ( bmi 2529.9 ) , whereas moderately obese individuals had only a modest increase in mortality . this finding is in line with several previous studies of elderly individuals25 and has been described as the obesity paradox.24 it has also been observed that moderate overweight hospitalised elderly patients have reduced mortality.26 these findings are important because almost half of the elderly population are overweight ( bmi 2529.9 ) and it is frequently assumed that these individuals have increased mortality.24 one strength of the present study is the strictly population - based design with inclusion of individuals from both urban and rural areas . some previous studies in this field have used cohorts based on particular professional affiliations or health insurance membership , introducing the possibility of bias.13 furthermore , in the present study , height , weight and wc were measured rather than self - reported . the participation rate was relatively high ( 70% ) , but participation required the ability to independently visit a research centre . it is probable that the non - participating individuals were frailer and had more co - morbidities ; any potential bias would probably be in the direction of more conservative estimates . the impact of a decrease in bmi in the lower bmi range was twice the impact of the corresponding increase in bmi in the higher bmi range ( figure 2 ) . however , the most striking finding was the relative increase in mortality found in all normal weight individuals in the bmi categories below 25 . mortality from respiratory diseases explained about 40% of the increased total mortality found in men in the lower bmi range . previous studies of elderly individuals have demonstrated increased mortality from respiratory diseases in selected groups with a bmi below 2022.7.12 13 we also found substantially increased mortality from respiratory diseases in the 2022.4 and 22.524.9 bmi categories , which are generally regarded as healthy weight . asthma and chronic bronchitis are prevalent conditions associated with the lower bmi categories in elderly men and women.14 when we analysed subjects without these conditions , the impact of bmi on mortality in the lower range persisted . smoking is a major risk factor for both premature death15 and malnutrition16 in elderly individuals , and we adjusted all analyses for smoking status . our results suggest that the impact of bmi 25 on mortality was greater in current smoking women than in women who had never smoked . a number of studies have reported increased mortality among individuals in the lowest bmi categories.24 9 17 mortality tends to increase when bmi falls below the range of 1923 . very few studies of elderly men and women , with information about smoking habits included , have been able to demonstrate a significant increase in mortality in the normal bmi range of 22.524.9 compared to moderately overweight subjects . for some of the studies , this may be explained by a focus on obesity , the use of wide reference categories ( bmi 18.525 ) or a limited number of participants . being underweight is associated with loss of both peripheral and respiratory muscles,18 and this association may partly explain the increases in both total mortality and mortality from respiratory diseases observed in this study among underweight participants . furthermore , a low bmi may increase vulnerability to acute diseases . an elevated in - hospital case - fatality rate among underweight patients has been found for several conditions.19 it has been shown that the immune response is decreased in elderly malnourished individuals,20 which may increase the relative mortality rate among underweight elderly subjects during intercurrent diseases . reverse causation that is , the possibility that pre - existing illnesses or conditions associated with increased mortality lead to loss of body weight is a concern in studies of lower bmi and mortality.21 22 to overcome this problem in our analyses , we used several methods . second , we repeated the analyses of mortality excluding participants with a history of cancer at baseline as these patients might have reduced weight because of the disease . however , the relation between low bmi and mortality was not influenced by this exclusion . finally , to explore the effect of underweight - associated pulmonary disease , we analysed both total mortality and mortality from respiratory diseases in individuals not reporting asthma / chronic bronchitis at baseline . in addition , we stratified the analyses for smoking habits ( figure 2 ) and found that the increased total mortality in subjects with low bmi was not due to smoking . nevertheless , some unknown bias from pre - existing diseases or conditions might have influenced the bmi mortality relations . the prevalence of obesity is rising in elderly individuals and the proportion of obese individuals in the present study ( 20.7% ) was almost at the level found in us populations in the same period.23 we found moderately increased mortality in obese individuals more so in men than in women . most previous studies have found increased mortality in obese elderly individuals , but the increase was somewhat less pronounced than in middle - aged adults.4 using bmi alone for assessment of obesity may be a limitation because it provides only an indirect estimate of abdominal adiposity . in the present study , wc as a measure of abdominal obesity however , overall , wc was not a stronger risk factor for total mortality than bmi in the higher weight categories . this finding is in line with the results of a recent study by flegal et al.8 in the lower weight categories , bmi was a stronger risk factor than wc . several explanations have been proposed for the limited impact of overweight or obesity on mortality in this age group.3 possible explanations include a healthy survival effect in obese individuals meaning that those most vulnerable to the effects of obesity have already died before reaching older age . furthermore , elderly people have a shorter life expectancy . obesity - related consequences develop slowly and before these consequences appear , individuals who become obese later in life may die from other diseases.24 we found the optimal weight with the lowest mortality to be in the overweight categories ( bmi 2529.9 ) , whereas moderately obese individuals had only a modest increase in mortality . this finding is in line with several previous studies of elderly individuals25 and has been described as the obesity paradox.24 it has also been observed that moderate overweight hospitalised elderly patients have reduced mortality.26 these findings are important because almost half of the elderly population are overweight ( bmi 2529.9 ) and it is frequently assumed that these individuals have increased mortality.24 one strength of the present study is the strictly population - based design with inclusion of individuals from both urban and rural areas . some previous studies in this field have used cohorts based on particular professional affiliations or health insurance membership , introducing the possibility of bias.13 furthermore , in the present study , height , weight and wc were measured rather than self - reported . the participation rate was relatively high ( 70% ) , but participation required the ability to independently visit a research centre . it is probable that the non - participating individuals were frailer and had more co - morbidities ; any potential bias would probably be in the direction of more conservative estimates . our results show that in elderly men and women , a bmi below 25 was associated with increased total mortality . in men , this finding could be explained to some extent by an increased risk of death from respiratory diseases . a modest increase in mortality was found with increasing bmi for obese elderly men and women . the body mass index ( bmi)mortality relationship is u - shaped in both adult and elderly individuals and shifted to the right in the older age categories . results from previous studies in elderly individuals are conflicting about the upper and especially the lower bmi cut - off points for optimal weight with regard to mortality . there is a lack of data concerning the relationship between bmi and mortality , particularly cause - specific mortality , in the elderly and the impact of mortality from respiratory diseases on the bmi mortality relationship . in the present large study of elderly individuals from norway , mortality was significantly increased in all body mass index ( bmi ) categories below 25 , including the the impact of decreases in bmi in the lower bmi range was twice the impact of the corresponding increases in bmi in the higher bmi range . in men , waist circumference and bmi performed approximately equally in identifying individuals in the higher weight categories with increased mortality . more attention should be given to elderly individuals in the lower bmi categories and moderately increased bmi ( 2529.9 ) should not be a concern with regard to mortality in this age group .

backgroundthe impact of body mass index ( bmi ; kg / m2 ) and waist circumference ( wc ) on mortality in elderly individuals is controversial and previous research has largely focused on obesity.methodswith special attention to the lower bmi categories , associations between bmi and both total and cause - specific mortality were explored in 7604 men and 9107 women aged 65 years who participated in the troms study ( 19941995 ) or the north - trndelag health study ( 19951997 ) . a cox proportional hazards model adjusted for age , marital status , education and smoking was used to estimate hrs for mortality in different bmi categories using the bmi range of 2527.5 as a reference . the impact of each 2.5 kg / m2 difference in bmi on mortality in individuals with bmi<25.0 and bmi25.0 was also explored . furthermore , the relations between wc and mortality were assessed.resultswe identified 7474 deaths during a mean follow - up of 9.3 years . the lowest mortality was found in the bmi range 2529.9 and 2532.4 in men and women , respectively . mortality was increased in all bmi categories below 25 and was moderately increased in obese individuals . u - shaped relationships were also found between wc and total mortality . about 40% of the excess mortality in the lower bmi range in men was explained by mortality from respiratory diseases.conclusionsbmi below 25 in elderly men and women was associated with increased mortality . a modest increase in mortality was found with increasing bmi among obese men and women . overweight individuals ( bmi 2529.9 ) had the lowest mortality .

Introduction Methods Study population BMI, WC and other characteristics Follow-up and endpoints Statistical analyses Results Baseline characteristics by mortality status during follow-up Total mortality and BMI Cause-specific mortality and BMI Discussion Lower BMI range Why is mortality higher in the lower BMI range? Higher BMI range Optimal body weight Conclusion

the impact of body mass index ( bmi ; weight ( kg)/height ( m ) ) and waist circumference ( wc ) on mortality in the growing elderly population is still controversial . previous studies on body weight and mortality in elderly individuals have often focused on obesity and mainly assessed total mortality . we explored the associations between bmi , wc and mortality in elderly men and women based on two large population - based norwegian studies . the fourth troms study was carried out in 19941995 and the second north - trndelag health study ( hunt ) in 19951997 . a total of 19 515 men and women ( 15 250 from north - trndelag and 4265 from troms ) aged 65 years and older participated and the overall participation rate was 70.0% . we excluded participants with a follow - up time under 1 year ( 425 participants ) and those missing information about cause - specific mortality ( 5 participants ) or questionnaire data concerning smoking , marital status or level of education ( 2374 participants ) . in the mortality analysis , we first divided the participants into nine categories based on bmi values ( < 18.5 , 18.519.9 , 20.022.4 , 22.524.9 , 25.027.4 , 27.529.9 , 30.032.4 , 32.534.9 and 35 ) ; thereby including the standard definitions of overweight ( bmi 25.029.9 ) and obesity ( bmi30 ) ( who , 2000).5 in the analyses of cause - specific mortality , we merged all bmi categories below 20 and above 30 due to the limited number of deaths for some of the disease categories . in a separate set of analyses , we modelled bmi as a continuous variable within the < 25.0 ( lower bmi ) and 25.0 ( higher bmi ) ranges yielding hrs for each 2.5 kg / m difference in bmi . for ease of understanding , the hrs for the lower bmi range indicate increased risk with decreasing bmi , whereas the hrs in the higher bmi range indicate increased risk with increasing bmi . both total and cause - specific mortality a similar method of modelling bmi was applied in a recent large study of bmi and mortality.9 we linked each participant to data from the norwegian causes of death registry using a personal identification number to identify vital status . hrs for both bmi categories and bmi modelled as a continuous variable were determined using a cox proportional hazards regression model controlling for potential confounders . participants from the troms ( 19941995 ) and hunt ( 19951997 ) studies p value for differences between groups using the t test or test . a total of 19 515 men and women ( 15 250 from north - trndelag and 4265 from troms ) aged 65 years and older participated and the overall participation rate was 70.0% . we excluded participants with a follow - up time under 1 year ( 425 participants ) and those missing information about cause - specific mortality ( 5 participants ) or questionnaire data concerning smoking , marital status or level of education ( 2374 participants ) . in the mortality analysis , we first divided the participants into nine categories based on bmi values ( < 18.5 , 18.519.9 , 20.022.4 , 22.524.9 , 25.027.4 , 27.529.9 , 30.032.4 , 32.534.9 and 35 ) ; thereby including the standard definitions of overweight ( bmi 25.029.9 ) and obesity ( bmi30 ) ( who , 2000).5 in the analyses of cause - specific mortality , we merged all bmi categories below 20 and above 30 due to the limited number of deaths for some of the disease categories . in a separate set of analyses , we modelled bmi as a continuous variable within the < 25.0 ( lower bmi ) and 25.0 ( higher bmi ) ranges yielding hrs for each 2.5 kg / m difference in bmi . for ease of understanding , the hrs for the lower bmi range indicate increased risk with decreasing bmi , whereas the hrs in the higher bmi range indicate increased risk with increasing bmi . both total and cause - specific mortality a similar method of modelling bmi was applied in a recent large study of bmi and mortality.9 we linked each participant to data from the norwegian causes of death registry using a personal identification number to identify vital status . participants from the troms ( 19941995 ) and hunt ( 19951997 ) studies p value for differences between groups using the t test or test . during a mean 9.3 years of follow - up , 51.5% of the 7604 men and 39.1% of the 9107 women aged 65 years died . the crude total mortality rates per 1000 person - years were 57.1 and 39.4 in men and women , respectively . in both men and women , individuals in the 2529.9 bmi category had the lowest mortality ( table 2 and figure 1 ) . in all bmi categories below 25.0 , adjustment for smoking status , educational level and marital status had only a minor impact on the risk estimates . adjusted hrs for total mortality by category of body mass index ( bmi ) and waist circumference ( wc ) in elderly men and women . participants from the troms ( 19941995 ) and hunt ( 19951997 ) studies adjusted for initial age and study site . adjusted hrs ( 95% ci ) * for total mortality rate by body mass index ( bmi ; kg / m ) category in elderly men and women . in the upper categories , the hrs were statistically significantly increased in the three highest categories in men and the highest category in women . when modelling bmi as a continuous variable ( figure 2 ) , we found a 20% increase in mortality per 2.5 kg / m decrease in bmi in the lower bmi range ( < 25 ) . in the upper bmi range ( 25 ) , we found a 79% increase in mortality per 2.5 kg / m increase in bmi . adjusted hrs * for total mortality for every 2.5 kg / m difference in body mass index ( bmi ) in elderly men and women . we repeated the analyses of total mortality and cause - specific mortality with adjustment for frequency of alcohol intake and physical activity , respectively , in the subpopulations with information concerning these two variables . the interaction between bmi and smoking status in the analysis of total mortality was significant only for women in the higher bmi range ( p=0.047 ) ; it was non - significant for men in the higher bmi range and for both men and women in the lower bmi range ( figure 2 ) . about 1 in 10 deaths was from respiratory diseases and the corresponding mortality was three to four times higher in individuals with a bmi below 20 compared to the reference category ( table 3 and figure 3 ) . when bmi was modelled as a continuous variable ( figure 4 ) , a 5474% increase in mortality from respiratory diseases was observed per 2.5 kg / m lower bmi below 25 . for men in the lower bmi range who had never smoked , there was no relation between bmi and respiratory diseases mortality . adjusted hrs * for cause - specific mortality by body mass index ( bmi ) category in elderly men and women . participants from the troms ( 19941995 ) and hunt ( 19951997 ) studies adjusted for smoking status , age , marital status , educational level and study site . adjusted hrs * for cause - specific mortality by body mass index ( bmi ; kg / m ) category in elderly men and women . adjusted hrs * for cause - specific mortality for every 2.5 kg / m difference in body mass index ( bmi ) in elderly men and women . to further explore mortality from respiratory diseases , we analysed the associations between bmi and respiratory disease mortality in participants without lung disease at baseline . in the lower bmi range , we found that the hr for a 2.5 kg / m decline in bmi below 25 decreased from 1.21 to 1.13 in men . in women , for the categorical analyses ( table 2 ) , the overall pattern of the bmi however , the hrs for mortality in the lower bmi categories were somewhat reduced , especially in men . in both men and women , individuals in the 2529.9 bmi category had the lowest mortality ( table 2 and figure 1 ) . in all bmi categories below 25.0 , adjustment for smoking status , educational level and marital status had only a minor impact on the risk estimates . adjusted hrs for total mortality by category of body mass index ( bmi ) and waist circumference ( wc ) in elderly men and women . participants from the troms ( 19941995 ) and hunt ( 19951997 ) studies adjusted for initial age and study site . adjusted hrs ( 95% ci ) * for total mortality rate by body mass index ( bmi ; kg / m ) category in elderly men and women . in the lower wc categories , the hrs were statistically significantly increased in the three highest categories in men and the highest category in women . when modelling bmi as a continuous variable ( figure 2 ) , we found a 20% increase in mortality per 2.5 kg / m decrease in bmi in the lower bmi range ( < 25 ) . in the upper bmi range ( 25 ) , we found a 79% increase in mortality per 2.5 kg / m increase in bmi . adjusted hrs * for total mortality for every 2.5 kg / m difference in body mass index ( bmi ) in elderly men and women . we repeated the analyses of total mortality and cause - specific mortality with adjustment for frequency of alcohol intake and physical activity , respectively , in the subpopulations with information concerning these two variables . the interaction between bmi and smoking status in the analysis of total mortality was significant only for women in the higher bmi range ( p=0.047 ) ; it was non - significant for men in the higher bmi range and for both men and women in the lower bmi range ( figure 2 ) . about 1 in 10 deaths was from respiratory diseases and the corresponding mortality was three to four times higher in individuals with a bmi below 20 compared to the reference category ( table 3 and figure 3 ) . when bmi was modelled as a continuous variable ( figure 4 ) , a 5474% increase in mortality from respiratory diseases was observed per 2.5 kg / m lower bmi below 25 . adjusted hrs * for cause - specific mortality by body mass index ( bmi ) category in elderly men and women . participants from the troms ( 19941995 ) and hunt ( 19951997 ) studies adjusted for smoking status , age , marital status , educational level and study site . adjusted hrs * for cause - specific mortality by body mass index ( bmi ; kg / m ) category in elderly men and women . adjusted hrs * for cause - specific mortality for every 2.5 kg / m difference in body mass index ( bmi ) in elderly men and women . to further explore mortality from respiratory diseases , we analysed the associations between bmi and respiratory disease mortality in participants without lung disease at baseline . in the lower bmi range , we found that the hr for a 2.5 kg / m decline in bmi below 25 decreased from 1.21 to 1.13 in men . however , the hrs for mortality in the lower bmi categories were somewhat reduced , especially in men . increased cvd mortality was found in the higher bmi range ; the effect was most pronounced in men with a bmi 30 ( table 3 ; figure 3 ) . in this population - based study of elderly men and women , we found increased total mortality in elderly individuals with a bmi below 25 . no excess mortality was found in overweight individuals ( bmi 2529.9 ) and only a moderate increase in mortality was observed with increasing bmi in obese individuals ( bmi30 ) . the impact of a decrease in bmi in the lower bmi range was twice the impact of the corresponding increase in bmi in the higher bmi range ( figure 2 ) . however , the most striking finding was the relative increase in mortality found in all normal weight individuals in the bmi categories below 25 . mortality from respiratory diseases explained about 40% of the increased total mortality found in men in the lower bmi range . previous studies of elderly individuals have demonstrated increased mortality from respiratory diseases in selected groups with a bmi below 2022.7.12 13 we also found substantially increased mortality from respiratory diseases in the 2022.4 and 22.524.9 bmi categories , which are generally regarded as healthy weight . asthma and chronic bronchitis are prevalent conditions associated with the lower bmi categories in elderly men and women.14 when we analysed subjects without these conditions , the impact of bmi on mortality in the lower range persisted . very few studies of elderly men and women , with information about smoking habits included , have been able to demonstrate a significant increase in mortality in the normal bmi range of 22.524.9 compared to moderately overweight subjects . for some of the studies , this may be explained by a focus on obesity , the use of wide reference categories ( bmi 18.525 ) or a limited number of participants . being underweight is associated with loss of both peripheral and respiratory muscles,18 and this association may partly explain the increases in both total mortality and mortality from respiratory diseases observed in this study among underweight participants . reverse causation that is , the possibility that pre - existing illnesses or conditions associated with increased mortality lead to loss of body weight is a concern in studies of lower bmi and mortality.21 22 to overcome this problem in our analyses , we used several methods . finally , to explore the effect of underweight - associated pulmonary disease , we analysed both total mortality and mortality from respiratory diseases in individuals not reporting asthma / chronic bronchitis at baseline . nevertheless , some unknown bias from pre - existing diseases or conditions might have influenced the bmi the prevalence of obesity is rising in elderly individuals and the proportion of obese individuals in the present study ( 20.7% ) was almost at the level found in us populations in the same period.23 we found moderately increased mortality in obese individuals more so in men than in women . several explanations have been proposed for the limited impact of overweight or obesity on mortality in this age group.3 possible explanations include a healthy survival effect in obese individuals meaning that those most vulnerable to the effects of obesity have already died before reaching older age . obesity - related consequences develop slowly and before these consequences appear , individuals who become obese later in life may die from other diseases.24 we found the optimal weight with the lowest mortality to be in the overweight categories ( bmi 2529.9 ) , whereas moderately obese individuals had only a modest increase in mortality . this finding is in line with several previous studies of elderly individuals25 and has been described as the obesity paradox.24 it has also been observed that moderate overweight hospitalised elderly patients have reduced mortality.26 these findings are important because almost half of the elderly population are overweight ( bmi 2529.9 ) and it is frequently assumed that these individuals have increased mortality.24 one strength of the present study is the strictly population - based design with inclusion of individuals from both urban and rural areas . the impact of a decrease in bmi in the lower bmi range was twice the impact of the corresponding increase in bmi in the higher bmi range ( figure 2 ) . however , the most striking finding was the relative increase in mortality found in all normal weight individuals in the bmi categories below 25 . mortality from respiratory diseases explained about 40% of the increased total mortality found in men in the lower bmi range . previous studies of elderly individuals have demonstrated increased mortality from respiratory diseases in selected groups with a bmi below 2022.7.12 13 we also found substantially increased mortality from respiratory diseases in the 2022.4 and 22.524.9 bmi categories , which are generally regarded as healthy weight . asthma and chronic bronchitis are prevalent conditions associated with the lower bmi categories in elderly men and women.14 when we analysed subjects without these conditions , the impact of bmi on mortality in the lower range persisted . very few studies of elderly men and women , with information about smoking habits included , have been able to demonstrate a significant increase in mortality in the normal bmi range of 22.524.9 compared to moderately overweight subjects . for some of the studies , this may be explained by a focus on obesity , the use of wide reference categories ( bmi 18.525 ) or a limited number of participants . being underweight is associated with loss of both peripheral and respiratory muscles,18 and this association may partly explain the increases in both total mortality and mortality from respiratory diseases observed in this study among underweight participants . reverse causation that is , the possibility that pre - existing illnesses or conditions associated with increased mortality lead to loss of body weight is a concern in studies of lower bmi and mortality.21 22 to overcome this problem in our analyses , we used several methods . finally , to explore the effect of underweight - associated pulmonary disease , we analysed both total mortality and mortality from respiratory diseases in individuals not reporting asthma / chronic bronchitis at baseline . the prevalence of obesity is rising in elderly individuals and the proportion of obese individuals in the present study ( 20.7% ) was almost at the level found in us populations in the same period.23 we found moderately increased mortality in obese individuals more so in men than in women . several explanations have been proposed for the limited impact of overweight or obesity on mortality in this age group.3 possible explanations include a healthy survival effect in obese individuals meaning that those most vulnerable to the effects of obesity have already died before reaching older age . obesity - related consequences develop slowly and before these consequences appear , individuals who become obese later in life may die from other diseases.24 we found the optimal weight with the lowest mortality to be in the overweight categories ( bmi 2529.9 ) , whereas moderately obese individuals had only a modest increase in mortality . this finding is in line with several previous studies of elderly individuals25 and has been described as the obesity paradox.24 it has also been observed that moderate overweight hospitalised elderly patients have reduced mortality.26 these findings are important because almost half of the elderly population are overweight ( bmi 2529.9 ) and it is frequently assumed that these individuals have increased mortality.24 one strength of the present study is the strictly population - based design with inclusion of individuals from both urban and rural areas . our results show that in elderly men and women , a bmi below 25 was associated with increased total mortality . a modest increase in mortality was found with increasing bmi for obese elderly men and women . the body mass index ( bmi)mortality relationship is u - shaped in both adult and elderly individuals and shifted to the right in the older age categories . there is a lack of data concerning the relationship between bmi and mortality , particularly cause - specific mortality , in the elderly and the impact of mortality from respiratory diseases on the bmi mortality relationship . in the present large study of elderly individuals from norway , mortality was significantly increased in all body mass index ( bmi ) categories below 25 , including the the impact of decreases in bmi in the lower bmi range was twice the impact of the corresponding increases in bmi in the higher bmi range . more attention should be given to elderly individuals in the lower bmi categories and moderately increased bmi ( 2529.9 ) should not be a concern with regard to mortality in this age group .

beyond anecdotal , literary and even lyrical acknowledgments , a rapidly expanding corpus of scientific evidence supports a causal role for sleep in memory processing . this review aims to provide a synthesis of recent findings in humans , with a focus on hippocampal - dependent memory . our goal is to extract consistent themes across domains of memory function that appear to be regulated by sleep , and provide a framework of sleep - dependent hippocampal memory processing within which these findings can be understood . we examine two stages of memory processing ( 1 ) a role for sleep before learning in preparing the hippocampus for initial formation or encoding of new experiences , and ( 2 ) a role for sleep after learning in both non - specific and specific offline consolidation of recently encoded experiences . some of the earliest evidence describing the impact of prior sleep loss on subsequent learning of declarative memories was described by morris et al . ( 1960 ) , and later by harrison and horne ( 2000 ) , demonstrating impairment in encoding and retention of temporal memory ( memory for when events occur ) . in the latter study , significant impairments in memory were evident even in a subgroup that received caffeine to overcome non - specific effects of lower alertness . interestingly , sleep - deprived subjects displayed significantly worse insight into their memory encoding performance , resulting in lower predictive ability of performance , a function itself that may also rely in part upon the hippocampus ( huijbers et al . , 2011 ) ( 2000 ) has examined the neural basis of similar memory impairments using fmri , investigating the effects of 35 h of total sleep deprivation on verbal learning . in those who were sleep - deprived , regions of the temporal lobe were significantly less active during learning , relative to a control group that had slept , while the prefrontal cortex actually expressed greater activation . most interesting , the parietal lobes , which were not activated in the control group during learning , were significantly more active in the deprivation group . such findings suggest that sleep loss prior to learning ( at least following one night ) produces bi - directional changes in verbal encoding activity , involving the inability of the temporal lobe regions to engage normally during learning , combined with potential compensation attempts by higher cortical regions ( drummond et al . , the impact of sleep loss on the specific neural dynamics of hippocampal memory encoding have been examined using event - related fmri ( yoo et al . , 2007a ) . in addition to performance impairments under conditions of sleep deprivation , and relative to a control group that slept , a highly significant yet selective deficit was identified in bilateral regions of the posterior hippocampus , known to be critical for learning new episodic information ( figure 1 ) . ( 2009 ) , demonstrating that selective slow wave sleep deprivation alone is sufficient to impair hippocampal memory encoding ability . taken together , this collection of findings indicate that sleep disruption as well as total sleep deprivation prior to learning compromises the function of human hippocampus to effectively commit new human experiences to memory . regions of decreased encoding activation in the sleep deprivation group relative to the sleep control group in bilateral posterior hippocampus , together with a histogram of parameter estimates ( effect size ) of averaged hippocampal activity in each group . the impact of sleep deprivation on memory formation does not appear to be universal , but instead , may differ on the basis of factors such as emotionality . we have examined the encoding of emotionally negative , positive , and neutral words ( walker , unpublished results ) . across all stimulus types combined , sleep deprivation imposed a 40% reduction in the ability to form new memories , relative to sleep rested individuals ( figure 2a ) . however , when these data were separated into the three affective categories ( negative , positive , or neutral ) , the magnitude of encoding impairment differed ( figure 2b ) . in those that had slept , both positive and negative stimuli were associated with superior retention levels relative the neutral condition , consonant with the notion that emotion facilitates memory encoding ( cahill and mcgaugh , 1998 ) . however , there was severe disruption of encoding and hence later retention for neutral and especially positive emotional memory in the sleep - deprived group . in contrast , a relative resistance of negative emotional memory was observed in the deprivation group . these data suggest that , while the effects of sleep deprivation are directionally consistent across memory subcategories , the most profound impact is on the encoding of positive emotional stimuli , and to a lesser degree , emotionally neutral stimuli . in contrast , the encoding of negative memory appears to be more resistant to the effects of one night of total sleep deprivation . sleep deprivation and encoding of emotional and non - emotional declarative memory . effects of 38 h of total sleep deprivation on encoding of human declarative memory ( a ) when combined across all emotional and non - emotional categories ; ( b ) when separated by emotional ( positive and negative valence ) and non - emotional ( neutral valance ) categories . when comparing between positive and negative words , within group , no significant difference was found for the sleep group while those that were deprived demonstrated greater recall for negative items ( p < 0.05 ) . p < 0.08 , * p < 0.05 , * * p < 0.01 , error bars represent s.e.m . while such evidence describes the detrimental impact of a lack of sleep , recent work has conversely demonstrated the proactive benefit of sleep , and specific sleep physiology , in restoring episodic memory encoding ability ( mander et al . , 2011 ) . following the first learning session at 12:00 , half of the subjects remained awake , while the other half obtained a 100 min nap opportunity . hippocampal - dependent learning deteriorated across the day in those who remained awake ( figure 3a ) . in contrast , sleep not only blocked this deterioration in learning capacity , but triggered a numeric enhancement in hippocampal - dependent encoding ability . within the nap group , the extent of learning restoration ( pre- to post - sleep ) was positively correlated with both the amount of stage-2 nrem , and specifically the number of fast sleep spindles over the left prefrontal cortex ( figures 3b d ) . moreover , and motivated by the role of the lateral prefrontal cortex and medial temporal lobe structures in episodic memory , eeg source analysis of these left prefrontal spindles was performed , revealing an oscillation of activity throughout the spindle event looping through the left temporal lobe ( figure 3e ) . such findings can parsimoniously be accounted for within a hippocampal - neocortical framework of memory processing ( discussed in the following section ) , predicting decreased episodic learning capacity with continued waking experience ; a potential limitation of sparse hippocampal representational coding ( treves et al . , 1996 ) . consequently , nrem sleep spindles , which co - occur with hippocampal sharp - wave ripples ( siapas and wilson , 1998 ; clemens et al . 2009 ) , are proposed to support a proactive shift from hippocampal- to increasing cortical - dependence of previously encoded representations , thereby restoring post - sleep episodic encoding ability . ( a ) episodic learning ability ( % face - name pairs encoded ) in the nap and non - nap group at 12:00 and 18:00 ( left ) , and the change in episodic learning ability between sessions ( 18:0012:00 ; right ) . episodic learning ability in a subset of nap and no - nap subjects ( n = 10 per group ) , matched on initial 12:00 performance , is presented in the center box . comparisons ( line across bars ) in both plots reflect significance * at : p < 0.05 . ( b ) within the nap group , correlations with stage-2 nrem sleep and episodic learning ability at 12:00 ( left - panel ) , 18:00 ( middle - panel ) , and the change in learning ability between session ( 18:0012:00 ; right - panel ) . ( c ) topographic correlations ( color - bar indicates pearson s correlation strength ) in the nap group between fast sleep spindles and episodic learning ability at 18:00 ( post - nap ) , significant in derivations f3 , f4 , fz , f7 , f8 , fp1 , and fp2 over pfc , maximal at f7 ( r = 0.536 , p = 0.018 ) . ( d ) of these derivations , the change in episodic learning ability ( 18:0012:00 ) , significantly and conjointly correlated with fast spindles at derivation f7 over left pfc ( r = 0.535 , p = 0.018 ) . ( e ) sloreta source time - series of fast sleep spindles identified by onset at f7 , demonstrating a current - density loop recurring in left temporal lobe , proceeding the peak of the spindle . modified from mander et al . ( 2011 ) . of note , neither the impairments in encoding following deprivation ( yoo et al . , 2007a ) nor the restoration of encoding following a nap ( mander et al . , 2011 ) appear to be parsimoniously accounted for on the basis of changes in basic alertness or attention . specifically , response times during encoding and testing in both studies , often used as an indexing of alertness , did not predict learning ability or associated encoding brain activity , and were not different between the respective control groups in either study . moreover , in the nap study ( mander et al . , 2011 ) , no differences were observed between the sleep and no sleep groups on a specific alertness control task . one consideration when interpreting the findings of studies that compare between evening and morning learning and testing phases are the inherent changes in alertness and fatigue at these different times , as well as changes in circadian clock time . in addition to measuring objective and subject alertness and attention as potential confounding factors , a nap - paradigm can also be advantageous in this context ( e.g. , mander et al . , 2011 ) , allowing for a manipulation of sleep and wakefulness whilst holding timing ( both clock and circadian ) of learning and testing constant between conditions . a robust and consistent literature has demonstrated the need for sleep after learning in the subsequent consolidation of hippocampal - dependent memory ( gais et al . , 2002 ; graves et al . , 2003 ; rauchs et al . , 2004 ellenbogen et al . , 2006 ; ferrara et al . , 2006 ; fogel and smith , 2006 ; gais et al . , 2006 ; marshall et al . , 2006 ; wagner et al . , 2006 ; gais et al . , 2007 ; , 2007 ; alvarenga et al . , 2008 ; axmacher et al . , 2008 ; , 2008 ; landsness et al . , 2009 ; ramadan et al . , 2009 ; , 2010 ; wamsley et al . , 2010 ) . in humans , arguably the first experimental description of a beneficial role of sleep for memory stabilization was provided by jenkins and dallenbach ( 1924 ) , indicating a protective benefit of sleep ( after approximately 4 h ) in preventing the normal decay - curve of forgetting that develops across time spent awake . ( 2006 ) , have since revealed the extent of sleep s ability to protect declarative memories from forgetting by systematically manipulating interference , employing an a b a c paradigm . in this paradigm , participants first learned unrelated word - pair associates , designated as list a b ( e.g. , leaf - wheel etc . ) . after sleep at night , or wakefulness during the day , half of the subjects in each group learned a new , interfering list containing a new associate paired with the first word , designated as list a c ( e.g. , leaf nail etc ) , before being tested on the original a b list ( e.g. , leaf wheel etc ) . in the groups that did not experience the interfering challenge simply being trained and then tested on list a however , when testing the groups that were exposed to interfering list learning ( list a b ) , a large and significant protective benefit was seen in those that slept ( figure 4 ) . therefore , sleep conferred a consolidation benefit that rendered recently encoded episodic memories resistant to the potentially aggravated effects of new learning of somewhat overlapping ( hence potentially competing ) memory representations the next day . yet , it was only by using an interfering challenge , the a c list , that the true benefit of sleep s protection of memory was revealed ; a benefit that would not necessarily have been evident in a standard study - test memory paradigm . such evidence would favor a mechanism by which sleep either ( 1 ) solidified the original ( a b ) hippocampal representations , forcing encoding of related representations ( a c ) in non - overlapping hippocampal networks , or ( 2 ) the transformation of the original ( a b ) representations to a less hippocampally dependent state , allowing for hippocampal encoding of related new ( a c ) representations that overlap with those previously coding the original ( a b ) representations , but without detrimental interference . percent correct recall for b words from the original a b pair after a 12 h retention interval of either wake or sleep following no interference or interference learning ( list a c ) . p < 0.10 , * p < 0.05 , * * p < 0.001 ; error bars indicate s.e.m . modified from ellenbogen et al . several reports by born and colleagues have shown offline improvement on hippocampus - dependent memory following post - learning sleep attributed to early night sleep , rich in sws ( diekelmann and born , 2010 ) . evidence for such an effect comes first from studies demonstrating that partial deprivation of early sws - rich sleep impairs recall of memories learned prior to sleep ( gais et al . , 2000 ; wagner et al . , 2001 ; wagner et al . , 2002 ; gais and born , 2004 ; fischer et al . , 2011 ) . the same group has also recently demonstrated a causal role for the slow cortical oscillation ( < 1 hz ) of nrem sleep in beneficially enhancing the consolidation of declarative memories by increasing activity in this frequency range . following learning of a word - pair list , transcranial direct current stimulation was applied over the prefrontal cortex of participants during early night sws , inducting slow oscillation - like field potentials ( in this case , at 0.75 hz ; marshall et al . , 2004 , 2006 ) . consequently , a greater benefit on overnight retention was observed for the set of information learned prior to sleep . direct current stimulation not only increased the amount of slow oscillation activity , as well as sleep spindle frequency activity , during the simulation period ( and for some time after ) , but also enhanced next - day word - pair retention . these findings were interpreted in the context of slow oscillations potentially triggering spindle - regulated plasticity in cortex . one mechanism proposed to underlie the benefit of sleep on hippocampal - dependent learning is the reactivation of previously encoded hippocampal memory representations during subsequent nrem sleep . in animals , the signature firing patterns of these hippocampal ( as well as cortical ) networks expressed during waking performance of spatial tasks and novel experiences appear to be replayed during subsequent nrem sleep ( and in some studies , also rem ; wilson and mcnaughton , 1994 ; skaggs and mcnaughton , 1996 ; dave et al . , 1998 ; dave and margoliash , 2000 ; poe et al . , 2000 ; louie and wilson , 2001 ; ribeiro and nicolelis , 2004 ; jones , 2005 ; ji and wilson , 2007 ) . extending these findings , related evidence has been reported in the human brain using a virtual maze task in combination with positron emission tomography ( pet ) scanning ( peigneux et al . , 2004 ) . then , during post - training sleep , there was a re - emergence of hippocampal activation , specifically during sws . most compelling , however , the amount of sws reactivation in the hippocampus was proportional to the amount of next - day task improvement , suggesting that this reactivation is associated with offline memory improvement . building on the framework that memories , particularly those involving the hippocampus , are reactivated at night during sleep , recent work has taken advantage of the classical psychology effect of cue - dependent recall , and translated it into a sleep - dependent consolidation paradigm rasch et al . following learning of a spatial memory task that was paired with a rose sent cue , the same rose odor was re - presented during subsequent sws that night a time when consolidation was presumed to be occurring . relative to a control condition where the odor was not presented again during sws , the re - perfusion of the rose scent at night resulted in significantly improved recall the following day ( when recall occurred without any odor cue ) . moreover , the representation of the odor during sws when previously paired with learning during encoding resulted in greater ( re)activation of the hippocampus during sws , as measured with fmri . these findings support a role for sws in the consolidation of declarative memory in relation to a prior context ( here , odor ) , and may indicate an active reprocessing of initially hippocampal - dependent information during sws . building on these findings , rudoy et al . ( 2009 ) have recently demonstrated similar sleep - dependent reactivation of memory using auditory rather than olfactory cues , describing a selective ability to manipulate individual item memory consolidation during nrem . determining the neural mechanisms that promote sleep - dependent human memory consolidation remains an active topic of research , and debate . it is perhaps unlikely that multiple different memory systems , involving diverse cortical and/or subcortical networks , require the same underlying neural mechanisms for their modulation . even if they do , it is not clear that this process would rely on just one type of sleep - stage physiology . multiple models of sleep - dependent memory have been offered to account for the overnight facilitation of recall , which build on different aspects of neural activity during sleep . a systems - level model of sleep - dependent memory processing ( for discussion of a homeostatic cortical model of sleep - regulated plasticity , see tononi and cirelli , 2003 ) can be considered that involves reciprocal hippocampal - neocortical communication , with potential benefits not only for offline consolidation , but also renewed hippocampal encoding capacity upon awakening . within this framework ( marr , 1971 ; squire and alvarez , 1995 ; squire et al . , 2004 ; frankland and bontempi , 2005 ) , the hippocampus initially binds cortical elements of an experience , creating a holistic episodic memory by way of hippocampal - cortical connections ( figure 5 ) . over time , and by way of iterative offline processes , increasing cortico - cortical connections develop , associated with decreasing dependence on the hippocampus . therefore , the classical model of memory consolidation holds that neocortical structures become increasingly important for the representation of consolidated episodic memories , while the corresponding contribution of the hippocampus progressively decreases . in addition to its role in binding distributed cortical memory components , the hippocampus plays a critical role in reactivating these networks , specifically during sleep . this process of reactivation , assumed to occur over multiple sleep cycles across a night and/or multiple occurrences of sleep over many nights , is proposed to gradually strengthen the initially weak connections between neocortical sites , thereby reinforcing them . eventually , this strengthening is suggested to allow the original information to be engaged in the cortex , largely independent of the hippocampus . ( b ) successive sleep - dependent reactivation of this hippocampal - cortical network leads to progressive strengthening of cortico - cortical connections , ( c ) which over time , allow these memories to become independent of the hippocampus and gradually integrated with pre - existing cortical memories . modified from frankland and bontempi , 2005 . it has been posited that such a sleep - dependent process can offer two symbiotic benefits ( walker , 2009 ) . the first is that episodic memories from the day prior should be more resistant to interference from new hippocampal learning the next day , due to the increased cortico - cortical connections formed during overnight consolidation ( figure 5 ) . it is precisely this behavioral effect that was reported in the study by ellenbogen et al . ( 2006 ) , showing greater post - sleep resistance to interference , using the a in addition to consolidation , however , the second suggested benefit of this sleep - dependent dialog is the post - sleep reinstatement of sparse encoding capacity within the hippocampus , restoring the efficient ability for renewed next - day episodic encoding ( figure 5 ) . this second premise appears to accurately explain the findings previously discussed describing a reduction in hippocampal encoding ability without intervening sleep ( yoo et al . , 2007a ; van der werf et al . , 2009 ) , and conversely , the restoration of hippocampal - dependent learning following the presence of sleep ( mander et al . , 2011 ) several reports have provided evidence supportive of this sleep - dependent dialog and neural transformation of declarative memory . in the first such report , takashima et al . in addition to a long - term evaluation of memory over 3 months , there was also a short - term evaluation of memory across the first day , which included an intervening nap period ( 90 min ) between training and testing of the original studied ( remote ) stimuli . the duration of nrem sws during the intervening nap correlated positively with later recognition memory performance , yet negatively with retrieval - related activity in the hippocampus , consistent with the notion of episodic representations becoming less dependent on the hippocampus , post - sleep . extending these findings , maquet and colleagues have since demonstrated that one night of post - training sleep deprivation , even following recovery sleep , significantly compromises these hippocampal - neocortical neural dynamics and associated memory recollection ( gais et al . , 2007 ) . beyond the contribution of nrem slow waves , a number of reports have described an association between nrem sleep spindle events and memory , including hippocampal - dependent learning . these short ( 1 s ) synchronous bursts of activity are expressed in the eeg in the 1115 hz frequency range ( sejnowski and destexhe , 2000 ; steriade , 2001 ; smith et al . , 2004 ) , and coincide with hippocampal sharp waves and ripples ( siapas and wilson , 1998 ; sirota et al . , 2009 ; diekelmann and born , 2010 ) , possibly reflecting reactivation of learned memory representations ( mlle et al . , 2006 ; born , 2010 ; oneill et al . , 2010 ) . ( 2002 ) , have shown significantly higher sleep spindle density following daytime episodic learning session ( encoding of word - pair associates ) . ( 1999 ) , who reported a similar increase in spindles following learning of a hippocampally dependent maze task , and by clemens et al . ( 2005 ) who have since identified a correlation between spindle density and overnight verbal memory retention ( although interesting , not memory for faces ) . that such results reflect spindle - related memory processing is supported by recent data demonstrating that the same cortical areas active during learning are also ( re)activated during post - encoding sleep spindle events this learning - dependent change in spindle - related activity extends that of use - dependent changes in eeg slow waves that may reflect homeostatic process ( kattler et al . , 1994 ; huber et al . , 2006 ) . continued evidence suggests that sleep spindles can be separated into two subtypes based on frequency : slow ( 1315 hz ; werth et al . , 1997 ; zeitlhofer et al . , the relevance of this separation from a memory consolidation perspective is highlighted by neuroimaging findings demonstrating that fast spindles are associated with , amongst other regions , significantly greater activation within the hippocampal complex ( schabus et al . moreover , recent fmri data have demonstrated that the occurrence of fast sleep spindles coincide with moments of increased functional connectivity between the hippocampus and areas of neocortex ( andrade et al . , 2011 ) , supporting a postulated component of the hippocampal - neocortical model of sleep - dependent memory consolidation . indeed , a growing number of reports describe select associations between episodic memory and fast- but not slow - spindle activity ( tamaki et al . , 2008 ; mander et al . , 2011 ; saletin et al . nevertheless , the role of specific spindle frequency subtypes , and their local topographic influence , in hippocampal- and extra - hippocampal - dependent memory processing represents a target for future research , with early discriminatory findings beginning to emerge ( van der helm et al . , 2011 ) . secondly , while the role for sleep spindles in declarative memory processing is increasingly clear , there remains the need to dissociate which feature or combination of features of the spindle oscillation govern these memory processing advantages ( e.g. , number clemens et al . , 2005 , density saletin et al . , 2011 , or amplitude / power schabus et al . , 2008 ) a consensus on the differential role of slow waves and spindles has also yet to be reached , with reports implicating sleep spindles ( e.g. , gais et al . , 2002 ; schabus et al . , 2004 ; clemens et al . , 2006 ; nishida and walker , 2007 ; genzel et al . , 2009 ; saletin et al . , 2011 ) or however , what , if any , inter - relationships exist between spindles and slow waves remains largely uncharacterized ( molle et al . , 2011 ) and will require studies capable of distinguishing between spindle - driven and slow wave - driven memory mechanisms , or identifying their combined influence . moreover , the potential role for rem sleep and its associated physiological features in declarative memory processing remains relevant ( rauchs et al . , 2004 ; walker , 2009 , see below discussion ) . by way of such future investigations , an increasingly nuanced characterization of dissociable sleep - dependent memory mechanisms can be reached . while evidence reviewed above focuses on nrem sleep in declarative memory consolidation , rem sleep should not be discounted . early work reported that the overnight retention of emotional details of a narrative story , relative to emotionally neutral details , was superior following late - night sleep ( a time period rich in rem sleep ; wagner et al . , 2001 ) . it has subsequently been demonstrated that the speed of recognizing emotional face expressions presented prior to sleep is significantly improved the next day , the amount of which positively correlated with the amount of intervening rem sleep ( wagner et al . , 2007 ) . moreover , not only does the amount of time and speed of entry into rem sleep predict the degree of subsequent strengthening and hence offline consolidation of emotional ( and not neutral ) memory , but specifically the amount of rem sleep theta eeg activity ( 47 hz ) expressed over the prefrontal cortex that predicts memory retention ( nishida et al . these findings have lead to the proposal that rem sleep represents a neurobiological brain - state particularly amenable to emotional memory processing ( pare et al . , 2002 ; hu et al . , 2006 ; walker , 2009 ; walker and van der helm , 2009 ) , with theta oscillations proposed as a carrier frequency that potentially allows disparate brain regions that initially encode information to selectively interact offline . by doing so , rem sleep theta may afford the ability to strengthen distributed aspects of specific memory representations across related but different anatomical networks , and/or promote their integration into pre - existing autobiographical memory networks ( cahill , 2000 ; jones and wilson , 2005 ) . evidence reviewed to this point implicates sleep in the consolidation and hence superior offline retention of declarative memory , relative to equivalent time awake . however , the capacity to forget can , in certain contexts , be as important as the need for memory retention , both in day - to - day life ( e.g. , forgetting last week s parking spot in preference for today s ) , and clinically ( e.g. , post - traumatic stress disorder and addiction ) . considered to be adaptive , selective forgetting has been shown to decrease neural resources required for targeted remembering ( levy and anderson , 2002 ; kuhl et al . , 2007 ) , and as a consequence , may afford improved efficiency of subsequent recall of select information ( block , 1971 ; anderson et al . , 2004 ; levy and anderson , 2008 ) . building on such evidence , here we offer the thesis that the goal of offline sleep - dependent memory processing is not the verbose , ubiquitous , and non - selective consolidation of all information recently encoded . nor instead , we suggest offline sleep - dependent memory processing may involve a discriminatory mechanism , affording a balance of retention , and forgetting , with item selection determined by salience cues present during wake , such as novelty , emotionality , reward value , and explicit conscious instruction ( figure 6 ) . new selective model of sleep - dependent hippocampal - neocortical memory consolidation . ( a ) at encoding the hippocampus rapidly integrates information within distributed cortical modules , however certain memories ( colored nodes ) are weighted over others by means of relevant information ( e.g. , emotion , reward , or intention ) , whereas other memories lack this relevance tag ( gray nodes ) . ( b ) successive sleep - dependent reactivation of this hippocampal - cortical network , by way of synchronous nrem ( sleep spindle and slow wave ) as well as rem ( theta ) oscillation , leads to progressive strengthening of cortico - cortical connections , though only for those memories deemed relevant by associated encoding information ( c ) which over time , leads to a selective consolidation of certain learned items , whereas other items , not given a benefit of selectivity , are allowed to dissipate at both hippocampal and neocortical levels . modified from frankland and bontempi ( 2005 ) . emerging reports have begun to support this more nuanced , discriminatory framework of sleep - dependent memory processing . , 2006 ; atienza and cantero , 2008 ) , and specifically rem sleep ( wagner et al . , 2001 ; nishida et al . , 2009 ) , has been demonstrated to target the selective consolidation of emotional relative to non - emotional experiences . moreover , sleep appears capable of separating episodic experiences into component parts , preferentially consolidating those of greatest affective salience ( payne et al . , 2008 ) . beyond emotion , sleep has recently been demonstrated to selectively enhance information based on waking knowledge of potential monetary reward prior to sleep ( fischer and born , 2009 ) , even when such knowledge of a reward was announced after initial learning ( but before sleep ) . further , the mere knowledge that some learned items will be subject to later post - sleep testing , presumably involving a difference in salient relevance , amongst other things , can selectively increase the offline sleep - dependent consolidation benefit ( wilhelm et al . , 2011 ) . explicit conscious instruction at the time of encoding also appears to represent a powerful modulatory influence on subsequent sleep - dependent consolidation . using a directed forgetting paradigm , the impact of cued instruction during learning to either remember or forget , prior to sleep , on subsequent differential retention of memory after sleep has recently been examined ( saletin et al . , 2011 ) . relative to time awake , sleep subsequently and selectively ignored the facilitation of items previously cued to be forgotten , yet preferentially enhance recall for items cued to be remembered ( figure 7a ) . this effect was further characterized as a difference - score ( remember words forget words ) . this difference measure , reflecting the efficiency of accomplishing targeted remembering and forgetting , demonstrating a greater separation between recall of remember relative to forget items following sleep ( figure 7b ) . thus , sleep did not benefit all memories universally , instead being selective for those items cued for remembering and not those cued to be forgotten , indicative of specificity based on prior waking instruction . moreover , the magnitude of this directed forgetting effect was positively predicted by fast spindles over left parietal cortex , suggesting their potential role in differential gating of offline consolidation , governing the directional fate of each memory item class ( remember , forget ; figure 7c ) . additionally , the same parietal fast spindles differentially and bi - directionally modulated each word class individually ; positively predicting recall for remember words alone while , conversely , negatively predicting recall for forget words ( figure 7d , e ) . this evidence favors a sleep mechanism that not only promotes remembering but also actively instigates forgetting . offering deeper anatomical insights , eeg source analyses revealed an loop of activity occurring during these parietal fast spindle events , consisting of areas previously implicated in differential remembering and forgetting memory ( wylie et al . , 2008 ) : prefrontal , medial temporal , and posterior parietal cortices . such a network may support memory modulation whereby top - down cues of instructed intent ( prefrontal cortex ; remember , forget ) are coordinated and utilized during offline processing of bottom - up item memory ( medial temporal lobe ) , and potentially integrated into association regions ( parietal cortex ) for consolidation ( shimamura , 2011 ) . related findings have been reported using an episodic memory paradigm involving the intentional suppression and contemplation of word items prior to sleep , with those intentionally suppressed during initial exposure showing significantly less offline sleep - dependent memory benefit ( fischer et al . , 2011 ) . adding to these findings , post - encoding sleep deprivation can further impair the subsequent offline development of these differential remember and forget consolidation effects , dependent , in part , on hippocampal signals at the time of encoding ( rauchs et al . ( a ) memory performance : number of words recalled based on prior cue instruction ( remember , r - words ; forget , f - words ) in the nap and no - nap groups and the ( b ) efficiency measure of directed forgetting , calculated as the subtraction of these scores ( r f ; expressed as a proportion of total recall ) . between group comparisons ( line across bars ) reflect significance at : * p < 0.05 and * * p < 0.01 . relationship between memory performance and sleep spindles : ( c ) scatter - plot and linear regression of the relationship between fast sleep - spindle density at p3 and the r ( c d ) scatterplots demonstrating that the same p3 fast spindles ( d ) positively predict r - words and ( e ) negatively predict f - words , though neither correlation is as strong as the r f difference measure , suggesting a relative selectivity of sleep - dependent memory consolidation . modified from saletin et al . ( 2011 ) . it is of note that the presentation of olfactory cues during sleep for memory reactivation described earlier only resulted in facilitated overnight consolidation when the same odor was initially presented at the time of learning ( rasch et al . , 2007 ) . odor presented during sleep in isolation , without prior waking association with learning , conferred neither an overnight memory consolidation advantage nor elevated hippocampal reactivation during sleep . this finding appears consistent with the proposed model of selective memory consolidation , suggesting that even the success of memory reactivation during sleep critically depends on associating those memories with the cues during initial encoding , prior to sleep . such associations would effectively label specific memory representations at the time of learning with contextually salient tags ; offering the capacity for selective reactivation , and subsequent consolidation , during sleep . a further circumstance that may determine selective memory consolidation is the relationship between newly learned representations and pre - existing autobiographical memory schemata ( lewis and durrant , 2011 ) . experiences that hold relevance to the organism s autobiographical past have the potential for being registered , as a consequence , with superior salience , and hence undergo preferential overnight consolidation . similarly , employing learning strategies such as mnemonic techniques that assist in elaboration on , and integration amongst , new learning ( or even pre - existing information ; wang and thomas , 2000 ) may similarly promote selective offline consolidation during sleep . when taken together , this collection of studies suggest that sleep does not universally consolidate all episodic memories learned during the prior day(s ) . instead , sleep appears to be more ecologically attuned to qualitative aspects of encoded experiences at the time of initial learning e.g. emotionality ( hu et al . , 2006 ; wagner et al . , 2006 , 2007 ; payne et al . , 2008 ; walker and van der helm , 2009 ; payne and kensinger , 2011 ) , reward potential ( fischer and born , 2009 ; wilhelm et al . , 2011 ) , or explicit cue instructions and intentions ( fischer et al . , 2011 ; rauchs et al . , 2011 ; saletin et al . , 2011 ) , resulting in discriminatory offline memory processing . such findings additionally offer key anatomical targets for the understanding of sleep - dependent memory processing , focusing on those regions whose interactions with the medial temporal lobe support selective memory processing e.g. , amygdala for emotional memories ( hu et al . , 2006 ; yoo et al . , 2007b ; nishida et al . , 2009 ; walker and van der helm , 2009 ; payne and kensinger , 2011 ) , striatum for reward signals ( knutson and adcock , 2005 ) and the prefrontal cortex for directed intentional cues ( anderson et al . , 2004 ; parenthetically , this selective sleep benefit , including the bi - directional influence of spindles on remembering and forgetting , seems difficult to reconcile within simple opportunistic frameworks of offline consolidation ( mednick et al . , 2011 ) . instead , such evidence appears to warrant a shift in perspective from notions of sleep affording a universal consolidation benefit to recently encoded information ( figure 4 ) , to one in which unique and specific sleep - stage physiologies select item information for preferential retention , based on prior waking cues ( figure 6 ) ; a process conceptually similar to a sleep - slave consolidation system , governed by prior waking cues , and yielding discriminatory long - term memory consolidation . while remaining speculative , potentially novel clinical and therapeutic implications stem from this revised model of selective memory consolidation . perhaps most prominent are the circumstance of anxiety disorders as well as major depression , both of which express dysfunction in memory as well as sleep abnormalities ( armitage , 1995 ; van wingen et al . , 2010 ; dickie et al . , 2011 ; harvey , 2011 ) . in major depression , disproportionate rumination of past negative autobiographical events can be prevalent , contributed to by the proposal of a bias in initial encoding of negative experiences ( taconnat et al . , 2010 ; van wingen et al . , 2010 ) . in the context of our model , this initial encoding bias may lead to the maladaptive further potentiation of those selectively negative experiences during sleep , the consequence of which would be their persistent dominance in long - term memory , further encouraging negative rumination . in anxiety disorders , including post - traumatic stress disorder and phobia , a similarly biased associative encoding of episodic experiences with strong negative aversive emotion ( mitte , 2008 ; dickie et al . , 2011 ) may tag those experiences for disproportional selective consolidation , potentiating the trauma experiences or phobic associations ( and see walker and van der helm , 2009 for discussion of emotion processing of memory in ptsd ) . conversely , however , these same mechanisms could be considered for use in therapeutic intervention . in both conditions , targeting the problematic memory sources during wake , by recollection and hence reactivation ( lee et al . , 2004 ; stickgold and walker , 2005 ; tronson and taylor , 2007 ) , can offer the capacity for developing new positive ( rather than negative ) , or simply neutral , associations , pre - sleep . subsequent selective consolidation of these new positive or neutral associations in sleep , put in place during prior wake , could help foster a revised and therapeutically advantageous collection of memory representations in the individual s autobiographical history . finally , similar bi - directional notions of memory modulation may also be relevant in addiction , both in the maladaptive potentiation of addiction associations , but also their potential targeted reversal leading to extinction of acquired associations . while not fully complete , a new taxonomy of sleep - dependent memory processing is emerging . this revised view describes a symbiotic role for sleep both before and after learning in optimally coordinating the initial encoding and subsequent consolidation of hippocampal - dependent memory , respectively . by way of such a mechanism , sleep appears capable of maximizing retention only of the most salient , relevant hippocampal memories , while forgetting ( perhaps actively ) , those non - essential to the organism an optimal algorithm for equilibrium within memory networks . 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 .

as critical as waking brain function is to learning and memory , an established literature now describes an equally important yet complementary role for sleep in information processing . this overview examines the specific contribution of sleep to human hippocampal memory processing ; both the detriments caused by a lack of sleep , and conversely , the proactive benefits that develop following the presence of sleep . first , a role for sleep before learning is discussed , preparing the hippocampus for initial memory encoding . second , a role for sleep after learning is considered , modulating the post - encoding consolidation of hippocampal - dependent memory . third , a model is outlined in which these encoding and consolidation operations are symbiotically accomplished , associated with specific nrem sleep physiological oscillations . as a result , the optimal network outcome is achieved : increasing hippocampal independence and hence overnight consolidation , while restoring next - day sparse hippocampal encoding capacity for renewed learning ability upon awakening . finally , emerging evidence is considered suggesting that , unlike previous conceptions , sleep does not universally consolidate all information . instead , and based on explicit as well as saliency cues during initial encoding , sleep executes the discriminatory offline consolidation only of select information . consequently , sleep promotes the targeted strengthening of some memories while actively forgetting others ; a proposal with significant theoretical and clinical ramifications .

Introduction Sleep before Learning for Memory Encoding Sleep after Learning, for Memory Consolidation Consolidation: Selective or Universal Sleep Benefits? Conclusions Conflict of Interest Statement

beyond anecdotal , literary and even lyrical acknowledgments , a rapidly expanding corpus of scientific evidence supports a causal role for sleep in memory processing . this review aims to provide a synthesis of recent findings in humans , with a focus on hippocampal - dependent memory . our goal is to extract consistent themes across domains of memory function that appear to be regulated by sleep , and provide a framework of sleep - dependent hippocampal memory processing within which these findings can be understood . we examine two stages of memory processing ( 1 ) a role for sleep before learning in preparing the hippocampus for initial formation or encoding of new experiences , and ( 2 ) a role for sleep after learning in both non - specific and specific offline consolidation of recently encoded experiences . ( 1960 ) , and later by harrison and horne ( 2000 ) , demonstrating impairment in encoding and retention of temporal memory ( memory for when events occur ) . interestingly , sleep - deprived subjects displayed significantly worse insight into their memory encoding performance , resulting in lower predictive ability of performance , a function itself that may also rely in part upon the hippocampus ( huijbers et al . , the impact of sleep loss on the specific neural dynamics of hippocampal memory encoding have been examined using event - related fmri ( yoo et al . in addition to performance impairments under conditions of sleep deprivation , and relative to a control group that slept , a highly significant yet selective deficit was identified in bilateral regions of the posterior hippocampus , known to be critical for learning new episodic information ( figure 1 ) . taken together , this collection of findings indicate that sleep disruption as well as total sleep deprivation prior to learning compromises the function of human hippocampus to effectively commit new human experiences to memory . the impact of sleep deprivation on memory formation does not appear to be universal , but instead , may differ on the basis of factors such as emotionality . in those that had slept , both positive and negative stimuli were associated with superior retention levels relative the neutral condition , consonant with the notion that emotion facilitates memory encoding ( cahill and mcgaugh , 1998 ) . however , there was severe disruption of encoding and hence later retention for neutral and especially positive emotional memory in the sleep - deprived group . these data suggest that , while the effects of sleep deprivation are directionally consistent across memory subcategories , the most profound impact is on the encoding of positive emotional stimuli , and to a lesser degree , emotionally neutral stimuli . while such evidence describes the detrimental impact of a lack of sleep , recent work has conversely demonstrated the proactive benefit of sleep , and specific sleep physiology , in restoring episodic memory encoding ability ( mander et al . following the first learning session at 12:00 , half of the subjects remained awake , while the other half obtained a 100 min nap opportunity . hippocampal - dependent learning deteriorated across the day in those who remained awake ( figure 3a ) . in contrast , sleep not only blocked this deterioration in learning capacity , but triggered a numeric enhancement in hippocampal - dependent encoding ability . within the nap group , the extent of learning restoration ( pre- to post - sleep ) was positively correlated with both the amount of stage-2 nrem , and specifically the number of fast sleep spindles over the left prefrontal cortex ( figures 3b d ) . moreover , and motivated by the role of the lateral prefrontal cortex and medial temporal lobe structures in episodic memory , eeg source analysis of these left prefrontal spindles was performed , revealing an oscillation of activity throughout the spindle event looping through the left temporal lobe ( figure 3e ) . such findings can parsimoniously be accounted for within a hippocampal - neocortical framework of memory processing ( discussed in the following section ) , predicting decreased episodic learning capacity with continued waking experience ; a potential limitation of sparse hippocampal representational coding ( treves et al . consequently , nrem sleep spindles , which co - occur with hippocampal sharp - wave ripples ( siapas and wilson , 1998 ; clemens et al . ( a ) episodic learning ability ( % face - name pairs encoded ) in the nap and non - nap group at 12:00 and 18:00 ( left ) , and the change in episodic learning ability between sessions ( 18:0012:00 ; right ) . ( b ) within the nap group , correlations with stage-2 nrem sleep and episodic learning ability at 12:00 ( left - panel ) , 18:00 ( middle - panel ) , and the change in learning ability between session ( 18:0012:00 ; right - panel ) . ( c ) topographic correlations ( color - bar indicates pearson s correlation strength ) in the nap group between fast sleep spindles and episodic learning ability at 18:00 ( post - nap ) , significant in derivations f3 , f4 , fz , f7 , f8 , fp1 , and fp2 over pfc , maximal at f7 ( r = 0.536 , p = 0.018 ) . ( d ) of these derivations , the change in episodic learning ability ( 18:0012:00 ) , significantly and conjointly correlated with fast spindles at derivation f7 over left pfc ( r = 0.535 , p = 0.018 ) . specifically , response times during encoding and testing in both studies , often used as an indexing of alertness , did not predict learning ability or associated encoding brain activity , and were not different between the respective control groups in either study . one consideration when interpreting the findings of studies that compare between evening and morning learning and testing phases are the inherent changes in alertness and fatigue at these different times , as well as changes in circadian clock time . a robust and consistent literature has demonstrated the need for sleep after learning in the subsequent consolidation of hippocampal - dependent memory ( gais et al . yet , it was only by using an interfering challenge , the a c list , that the true benefit of sleep s protection of memory was revealed ; a benefit that would not necessarily have been evident in a standard study - test memory paradigm . several reports by born and colleagues have shown offline improvement on hippocampus - dependent memory following post - learning sleep attributed to early night sleep , rich in sws ( diekelmann and born , 2010 ) . the same group has also recently demonstrated a causal role for the slow cortical oscillation ( < 1 hz ) of nrem sleep in beneficially enhancing the consolidation of declarative memories by increasing activity in this frequency range . consequently , a greater benefit on overnight retention was observed for the set of information learned prior to sleep . direct current stimulation not only increased the amount of slow oscillation activity , as well as sleep spindle frequency activity , during the simulation period ( and for some time after ) , but also enhanced next - day word - pair retention . one mechanism proposed to underlie the benefit of sleep on hippocampal - dependent learning is the reactivation of previously encoded hippocampal memory representations during subsequent nrem sleep . in animals , the signature firing patterns of these hippocampal ( as well as cortical ) networks expressed during waking performance of spatial tasks and novel experiences appear to be replayed during subsequent nrem sleep ( and in some studies , also rem ; wilson and mcnaughton , 1994 ; skaggs and mcnaughton , 1996 ; dave et al . then , during post - training sleep , there was a re - emergence of hippocampal activation , specifically during sws . most compelling , however , the amount of sws reactivation in the hippocampus was proportional to the amount of next - day task improvement , suggesting that this reactivation is associated with offline memory improvement . building on the framework that memories , particularly those involving the hippocampus , are reactivated at night during sleep , recent work has taken advantage of the classical psychology effect of cue - dependent recall , and translated it into a sleep - dependent consolidation paradigm rasch et al . moreover , the representation of the odor during sws when previously paired with learning during encoding resulted in greater ( re)activation of the hippocampus during sws , as measured with fmri . these findings support a role for sws in the consolidation of declarative memory in relation to a prior context ( here , odor ) , and may indicate an active reprocessing of initially hippocampal - dependent information during sws . determining the neural mechanisms that promote sleep - dependent human memory consolidation remains an active topic of research , and debate . multiple models of sleep - dependent memory have been offered to account for the overnight facilitation of recall , which build on different aspects of neural activity during sleep . a systems - level model of sleep - dependent memory processing ( for discussion of a homeostatic cortical model of sleep - regulated plasticity , see tononi and cirelli , 2003 ) can be considered that involves reciprocal hippocampal - neocortical communication , with potential benefits not only for offline consolidation , but also renewed hippocampal encoding capacity upon awakening . , 2004 ; frankland and bontempi , 2005 ) , the hippocampus initially binds cortical elements of an experience , creating a holistic episodic memory by way of hippocampal - cortical connections ( figure 5 ) . over time , and by way of iterative offline processes , increasing cortico - cortical connections develop , associated with decreasing dependence on the hippocampus . therefore , the classical model of memory consolidation holds that neocortical structures become increasingly important for the representation of consolidated episodic memories , while the corresponding contribution of the hippocampus progressively decreases . in addition to its role in binding distributed cortical memory components , the hippocampus plays a critical role in reactivating these networks , specifically during sleep . ( b ) successive sleep - dependent reactivation of this hippocampal - cortical network leads to progressive strengthening of cortico - cortical connections , ( c ) which over time , allow these memories to become independent of the hippocampus and gradually integrated with pre - existing cortical memories . ( 2006 ) , showing greater post - sleep resistance to interference , using the a in addition to consolidation , however , the second suggested benefit of this sleep - dependent dialog is the post - sleep reinstatement of sparse encoding capacity within the hippocampus , restoring the efficient ability for renewed next - day episodic encoding ( figure 5 ) . , 2009 ) , and conversely , the restoration of hippocampal - dependent learning following the presence of sleep ( mander et al . the duration of nrem sws during the intervening nap correlated positively with later recognition memory performance , yet negatively with retrieval - related activity in the hippocampus , consistent with the notion of episodic representations becoming less dependent on the hippocampus , post - sleep . extending these findings , maquet and colleagues have since demonstrated that one night of post - training sleep deprivation , even following recovery sleep , significantly compromises these hippocampal - neocortical neural dynamics and associated memory recollection ( gais et al . beyond the contribution of nrem slow waves , a number of reports have described an association between nrem sleep spindle events and memory , including hippocampal - dependent learning . that such results reflect spindle - related memory processing is supported by recent data demonstrating that the same cortical areas active during learning are also ( re)activated during post - encoding sleep spindle events this learning - dependent change in spindle - related activity extends that of use - dependent changes in eeg slow waves that may reflect homeostatic process ( kattler et al . , the relevance of this separation from a memory consolidation perspective is highlighted by neuroimaging findings demonstrating that fast spindles are associated with , amongst other regions , significantly greater activation within the hippocampal complex ( schabus et al . , 2011 ) , supporting a postulated component of the hippocampal - neocortical model of sleep - dependent memory consolidation . nevertheless , the role of specific spindle frequency subtypes , and their local topographic influence , in hippocampal- and extra - hippocampal - dependent memory processing represents a target for future research , with early discriminatory findings beginning to emerge ( van der helm et al . secondly , while the role for sleep spindles in declarative memory processing is increasingly clear , there remains the need to dissociate which feature or combination of features of the spindle oscillation govern these memory processing advantages ( e.g. moreover , the potential role for rem sleep and its associated physiological features in declarative memory processing remains relevant ( rauchs et al . by way of such future investigations , an increasingly nuanced characterization of dissociable sleep - dependent memory mechanisms can be reached . while evidence reviewed above focuses on nrem sleep in declarative memory consolidation , rem sleep should not be discounted . moreover , not only does the amount of time and speed of entry into rem sleep predict the degree of subsequent strengthening and hence offline consolidation of emotional ( and not neutral ) memory , but specifically the amount of rem sleep theta eeg activity ( 47 hz ) expressed over the prefrontal cortex that predicts memory retention ( nishida et al . evidence reviewed to this point implicates sleep in the consolidation and hence superior offline retention of declarative memory , relative to equivalent time awake . however , the capacity to forget can , in certain contexts , be as important as the need for memory retention , both in day - to - day life ( e.g. , 2007 ) , and as a consequence , may afford improved efficiency of subsequent recall of select information ( block , 1971 ; anderson et al . building on such evidence , here we offer the thesis that the goal of offline sleep - dependent memory processing is not the verbose , ubiquitous , and non - selective consolidation of all information recently encoded . nor instead , we suggest offline sleep - dependent memory processing may involve a discriminatory mechanism , affording a balance of retention , and forgetting , with item selection determined by salience cues present during wake , such as novelty , emotionality , reward value , and explicit conscious instruction ( figure 6 ) . new selective model of sleep - dependent hippocampal - neocortical memory consolidation . ( b ) successive sleep - dependent reactivation of this hippocampal - cortical network , by way of synchronous nrem ( sleep spindle and slow wave ) as well as rem ( theta ) oscillation , leads to progressive strengthening of cortico - cortical connections , though only for those memories deemed relevant by associated encoding information ( c ) which over time , leads to a selective consolidation of certain learned items , whereas other items , not given a benefit of selectivity , are allowed to dissipate at both hippocampal and neocortical levels . emerging reports have begun to support this more nuanced , discriminatory framework of sleep - dependent memory processing . further , the mere knowledge that some learned items will be subject to later post - sleep testing , presumably involving a difference in salient relevance , amongst other things , can selectively increase the offline sleep - dependent consolidation benefit ( wilhelm et al . using a directed forgetting paradigm , the impact of cued instruction during learning to either remember or forget , prior to sleep , on subsequent differential retention of memory after sleep has recently been examined ( saletin et al . thus , sleep did not benefit all memories universally , instead being selective for those items cued for remembering and not those cued to be forgotten , indicative of specificity based on prior waking instruction . moreover , the magnitude of this directed forgetting effect was positively predicted by fast spindles over left parietal cortex , suggesting their potential role in differential gating of offline consolidation , governing the directional fate of each memory item class ( remember , forget ; figure 7c ) . additionally , the same parietal fast spindles differentially and bi - directionally modulated each word class individually ; positively predicting recall for remember words alone while , conversely , negatively predicting recall for forget words ( figure 7d , e ) . related findings have been reported using an episodic memory paradigm involving the intentional suppression and contemplation of word items prior to sleep , with those intentionally suppressed during initial exposure showing significantly less offline sleep - dependent memory benefit ( fischer et al . adding to these findings , post - encoding sleep deprivation can further impair the subsequent offline development of these differential remember and forget consolidation effects , dependent , in part , on hippocampal signals at the time of encoding ( rauchs et al . ( a ) memory performance : number of words recalled based on prior cue instruction ( remember , r - words ; forget , f - words ) in the nap and no - nap groups and the ( b ) efficiency measure of directed forgetting , calculated as the subtraction of these scores ( r f ; expressed as a proportion of total recall ) . relationship between memory performance and sleep spindles : ( c ) scatter - plot and linear regression of the relationship between fast sleep - spindle density at p3 and the r ( c d ) scatterplots demonstrating that the same p3 fast spindles ( d ) positively predict r - words and ( e ) negatively predict f - words , though neither correlation is as strong as the r f difference measure , suggesting a relative selectivity of sleep - dependent memory consolidation . it is of note that the presentation of olfactory cues during sleep for memory reactivation described earlier only resulted in facilitated overnight consolidation when the same odor was initially presented at the time of learning ( rasch et al . this finding appears consistent with the proposed model of selective memory consolidation , suggesting that even the success of memory reactivation during sleep critically depends on associating those memories with the cues during initial encoding , prior to sleep . such associations would effectively label specific memory representations at the time of learning with contextually salient tags ; offering the capacity for selective reactivation , and subsequent consolidation , during sleep . experiences that hold relevance to the organism s autobiographical past have the potential for being registered , as a consequence , with superior salience , and hence undergo preferential overnight consolidation . similarly , employing learning strategies such as mnemonic techniques that assist in elaboration on , and integration amongst , new learning ( or even pre - existing information ; wang and thomas , 2000 ) may similarly promote selective offline consolidation during sleep . when taken together , this collection of studies suggest that sleep does not universally consolidate all episodic memories learned during the prior day(s ) . instead , sleep appears to be more ecologically attuned to qualitative aspects of encoded experiences at the time of initial learning e.g. , 2011 ) , resulting in discriminatory offline memory processing . such findings additionally offer key anatomical targets for the understanding of sleep - dependent memory processing , focusing on those regions whose interactions with the medial temporal lobe support selective memory processing e.g. instead , such evidence appears to warrant a shift in perspective from notions of sleep affording a universal consolidation benefit to recently encoded information ( figure 4 ) , to one in which unique and specific sleep - stage physiologies select item information for preferential retention , based on prior waking cues ( figure 6 ) ; a process conceptually similar to a sleep - slave consolidation system , governed by prior waking cues , and yielding discriminatory long - term memory consolidation . perhaps most prominent are the circumstance of anxiety disorders as well as major depression , both of which express dysfunction in memory as well as sleep abnormalities ( armitage , 1995 ; van wingen et al . in the context of our model , this initial encoding bias may lead to the maladaptive further potentiation of those selectively negative experiences during sleep , the consequence of which would be their persistent dominance in long - term memory , further encouraging negative rumination . while not fully complete , a new taxonomy of sleep - dependent memory processing is emerging . this revised view describes a symbiotic role for sleep both before and after learning in optimally coordinating the initial encoding and subsequent consolidation of hippocampal - dependent memory , respectively . by way of such a mechanism , sleep appears capable of maximizing retention only of the most salient , relevant hippocampal memories , while forgetting ( perhaps actively ) , those non - essential to the organism an optimal algorithm for equilibrium within memory networks .

most of the commonly used approaches to analyze gene regulatory interactions , such as epistasis analysis , rely on some implicit assumptions . as we will show , one common of such implicit assumptions is that genes are arranged in a hierarchical pattern of interactions in which each gene can either be upstream or downstream , but not both , as it occurs in feedback loops ( figures 1a , b ) . another commonly implicit assumption is that gene interactions are part of a single - path , in contrast to cases in which a given gene can regulate another gene via two different pathways at the same time , as it occurs in feed - forward loops ( figures 1c , d ) . the notion of hierarchical and single - path gene regulation is consistent with the search of pathways or cascades rather than networks ( greenspan , 2001 ; aylor and zeng , 2008 ) . however , experimental and theoretical work has demonstrated that biological molecular mechanisms contain regulatory feedback and feed - forward loops that do not fulfill the hierarchical and single - path assumptions , but are rather consistent with a network - based perspective . such traits of gene regulation are key for understanding gene regulatory dynamics of almost any biological process ( mangan and alon , 2003 ; brandman and meyer , 2008 ; jaeger et al . , 2008 ; kaplan et al . , 2008 ; mitrophanov and groisman , 2008 ) . hence , methods that consider regulatory feedback loops , feed - forward loops , and temporal dynamics at the same time will likely improve previous approaches . in this article we use epistasis analysis as an example , to explore the effect of these three aspects of gene regulation on the results and interpretation of gene interaction analyses . all nodes are either upstream , downstream , or at the same level . consequently x regulates y and z , but x is not regulated by either y or z. no gene can by upstream and downstream at the same time . in ( b ) two feedback loops are included , by assuming that y and z regulate x. hence , it is not possible to establish a hierarchy on gene regulation , since all genes can by upstream and downstream at the same time . in ( c ) ( d ) a feed - forward loop is incorporated yielding two alternative pathways starting at x : one is a direct regulation of z , and the other one implies an indirect regulation of z , via y. epistasis is ubiquitous within gene regulatory networks in living organisms ( tyler et al . , 2009 ) . as acknowledged by many authors , epistasis has important implications in a broad range of biological issues , from biomedicine to evolutionary studies ( see phillips , 2008 and references therein ) . is important to note that there are different notions of the term epistasis : the original one proposed by bateson in 1907 ( herein called classical epistasis ) that refers to the masking of the effect of one allele by another allele in a different locus , and a broader definition referring to gene interactions in general . we focus here on classical epistasis , but it is important to note that the different notions of epistasis are related . hence , some works have aimed to bring together these different notions of the term epistasis ( e.g. , moore and williams , 2005 ) , allowing elegant and improved analyses of classical epistasis for quantitative traits as well as qualitative discrete ones ( e.g. , aylor and zeng , 2008 ; phenix et al . , 2011 ) . in any case , the original analysis proposed for classical epistasis ( herein called classical epistasis analysis ) as described by avery and wasserman ( 1992 ) is still one of the most powerful and widely used tools in molecular biology to infer biological pathways and regulatory interactions among genes and to validate predictions derived from high - throughput experimental analysis . it is simple , very powerful , and relies on some explicit and implicit assumptions that , when met , allow this analysis to be taken almost as a recipe to order genes along control pathways ( avery and wasserman , 1992 ; huang and sternberg , 2006 ; roth et al . , 2009 ; compares the effect of each single mutant ( x and y single gene mutants ) against the double mutant ( xy double mutant ) of the genes under study on the phenotype of a trait ( in this paper , the trait represents a gene ) . the masking of the effect of one gene mutation in the double mutant is called epistasis . the explicit assumptions of the classical epistasis analysis are : ( 1 ) there is a signal or input that determines the state of the phenotype under analysis , ( 2 ) the signal also determines the state of the upstream gene , ( 3 ) the signal and the two genes are the only determinants of the phenotype , at least in the context of an experimental model , and finally , ( 4 ) the mutants analyzed are null or complete loss - of - function mutants ( based on avery and wasserman , 1992 ; huang and sternberg , 2006 ) . however , classical epistasis analysis also relies on the implicit assumptions of hierarchical and single - path gene regulation . as we will show , the accomplishment of the implicit assumptions is fundamental for the validity of the classical epistasis analysis . many authors have focused on diverse assumptions of classical epistasis analysis and discussed the implications of violating some of them ( e.g. , avery and wasserman , 1992 ; huang and sternberg , 2006 ; phenix et al . , this has motivated efforts to attain better interpretations , relax the assumption of epistasis analyses and expand its applicability ( see an excellent review in phillips , 2008 ) . anyhow , to our knowledge , no previous work has explored the joint effect of the hierarchical and single - path aspects of gene regulatory interactions on classical epistasis analysis . network - based approaches can almost naturally overcome many of the limitations of classical epistasis analysis and gene interaction analyses in general . partly because of this , most of the improvements of the epistasis analyses have relied on the use of networks . for example , systems biology is creating epistatic networks that take into account many gene interactions ( e.g. , tong et al . , 2004 ; st onge et al . , 2007 ; battle et al . , 2010 ) . these studies propose the use of network - based approaches applying modifications to the standard methods , with the incorporation of graph theory , bayesian networks , as well as statistical or probabilistic properties , among others , for the study of epistasis ( phillips , 2008 ; tyler et al . such approaches have allowed inferences of gene interactions with high statistical confidence levels , but the validation of the predicted gene interactions with such methods usually requires further confirmations with more detailed experiments because false interactions or wrong gene order can sometimes be inferred ( e.g. , battle et al . , 2010 ) . only a few of these reports have addressed the improvement of classical epistasis analysis in particular . however , improved classical epistasis analysis approaches are also available ( aylor and zeng , 2008 ; phenix et al . , 2011 ) . aylor and zeng ( 2008 ) present a method for experimentally estimating and interpreting classical epistasis that combines the approaches of classical and quantitative genetics , while phenix et al . ( 2011 ) present a quantitative method for interpreting classical epistasis and inferring pathways from vast sets of data . these previous publications have mainly explored how to overcome the single - path assumption or assumptions 2 , 3 , and the problem of how to use huge amounts of data to infer precise gene interactions . we specially focus on the use of boolean network formalism as an improvement of classical epistasis analysis . boolean networks have been shown to be useful tools to analyze discrete dynamic systems that rely on a pure logical formalism ( bornholdt , 2008 ) . interestingly , integration of experimental gene interaction data into boolean networks may be particularly useful for classical epistasis analysis because the latter also relies on a pure logical , discrete formalism . however , the boolean network approach does not imply the limiting assumptions that the classical epistasis analysis does ; indeed , a boolean approach is useful for analyzing and integrating much more information than classical epistasis analysis alone . importantly , boolean networks can be modified for more detailed analysis when noise ( bornholdt , 2008 ) or multivalued genes ( genes that can perform different activities depending on their level of expression ; didier et al . , 2011 ) furthermore , once a boolean network is validated it can be transformed into an equivalent continuous system ( wittmann et al . , 2009 ) , which can be amenable to further formal analyses . given that the boolean network formalism is very intuitive and there are a handful of freely available tools for its analysis ( e.g. , squad , di cara et al . , 2007 ; atalia , alvarez - buylla et al . , 2010 ; boolnet , mssel et al . , 2010 ; simboolnet , zheng et al . , 2010 ) , we therefore propose here the use of boolean networks for an easy , but more powerful analysis of classical epistasis experiments . after providing a historical perspective of hierarchical and single - path gene regulation , we will provide a detailed explanation of how classical epistasis analysis works , explore some of the implications of violating the hierarchical and single - path assumptions and discuss the importance of considering the temporal dynamics of gene interactions . we will show that classical epistasis analysis can be useful and precise , but that it can also conceal relevant information concerning the nature of gene interactions underlying biological processes . next , we will argue that complementary experiments can uncover the information that is hidden to epistasis analysis , namely , unknown non - hierarchical and non - single - path genetic interactions . then we will show that the use of a dynamical network - based approach can facilitate the access to this information . finally , we will review how boolean networks work and use experimental and theoretical examples to illustrate ways in which boolean networks can be used to complement and improve classical epistasis analysis . the single - path and hierarchical notions of gene regulation are not assumptions that were incorporated in classical epistasis analysis just for simplicity . these views are historically rooted and hence , they are not exclusive of classical epistasis analysis , but have permeated almost all of biological research . in fact , these assumptions affect the way biologists still design , analyze , and interpret experimental data in many areas of research . hence , we briefly review some of the historical roots of the single - path and hierarchical notions of gene regulation . at the beginning of the twentieth century , during the so - called eclipse of darwinism ( bowler , 1983 ) , genes were conceptualized as functional units of recombination ( here referred to as the functional gene , similar to longo and tendero , 2007 ) , and their phenotypic effects were inferred from hybridization experiments . dna structure was discovered many decades afterward ( watson and crick , 1953 ) revealing that genes were encoded in the double helix dna sequence ( here we refer to the coding dna as the structural gene ) , and thus provided an apparently clear material basis for the action of functional genes . before this , in the framework of the modern synthesis of evolutionary biology , it was suggested that inheritable phenotypic traits with modifications guided evolution and that all or most inheritable variable traits were encoded almost exclusively in the genes , without making a distinction between the functional and the structural gene ( mayr and provine , 1980 ) . based on this and other and historical issues ( for instance , experiments regarding the role of the homeotic genes showing a key control of genes over phenotypes ; morata and lawrence , 1977 ) , an apparently logical and immediate direct link between the functional and the structural conception of genes was made ( longo and tendero , 2007 ) . approach that assumed that phenotypic traits are almost completely determined by the information , or blueprint , contained in genes ( lorenz , 1965 ; nijouth , 1990 ) . consequently , during the decades that followed the modern synthesis , the research of many biological fields has focused almost exclusively on genetics and molecular research . however , all of this was done without a distinction between the functional and structural notions of genes , although in reality these could represent different units . little was known at that time about gene interactions or epigenetic mechanisms , and according to an extreme genocentric view , development and organismal organization could be explained through pivotal genes that regulate the activity of other downstream genes , which in turn regulate other further downstream genes , and so on ( i.e. , a single - path and hierarchical view ; e.g. , davidson and erwin , 2006 ) . under such a perspective , one could understand the order of gene action using straightforward genetic analyses , such as the classical epistasis analysis . however , as pervasive and useful as it has been , the extreme genocentric approach has been severely criticized ( e.g. , oyama , 1985 ; alberch , 1989 ; nijouth , 1990 ; griffiths and gray , 1994 ; goodwin , 2001 ; greenspan , 2001 ; gould , 2002 ; jablonka and lamb , 2005 ; salazar - ciudad , 2006 ; pigliucci and mller , 2010 ) . it has become evident that most phenotypes depend on highly non - linear regulatory interactions among multiple elements and therefore that single and direct causes are rare ( wagner , 1999 ; lewontin , 2000 ; robert , 2004 ; longo and tendero , 2007 ) . studies on the gene interactions underlying transcriptional regulatory networks ( e.g. , albert and othmer , 2003 ; espinosa - soto et al . , 2004 ; davidich and bornholdt , 2008 ) support this idea and show that many phenotypic traits depend on the distributed ( non - hierarchical ) action of many interacting genes and also on environmental and developmental factors ( e.g. , lewontin , 2000 ; greenspan , 2001 ; salazar - ciudad , 2006 ; gordon et al . , 2009 ) . hence , the assumption of single - path and hierarchical gene interactions often leads to oversimplified models , which are instrumental starting points for exploratory purposes , but that need to be improved later on . confronted with these kinds of criticism and the growing set of experimental evidence that challenges the genocentric view , the modern synthesis seems to be ready for at least an extension ( griffiths and gray , 1994 ; jablonka and lamb , 2005 ; pigliucci , 2007 , 2009 ; pigliucci and mller , 2010 ) . indeed , it is becoming generally accepted that we need to embrace an interactionist view and accept that development unfolds and emerges as a consequence of complex interactions among several genetic , organismal , and environmental factors ( oyama , 1985 ; robert , 2004 ) . yet , a closer inspection of the literature and some recent data show that , in practice , many experimental setups and analyses assume a single - pathway and hierarchical idea of gene regulation . this could be due to the persistence of the genocentric view , the assumption that the hierarchical action of genes is a necessary first step in tackling complex biological systems , and that the methods , techniques , and conceptual frameworks that enable going beyond a hierarchical view of development and evolution are still under construction . before we try to explore the effect of ubiquitous complex , non - hierarchical gene interactions , let us briefly explain how classical epistasis analysis is traditionally done ( see more detail in avery and wasserman , 1992 ; huang and sternberg , 2006 ; roth et al . , 2009 and references therein ) to use it as an example to illustrate the type of problems we can encounter if hierarchical and single - path interactions are assumed . classical epistasis analysis states that in a double mutant experiment , the two genes act in the same pathway if the phenotype of the double mutant is the same as that of organisms carrying a single mutation for one of the genes . the gene with the allele whose phenotype persists in the double mutant is called epistatic gene , while the other is the hypostatic gene . as mentioned above , if some assumptions regarding the nature of gene regulation are met , few simple rules allow the use of this information to order genes in a hierarchical way ( avery and wasserman , 1992 ; roth et al . , 2009 ) . the rules are as follows : in the double mutant , the epistatic gene is upstream and positively regulates the downstream gene when the two genes used in the double mutant display a characteristic single mutant phenotype under the same condition ( e.g. , both genes have certain mutant phenotype only when a signaling pathway is active or only when the pathway is inactive ) . in the double mutant , the epistatic gene is downstream and is negatively regulated by the upstream gene when the two genes display a characteristic single mutant phenotype under different conditions ( e.g. , one gene has a mutant phenotype when a signaling pathway is active and the other when the pathway is inactive ) . these simple rules are useful and applicable for many cases ( avery and wasserman , 1992 ; huang and sternberg , 2006 ; roth et al . , 2009 ) . but , what happens when the single - path and hierarchical assumptions are not met or if temporal dynamics are considered ? we use some examples to illustrate these cases . consider generic nodes x , y , and z to represent genes ( although they can represent other entities , see huang and sternberg , 2006 ) . if x positively controls the expression of y and y positively controls the expression of z ( figure 3a ) , then the single and double loss - of - function mutants yield the results shown in table 1 . applying the rules of the classical epistasis analysis to these results we correctly conclude that x is upstream in relation to y. results obtained from epistasis analysis of examples in figure 3 . the examples of figures 3b , c , d have some extra interactions than the one shown in figure 3a , but we could not detect these extra interactions with the epistasis analysis alone . is important to note , that even when huang and sternberg ( 2006 ) advise us that we can not order genes with the same phenotype in this kind of pathways , we can do it here with the table . this is because we observe that when x is mutated we obtain the same values for x , y , and z as in the double mutant of x and y. moreover , the presence of x in y mutant could indicate the presence of a substrate product of x activity ( as could be assumed in huang and sternberg , 2006 ) indicating that x is epistatic to y and hence is upstream of y. subgraphs to be inferred with epistasis analysis . inx ( input of x ) is included to meet assumptions 1 , 2 , and 3 of epistasis analysis , but it could be obviated , as it is commonly done . according to huang and sternberg ( 2006 ) we can call such cases to be substrate dependent pathways , and we can only order genes in this kind of pathways through epistasis when they do not display the same mutant phenotype . the logical rules for each motif are : ( a ) x = in , y = x , z = y , ( b ) x = inx , y = x , z = x y , z = y , ( c ) x = inx ( x y ) , y = x , z = x y , and ( d ) x = inx x , y = x , z = y. now let us see what happens in the same example if we add one more interaction . let us assume that x positively and directly regulates z as well ( feed - forward case , figure 3b ) . if we proceed to generate all loss - of - function mutant combinations we recover exactly the same results as in the previous case ( without x z ) . this simple example shows that there are categories of gene regulatory networks that render the same set of results in the single and double loss - of - function mutant analyses but that , nevertheless , have different regulatory interactions or architectures . it is important to note that the graph just described is well known and widely distributed in real gene networks ( milo et al . this subgraph is usually referred to as a coherent feed - forward loop ( mangan and alon , 2003 ; mangan et al . for instance , in the gene regulatory network of the radial root pattern of arabidopsis thaliana the transcription factor shortroot ( shr ) has been shown to positively regulate scarecrow ( scr ) gene transcription in the endodermis , and both shr and scr together regulate the expression of many other genes , including scr itself ( levesque et al . , 2006 ) . the feed - forward loop is not the only subgraph that can mimic the results of the subgraph in figure 3a , there are many others , and as larger gene networks are considered , more cases would render the same inference . if x positively regulates y , and both x and y positively regulate z , but at the same time y negatively regulates x ( creating a feedback loop between x and y ) and finally x positively self - regulates ( a second feedback loop ; figure 3c ) , the same inference as in the two previous examples is reached from the single and double loss - of - function mutants . in all of these cases , classical epistasis analysis would not be completely misleading , as it would suggest that x positively regulates y and y positively regulates z , which is true for all three examples , but it would not be able to yield information concerning the additional regulatory interactions included in figures 3b , c . indeed , such interactions can rarely be detected if we are not looking for them or if we assume that genes act in a hierarchical and single - path way . inx ( input of x ) is included to meet assumptions 1 , 2 and 3 of epistasis analysis , but it could be obviated , as it is commonly done ( see figure 2 ) . the logical rules for each motif are : ( a ) x = inx , y = x , z = y , ( b ) x = inx y , y = iny x , z = xxor y. in fact , in the last example , the notion of upstream and downstream gene does not make any sense . in most real systems the genes feed back to each other creating not a pathway , but a complex circuit or a subgraph that , in figure 3c corresponds to a very well studied and seemingly ubiquitous system known as activator inhibitor system ( gierer and meinhardt , 1972 ; meinhardt and gierer , 2000 ; kondo and miura , 2010 ) . actually , this system has been found to underlie developmental processes of several structures in many organisms ( meinhardt and gierer , 2000 ) . for instance , it is found in the regulation of stem cell pools in a. thaliana shoot apical meristem by clavata3 ( clv3 ) and wuschel ( wus ) genes where clv3 represses wus transcription while wus self - activates and activates clv3 ( schoof et al . , 2000 ; the activator inhibitor system has also been used as an example to challenge the linear causality often attributed to gene action ( goodwin , 2001 ) . in conclusion , classical epistasis analysis could not discern the topologies shown in figures 3a c . however if additional combinations of loss and gain - of - function lines , as well as all the gene expression patterns were available , these three topologies could be distinguished . this requires a considerable experimental effort that , as we will show , could be optimized by adopting a network approach . moreover , there are some topologies that even if the whole set of individual and combined loss - of - function and gain - of - function mutants were available , would still be indiscernible under a classical epistasis analysis . such an example is provided in figure 3d , which depicts a topology that can not be discerned from figure 3a even with an exhaustive set of mutants of the genes conforming the graph under analysis . however , if one could manipulate the input ( or inputs ) , one could , in principle , perform nested classical epistasis analysis based on complete sets of single and combined loss and gain - of - function mutants , in order to infer the correct topology . the fact that one can not distinguish the topologies in figures 3a , d with classical epistasis analysis is due to the presence of an input and a positive feedback loop acting on x. importantly , this appears to be a relatively common situation in real data sets ( see examples from the literature for examples below ) . we now consider a few additional graphs in which a classical epistasis analysis may be insufficient and could be somewhat misleading . let us assume that x negatively regulates y , and y negatively regulates z ( figure 4a ) . now assume another subgraph where x positively regulates y and negatively regulates z ( generating a feed - forward loop from x to z ) , while y positively regulates x and z ( generating a feedback loop between x and y ) , and both x and y nodes , have an input ( figure 4b ; since no gene is upstream or downstream , and since the input should be over the upstream gene , in this case both x and y have inputs ) . the results from the classical epistasis analysis of these two subgraphs are shown in table 2 . following the classical epistasis analysis rules to order gene interactions , we can conclude that gene y negatively regulates gene z , which is not true for both subgraphs . it can be argued that our assumption that y and x regulate z in the subgraph of figure 4b ( through an exclusive or ( xor ) rule , see below ) is a rare situation ( davidson , 2001 ) . however , this kind of examples , where we can be misguided without the xor rule , become frequent as the regulation of z becomes more complex , for instance , having several inputs . results of epistasis analysis for regulatory motifs in figures 4a , b , respectively . given that these are switch regulatory pathways , the presence of a substrate , as explained by huang and sternberg ( 2006 ) , is neglected and epistasis analysis may discern between them only with additional experiments . in addition to feedback and feed - forward loops , it is important to acknowledge the time it takes for genes to interact with other genes in the analysis ( i.e. , temporal dynamics ) . , 2006 ) . in cases where the single - path and hierarchical assumptions are not met , if temporal dynamics are not considered , classical epistasis analysis can also reach wrong inferences ( e.g. , faur et al . , 2006 ) . consider , for example , that x activates itself ( feedback loop ) and at the same time inhibits y and z ( feed - forward loop ) . correspondingly , y also activates itself ( second feedback loop ) and z , and it inhibits x ( third feedback loop ; figure 5a ) . we analyze two cases . in the first one , the network is updated synchronously . that is , the states of all genes in the subgraph at time t + 1 are updated at the same time and are determined by the gene states at time t. it is straightforward to check that if the network is initialized with x off and y on , the system will remain there , with x off , and y and z on ( figure 5b ) . on the other hand , if the updating is not synchronous , and for example , the state of x and y at time t are determined by the gene states at time t 1 and the state of z is determined by the gene states at time t ( i.e. , z regulation is faster than the expression of x and y ) , the same condition can lead to a periodic expression of genes ( figure 5b ) . here , a classical epistasis analysis would correctly infer that x inhibits y and y activates z , but would not render any useful information about the other subgraph interactions nor its behavior . as explained in the main text , these networks yield different results if we add a temporal dynamic analysis . if a synchronous updating of the gene states is assumed , the system reaches a fix - point attractor , while with an asynchronous updating the system reaches a periodic attractor . the logical rules of the motif are : ( a ) x = ( inx x) y , y = ( iny y) x , and z = x y. the above examples illustrate how if we do not consider the possible presence of feedback loops , feed - forward loops , and temporal dynamics , analysis like classical epistasis may help to infer some gene interactions and the order in which they occur , but can hide or even be misleading in other aspects of the regulatory system under consideration . network theory has been fruitfully applied to ask and address novel questions in the fields of evolution , development , and behavior ( see for examples von dassow et al . , 2000 ; aldana et al . , 2007 ; balleza et al . , 2008 ; wagner , 2009 ) . importantly , network - based approaches have been already applied for the study of epistasis ( e.g. , tyler et al . , 2009 ; battle et al . , 2010 ; jiang et al . , 2011 ; phenix et al . , 2011 ) . network models provide a formal framework for integrating detailed and high - quality experiments that , although extremely valuable , often remain isolated . the integration of such experimental data into dynamic network models can help discern among possible topologies among which classical epistasis analysis is unable to distinguish . furthermore , dynamic network models may be used to make novel predictions and provide integrative system - level explanations for the behavior of large data sets . in the last section we provided several examples in which certain sets of gene interactions involving the same elements ( genes ) may render the same phenotypes for single and double mutations , but that nevertheless may have different gene interaction topologies . dynamical network models provide a formal framework for integrating experiments that can help discriminate among alternative topologies yielding the same results when subject to a classical epistasis analysis . furthermore , this integration enables the specification of larger dynamic models that may feedback independent experiments and are helpful to validate a whole set of data . there are many ways in which we could use dynamical network models to improve classical epistasis analysis . for instance , we could keep a catalog of possible regulatory graphs that render the same results , as for the cases shown above . this would help us to bear in mind some of the possible topologies that are consistent with a set of genetic data , as well as to design experiments and crosses in order to discern among them . it is also possible to perform exhaustive ( if our network is finite , discrete , and deterministic ) computational simulations of the dynamic consequences of alternative regulatory graphs , enabling to test and compare their dynamics with available evidence . this has already been done for other purposes and in different ways ( e.g. , nochomovitz and li , 2006 ; giacomantonio and goodhill , 2010 ) , and its applicability is being studied now in the specific context of classical epistasis ( e. azpeitia and e. r. alvarez - buylla , unpublished data ) . also , using networks to predict experimental results or to systematically explore the perturbations that may affect a system can be very helpful ( e.g. , espinosa - soto et al . gathering data from additional related loss and gain - of - function lines , as well as from other types of molecular genetic experiments , and building larger network models can also help to discern among possible network topologies . stable networks states or configurations ( attractors ) can be obtained for a network grounded on several classical epistasis analyses and systematically test the different topological possibilities . in order to avoid circular explanations , the networks under study should reproduce the data with which they were built , as well as expression patterns or other results that were not fed into the model . ideally , the network being challenged should also lead to novel and testable predictions . however , such approach is limited because the number of possible network topologies and configurations greatly increase as a function of the number of nodes considered . nonetheless , it is possible to focus on relatively small subnetworks or modules that are relatively isolated from the rest of the network . all the above suggestions can be achieved with any kind of dynamic network approach , but we contend that boolean networks are particularly useful and easily applicable in the context of classical epistasis analysis because they use exactly the same logical formalism . several programs are available for boolean network analysis ( e.g. , atalia , alvarez - buylla et al . boolnet , mssel et al . , 2010 ; biocham , calzone et al . , 2006 ; antelope , arellano et al . , accepted ; among many others ) , and boolean networks have been successfully applied and validated in many systems , such as cell type determination in a. thaliana ( espinosa - soto et al . savage et al . , 2008 ) , body segmentation in drosophila melanogaster ( von dassow et al . , 2000 ; albert and othmer , 2003 ) , and yeast cell - cycle ( li et al . , 2004 ) , among others . these are discrete networks where nodes ( commonly representing genes ) can only attain two values , 1 when the gene is active and 0 when it is non - active . thus , a node s activation state changes according to the function : where xn represents the state of node n at the time ( t + ) ( representing a positive integer ) and { xn1(t),xn2(t),k , xnk(t ) } represents all of the k regulators of node xn at time t , and the set of states of all the nodes included in the network at a given time is referred to as the system configuration . since these are discrete and deterministic systems , the number of possible configurations is finite and configurations at posterior time steps can be assessed from previous ones . some network configurations ( represented as a vector of zeros and ones ) do not change once they are reached . kauffman ( 1969 ) proposed that attractors represent the experimentally observed stable configurations of gene activity that occur , for example , in an already determined cell type or that characterize a cell - fate . defining the logical function of each of the possible interaction sets according to available experimental evidence and following the dynamics of such sets can be very useful when analyzing experimental data . for example , consider one of the networks depicted in the previous section ( figure 3c ) . it is possible to derive transition tables for all genes , as shown in table 3 for x , y , and z , with the use of boolean equations . the boolean equations use the logical operators and , or , and not to formalize biological data regarding gene interactions . for instance , if a logical operator and is placed in a boolean equation it could represent that x and y form a dimer . this can be represented as the boolean function : z = x and y. in a similar way , the or and not operators can represent different kinds of gene interactions . once we have the complete set of boolean equations , a transition table that integrates all the boolean equations of the system , gives the system state that will follow at time ( t + t ) , given a system state at time t. then , it is possible to obtain attractors for each tested network and compare them with the expected equilibrium states for the system under study ( see methodological details in kauffman , 1969 ; de jong , 2002 ; alvarez - buylla et al . , 2007 , 2008 ; bornholdt , 2008 ) . this approach may seem insufficient for some instances , but almost all limitations can be resolved by introducing certain modifications . if , for example , experimental evidence suggests the existence of more than two gene activity states , nodes taking additional activity states could be used ( e.g. , espinosa - soto et al . , 2004 ; if non - determinism is not important for the system under study , probabilistic networks can be used ( e.g. , shmulevich and kauffman , 2004 ; bornholdt , 2008 ) . on the other hand , if quantitative data is available , continuous networks described by ordinary differential equations could be approximated , and so on ( wittmann et al . , 2009 ) . for the purposes of classical epistasis analysis discussed here , boolean networks are generally sufficient . conveniently , one only requires basic notions of logic to improve classical epistasis analysis with the use of boolean networks . suppose we want to explore the interactions between two genes for which we do not have any previous information . in order to find how they interact , we perform a classical epistasis analysis . yet , as an extension of the classical epistasis analysis we assume a non - hierarchical and multi - path organization of gene interactions . if we think that the genes under study may not be hierarchically organized , but all other epistasis assumptions are met , five new interactions are possible : ( 1 ) there can be an input for both genes , ( 2 ) feedback circuits where both genes regulate each other s expression , ( 3 and 4 ) either or both genes can self - regulate , and finally , ( 5 ) both genes can control the output ( figure 6 ) . all the possible ways in which x and y can control the output value based on a boolean approach are shown in table 4 , including the case in which neither x nor y are regulators of z. some topologies can be represented with different logical rules , and different logical rules can display the same behavior , which are then dynamically equivalent topologies . it is assumed that x , y , or x and y together can regulate z. an x or a y followed by a for a negative one . based on the results of the epistasis analysis mentioned in the main text and shown in table 5 we can discard all logical rules in which z activity is observed when x is mutated ( i.e. , we discard all logical functions in which z is 1 in both lines when x is 0 ) , and all logical rules where no z activity is observed when y is mutated or in the double mutant of xy nodes . is used to denote the final two options that we obtain when we know the attractors of the motif . all possible topologies in a motif where gene x and gene y regulate the state of a gene z. the edges represent possible regulatory interactions . the inclusion of an input over genes x and y is assumed because it is not clear which gene is upstream and which one is downstream . results obtained through epistasis analysis of the theoretical example described in the main text . * indicates an unknown value , this is because we are assuming non - hierarchical regulation and hence , self - regulation and feedback are allowed , which can result in different values for x and y in the y and x mutants , respectively . first , if we use the rules of classical epistasis analysis we can conclude that x is upstream of y , and that x negatively regulates y , which negatively regulates z. nevertheless , based on table 4 we can observe that not only y negatively regulating z can explain the results obtained from the epistasis analysis . using a discrete boolean formalism there are three different networks that could explain these results assuming that only x and y regulate z. the question that arises then , is how can we distinguish which of the possible explanations is the correct one and , equally important , how do x and y interact a further step would involve identifying the attractors needed in order to explain the results obtained in the classical epistasis analysis . if we do this we observe that depending on how z is regulated , different attractors are expected . hence , if the expression patterns of x and y are obtained , the possibilities are constrained . suppose that when the input is active , x is expressed and y is not expressed , and vice versa when the input is inactive . this will leave only two possibilities ( table 4 ) from which we can easily distinguish the correct one with one additional experiment . then again , we only need to know if the expression of y is stable or not under the possible regulatory graphs , both in a wild - type and mutant cases . then , we can compare the stability of y expression in each graph with that expected from available evidence ( e.g. , y activity is expected to be stable if its expression is observed in wild - type lines ) . if we do this we will find that there are nine possible ways to explain the observed gene y behavior . two of these possibilities are negligible since no regulation of gene x over y is inferred and the observed results in the classical epistasis analysis can not be explained this way . now , two experiments ( one to see if gene x positively or negatively regulates y , and another one to verify if gene y can self - regulate ) will be enough to distinguish the correct graph . the kind of dynamic analysis proposed here is doable even without a computer and it will only render non - trivial information if there are multiple - path or non - hierarchical interactions in the network architectures under analysis . however , as mentioned above , there are now several computational tools that are available to analyze the dynamics of larger networks . it would seem like networks could grow indefinitely before they can tell us something about a process . is it possible to learn something about a particular biological process , for instance , cell type determination during flower organ specification , or body segmentation , without considering every genetic and epigenetic regulatory interaction in the organism ? to answer this question one must turn to one of the central concepts in current biology and network studies , that of modularity . modules are characterized by their greater internal than external integration ( mller , 2007 ) . in the context of networks , modules are often defined as highly connected subsets ( wagner et al . , 2007 ) or as sets of nodes with more interactions among them modular organization seems to permeate biological systems at all levels : molecular , metabolical , structural , functional , developmental , etc . ( wagner et al . , 2007 ; callebaut and rasskin - gutman , 2009 ) . modularity is central to our discussion because the modular organization of networks and biological processes allows us to focus on a limited set of interacting elements that are relatively isolated from the rest . thus , modules have a relatively autonomous behavior with respect to the rest of the network . of course , the definition of modules does not precede the inference of networks , but one can aim to uncover networks that are necessary and sufficient for processes to take place and that , therefore , constitute a functional module . we have already discussed what kind of information could be hidden or even misinterpreted with classical epistasis analysis in several cases of non - hierarchical and non - single - path gene regulatory theoretical subgraphs . now we will describe some of the results , and the kinds of interactions found when complex discrete networks have been used to integrate available molecular information in particular experimental systems . first , we will briefly discuss a case in which one of the gene interactions was predicted by a network model , and it was later corroborated experimentally . importantly , this case involves a feedback loop or gene regulatory circuit , such as those likely overlooked in classical epistasis analyses . in the flower organ specification network a positive feedback loop was predicted for the gene agamous ( ag ; espinosa - soto et al . , 2004 ) . this seemed unlikely to occur given that in the ag-1 mutant plants , which produce a non - functional ag mrna , the pattern of ag mrna expression is as in wild - type arabidopsis lines ( gustafson - brown et al . , 1994 ) . however , these data could still be compatible with an ag positive feedback loop because in the ag-1 background the non - active mutant ag protein is unable to downregulate ag s activator wuschel ( wus ) . thus wus , in the ag-1 background , would permanently upregulate transcription of the non - functional ag mrna . to test the dynamic consequences of the ag positive feedback loop , a gene regulatory network model was used to simulate a network lacking the loop for ag . the results showed that in this case , some of the expected network features were lost . here , the whole set of data ( including many classical epistasis analyses ) helped to build a dynamic network and to predict a gene regulatory subgraph that had been overlooked and that was later verified by independent experiments in another laboratory ( gmez - mena et al . , 2005 ) . other experiments to test this were also suggested from the network analyses and included ectopic gus staining in an ag : gus 35s::ag cross . a similar case was found in the network underlying arabidopsis root epidermis cellular sub - differentiation . this system has been relatively well studied from experimental and theoretical perspectives and there are two non - exclusive models that aim to provide a dynamic account of said patterning process . one of these models is the mutual support model put forward by savage et al . ( 2008 ) and the other one is the so - called wer self - activation model put forward by bentez et al . as its name suggests , the latter relies on the self - activation of the gene wer ( see recent review in bentez et al . , 2011 ) . in order to help discern between these two models , savage and coworkers assessed the activity of the wer promoter in a wer loss - of - function line . in this line , wer is still present . if this gene were located on a linear regulatory pathway , this experiment would have sufficed to rule out the wer self - activation but since this gene is immersed in a complex network , this experiment is not conclusive . it is possible to picture a scenario in which wer has more than one possible input and therefore sustains its expression even when one of these inputs is lacking . a network - based study of this patterning system suggests that these two models act in a partially redundant manner during root development , conferring robustness to the overall system when both are considered ( see a more detailed discussion in alvarez - buylla et al . , 2011 ; further theoretical and empirical work will be required in order to establish how common the reinforcing action of partially redundant subgraphs is , in which types of regulatory networks they arise , and which experimental setups can help uncover them . there are other examples like the two reviewed here among the gene regulatory network literature ( li et al . , 2006 ; chickarmane and peterson , 2008 ; azpeitia et al . , 2010 ; they show that boolean network models are useful tools in integrating the reported experimental molecular data , as well as to detect missing interactions , postulate novel ones and design new crosses and experiments . in this article we have focused on the assumptions of hierarchical and single - path notions of gene regulation , as well as on the importance of considering temporal dynamics of gene regulation when performing a classical epistasis analysis . with the use of simple examples , we have shown how if we assume non - synchronous dynamics and complex non - hierarchical , multi - path gene interactions , more precise inferences of gene interactions can be reached . a network - based perspective not only complements classical epistasis analysis , but it also challenges the notion of a blueprint contained in genes , a linear relationship between genotype and phenotype , and the atomization of an organism s traits and cell types based on the premise that genes are particulate , stable , and separable and hence can be studied in isolation of other regulatory elements ( greenspan , 2001 ; newman et al . 2008 ) . besides the use of network modeling to address how genes map onto phenotypical traits and such developmental processes evolve ( albert and othmer , 2003 ; espinosa - soto et al . kwon and cho , 2008 ; wagner , 2009 ) , some authors have addressed the use of such models specifically for epistasis analyses ( e.g. , phillips , 2008 ; tyler et al . , 2009 ; battle et al . , 2010 ; jiang et al . , 2011 ; phenix et al . , 2011 ) in order to relax some of its assumptions , expand its applicability , and improve its inference capacity . we specifically argued that boolean network approaches , which like classical epistasis analysis use a logical approach , naturally complement it and provide more accurate inferences of gene interactions . boolean network modeling is intuitive and practical and has been validated for several biological systems . network approaches are contributing to the integration of complex interactions at the genetic and other levels of organization , creating a formal language to build up rigorous databases , and the creation of a novel set of terms and concepts for understanding biological research . we think that the use of network - based approaches is a promising field and its application in order to understand a wide range of biological systems is underway . 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 .

in this article we focus on how the hierarchical and single - path assumptions of epistasis analysis can bias the inference of gene regulatory networks . here we emphasize the critical importance of dynamic analyses , and specifically illustrate the use of boolean network models . epistasis in a broad sense refers to gene interactions , however , as originally proposed by bateson , epistasis is defined as the blocking of a particular allelic effect due to the effect of another allele at a different locus ( herein , classical epistasis ) . classical epistasis analysis has proven powerful and useful , allowing researchers to infer and assign directionality to gene interactions . as larger data sets are becoming available , the analysis of classical epistasis is being complemented with computer science tools and system biology approaches . we show that when the hierarchical and single - path assumptions are not met in classical epistasis analysis , the access to relevant information and the correct inference of gene interaction topologies is hindered , and it becomes necessary to consider the temporal dynamics of gene interactions . the use of dynamical networks can overcome these limitations . we particularly focus on the use of boolean networks that , like classical epistasis analysis , relies on logical formalisms , and hence can complement classical epistasis analysis and relax its assumptions . we develop a couple of theoretical examples and analyze them from a dynamic boolean network model perspective . boolean networks could help to guide additional experiments and discern among alternative regulatory schemes that would be impossible or difficult to infer without the elimination of these assumption from the classical epistasis analysis . we also use examples from the literature to show how a boolean network - based approach has resolved ambiguities and guided epistasis analysis . our article complements previous accounts , not only by focusing on the implications of the hierarchical and single - path assumption , but also by demonstrating the importance of considering temporal dynamics , and specifically introducing the usefulness of boolean network models and also reviewing some key properties of network approaches .

Introduction The Hierarchical Notion of Gene Regulation Classical Epistasis Analysis Epistasis and (Boolean) Networks Examples from the Literature Concluding Remarks Conflict of Interest Statement

most of the commonly used approaches to analyze gene regulatory interactions , such as epistasis analysis , rely on some implicit assumptions . another commonly implicit assumption is that gene interactions are part of a single - path , in contrast to cases in which a given gene can regulate another gene via two different pathways at the same time , as it occurs in feed - forward loops ( figures 1c , d ) . the notion of hierarchical and single - path gene regulation is consistent with the search of pathways or cascades rather than networks ( greenspan , 2001 ; aylor and zeng , 2008 ) . however , experimental and theoretical work has demonstrated that biological molecular mechanisms contain regulatory feedback and feed - forward loops that do not fulfill the hierarchical and single - path assumptions , but are rather consistent with a network - based perspective . in this article we use epistasis analysis as an example , to explore the effect of these three aspects of gene regulation on the results and interpretation of gene interaction analyses . in ( c ) ( d ) a feed - forward loop is incorporated yielding two alternative pathways starting at x : one is a direct regulation of z , and the other one implies an indirect regulation of z , via y. epistasis is ubiquitous within gene regulatory networks in living organisms ( tyler et al . is important to note that there are different notions of the term epistasis : the original one proposed by bateson in 1907 ( herein called classical epistasis ) that refers to the masking of the effect of one allele by another allele in a different locus , and a broader definition referring to gene interactions in general . we focus here on classical epistasis , but it is important to note that the different notions of epistasis are related . in any case , the original analysis proposed for classical epistasis ( herein called classical epistasis analysis ) as described by avery and wasserman ( 1992 ) is still one of the most powerful and widely used tools in molecular biology to infer biological pathways and regulatory interactions among genes and to validate predictions derived from high - throughput experimental analysis . it is simple , very powerful , and relies on some explicit and implicit assumptions that , when met , allow this analysis to be taken almost as a recipe to order genes along control pathways ( avery and wasserman , 1992 ; huang and sternberg , 2006 ; roth et al . , 2009 ; compares the effect of each single mutant ( x and y single gene mutants ) against the double mutant ( xy double mutant ) of the genes under study on the phenotype of a trait ( in this paper , the trait represents a gene ) . the explicit assumptions of the classical epistasis analysis are : ( 1 ) there is a signal or input that determines the state of the phenotype under analysis , ( 2 ) the signal also determines the state of the upstream gene , ( 3 ) the signal and the two genes are the only determinants of the phenotype , at least in the context of an experimental model , and finally , ( 4 ) the mutants analyzed are null or complete loss - of - function mutants ( based on avery and wasserman , 1992 ; huang and sternberg , 2006 ) . however , classical epistasis analysis also relies on the implicit assumptions of hierarchical and single - path gene regulation . as we will show , the accomplishment of the implicit assumptions is fundamental for the validity of the classical epistasis analysis . many authors have focused on diverse assumptions of classical epistasis analysis and discussed the implications of violating some of them ( e.g. anyhow , to our knowledge , no previous work has explored the joint effect of the hierarchical and single - path aspects of gene regulatory interactions on classical epistasis analysis . network - based approaches can almost naturally overcome many of the limitations of classical epistasis analysis and gene interaction analyses in general . partly because of this , most of the improvements of the epistasis analyses have relied on the use of networks . these studies propose the use of network - based approaches applying modifications to the standard methods , with the incorporation of graph theory , bayesian networks , as well as statistical or probabilistic properties , among others , for the study of epistasis ( phillips , 2008 ; tyler et al . such approaches have allowed inferences of gene interactions with high statistical confidence levels , but the validation of the predicted gene interactions with such methods usually requires further confirmations with more detailed experiments because false interactions or wrong gene order can sometimes be inferred ( e.g. only a few of these reports have addressed the improvement of classical epistasis analysis in particular . these previous publications have mainly explored how to overcome the single - path assumption or assumptions 2 , 3 , and the problem of how to use huge amounts of data to infer precise gene interactions . we specially focus on the use of boolean network formalism as an improvement of classical epistasis analysis . interestingly , integration of experimental gene interaction data into boolean networks may be particularly useful for classical epistasis analysis because the latter also relies on a pure logical , discrete formalism . however , the boolean network approach does not imply the limiting assumptions that the classical epistasis analysis does ; indeed , a boolean approach is useful for analyzing and integrating much more information than classical epistasis analysis alone . , 2010 ) , we therefore propose here the use of boolean networks for an easy , but more powerful analysis of classical epistasis experiments . after providing a historical perspective of hierarchical and single - path gene regulation , we will provide a detailed explanation of how classical epistasis analysis works , explore some of the implications of violating the hierarchical and single - path assumptions and discuss the importance of considering the temporal dynamics of gene interactions . we will show that classical epistasis analysis can be useful and precise , but that it can also conceal relevant information concerning the nature of gene interactions underlying biological processes . next , we will argue that complementary experiments can uncover the information that is hidden to epistasis analysis , namely , unknown non - hierarchical and non - single - path genetic interactions . then we will show that the use of a dynamical network - based approach can facilitate the access to this information . finally , we will review how boolean networks work and use experimental and theoretical examples to illustrate ways in which boolean networks can be used to complement and improve classical epistasis analysis . the single - path and hierarchical notions of gene regulation are not assumptions that were incorporated in classical epistasis analysis just for simplicity . these views are historically rooted and hence , they are not exclusive of classical epistasis analysis , but have permeated almost all of biological research . hence , we briefly review some of the historical roots of the single - path and hierarchical notions of gene regulation . dna structure was discovered many decades afterward ( watson and crick , 1953 ) revealing that genes were encoded in the double helix dna sequence ( here we refer to the coding dna as the structural gene ) , and thus provided an apparently clear material basis for the action of functional genes . under such a perspective , one could understand the order of gene action using straightforward genetic analyses , such as the classical epistasis analysis . however , as pervasive and useful as it has been , the extreme genocentric approach has been severely criticized ( e.g. , 2004 ; davidich and bornholdt , 2008 ) support this idea and show that many phenotypic traits depend on the distributed ( non - hierarchical ) action of many interacting genes and also on environmental and developmental factors ( e.g. hence , the assumption of single - path and hierarchical gene interactions often leads to oversimplified models , which are instrumental starting points for exploratory purposes , but that need to be improved later on . yet , a closer inspection of the literature and some recent data show that , in practice , many experimental setups and analyses assume a single - pathway and hierarchical idea of gene regulation . this could be due to the persistence of the genocentric view , the assumption that the hierarchical action of genes is a necessary first step in tackling complex biological systems , and that the methods , techniques , and conceptual frameworks that enable going beyond a hierarchical view of development and evolution are still under construction . before we try to explore the effect of ubiquitous complex , non - hierarchical gene interactions , let us briefly explain how classical epistasis analysis is traditionally done ( see more detail in avery and wasserman , 1992 ; huang and sternberg , 2006 ; roth et al . , 2009 and references therein ) to use it as an example to illustrate the type of problems we can encounter if hierarchical and single - path interactions are assumed . classical epistasis analysis states that in a double mutant experiment , the two genes act in the same pathway if the phenotype of the double mutant is the same as that of organisms carrying a single mutation for one of the genes . as mentioned above , if some assumptions regarding the nature of gene regulation are met , few simple rules allow the use of this information to order genes in a hierarchical way ( avery and wasserman , 1992 ; roth et al . but , what happens when the single - path and hierarchical assumptions are not met or if temporal dynamics are considered ? applying the rules of the classical epistasis analysis to these results we correctly conclude that x is upstream in relation to y. results obtained from epistasis analysis of examples in figure 3 . this is because we observe that when x is mutated we obtain the same values for x , y , and z as in the double mutant of x and y. moreover , the presence of x in y mutant could indicate the presence of a substrate product of x activity ( as could be assumed in huang and sternberg , 2006 ) indicating that x is epistatic to y and hence is upstream of y. subgraphs to be inferred with epistasis analysis . inx ( input of x ) is included to meet assumptions 1 , 2 , and 3 of epistasis analysis , but it could be obviated , as it is commonly done . this simple example shows that there are categories of gene regulatory networks that render the same set of results in the single and double loss - of - function mutant analyses but that , nevertheless , have different regulatory interactions or architectures . if x positively regulates y , and both x and y positively regulate z , but at the same time y negatively regulates x ( creating a feedback loop between x and y ) and finally x positively self - regulates ( a second feedback loop ; figure 3c ) , the same inference as in the two previous examples is reached from the single and double loss - of - function mutants . in all of these cases , classical epistasis analysis would not be completely misleading , as it would suggest that x positively regulates y and y positively regulates z , which is true for all three examples , but it would not be able to yield information concerning the additional regulatory interactions included in figures 3b , c . indeed , such interactions can rarely be detected if we are not looking for them or if we assume that genes act in a hierarchical and single - path way . inx ( input of x ) is included to meet assumptions 1 , 2 and 3 of epistasis analysis , but it could be obviated , as it is commonly done ( see figure 2 ) . however , if one could manipulate the input ( or inputs ) , one could , in principle , perform nested classical epistasis analysis based on complete sets of single and combined loss and gain - of - function mutants , in order to infer the correct topology . the fact that one can not distinguish the topologies in figures 3a , d with classical epistasis analysis is due to the presence of an input and a positive feedback loop acting on x. importantly , this appears to be a relatively common situation in real data sets ( see examples from the literature for examples below ) . the results from the classical epistasis analysis of these two subgraphs are shown in table 2 . following the classical epistasis analysis rules to order gene interactions , we can conclude that gene y negatively regulates gene z , which is not true for both subgraphs . given that these are switch regulatory pathways , the presence of a substrate , as explained by huang and sternberg ( 2006 ) , is neglected and epistasis analysis may discern between them only with additional experiments . in cases where the single - path and hierarchical assumptions are not met , if temporal dynamics are not considered , classical epistasis analysis can also reach wrong inferences ( e.g. on the other hand , if the updating is not synchronous , and for example , the state of x and y at time t are determined by the gene states at time t 1 and the state of z is determined by the gene states at time t ( i.e. the logical rules of the motif are : ( a ) x = ( inx x) y , y = ( iny y) x , and z = x y. the above examples illustrate how if we do not consider the possible presence of feedback loops , feed - forward loops , and temporal dynamics , analysis like classical epistasis may help to infer some gene interactions and the order in which they occur , but can hide or even be misleading in other aspects of the regulatory system under consideration . the integration of such experimental data into dynamic network models can help discern among possible topologies among which classical epistasis analysis is unable to distinguish . in the last section we provided several examples in which certain sets of gene interactions involving the same elements ( genes ) may render the same phenotypes for single and double mutations , but that nevertheless may have different gene interaction topologies . dynamical network models provide a formal framework for integrating experiments that can help discriminate among alternative topologies yielding the same results when subject to a classical epistasis analysis . this would help us to bear in mind some of the possible topologies that are consistent with a set of genetic data , as well as to design experiments and crosses in order to discern among them . it is also possible to perform exhaustive ( if our network is finite , discrete , and deterministic ) computational simulations of the dynamic consequences of alternative regulatory graphs , enabling to test and compare their dynamics with available evidence . , nochomovitz and li , 2006 ; giacomantonio and goodhill , 2010 ) , and its applicability is being studied now in the specific context of classical epistasis ( e. azpeitia and e. r. alvarez - buylla , unpublished data ) . gathering data from additional related loss and gain - of - function lines , as well as from other types of molecular genetic experiments , and building larger network models can also help to discern among possible network topologies . all the above suggestions can be achieved with any kind of dynamic network approach , but we contend that boolean networks are particularly useful and easily applicable in the context of classical epistasis analysis because they use exactly the same logical formalism . thus , a node s activation state changes according to the function : where xn represents the state of node n at the time ( t + ) ( representing a positive integer ) and { xn1(t),xn2(t),k , xnk(t ) } represents all of the k regulators of node xn at time t , and the set of states of all the nodes included in the network at a given time is referred to as the system configuration . it is possible to derive transition tables for all genes , as shown in table 3 for x , y , and z , with the use of boolean equations . this can be represented as the boolean function : z = x and y. in a similar way , the or and not operators can represent different kinds of gene interactions . for the purposes of classical epistasis analysis discussed here , boolean networks are generally sufficient . conveniently , one only requires basic notions of logic to improve classical epistasis analysis with the use of boolean networks . yet , as an extension of the classical epistasis analysis we assume a non - hierarchical and multi - path organization of gene interactions . based on the results of the epistasis analysis mentioned in the main text and shown in table 5 we can discard all logical rules in which z activity is observed when x is mutated ( i.e. first , if we use the rules of classical epistasis analysis we can conclude that x is upstream of y , and that x negatively regulates y , which negatively regulates z. nevertheless , based on table 4 we can observe that not only y negatively regulating z can explain the results obtained from the epistasis analysis . using a discrete boolean formalism there are three different networks that could explain these results assuming that only x and y regulate z. the question that arises then , is how can we distinguish which of the possible explanations is the correct one and , equally important , how do x and y interact a further step would involve identifying the attractors needed in order to explain the results obtained in the classical epistasis analysis . two of these possibilities are negligible since no regulation of gene x over y is inferred and the observed results in the classical epistasis analysis can not be explained this way . of course , the definition of modules does not precede the inference of networks , but one can aim to uncover networks that are necessary and sufficient for processes to take place and that , therefore , constitute a functional module . we have already discussed what kind of information could be hidden or even misinterpreted with classical epistasis analysis in several cases of non - hierarchical and non - single - path gene regulatory theoretical subgraphs . first , we will briefly discuss a case in which one of the gene interactions was predicted by a network model , and it was later corroborated experimentally . to test the dynamic consequences of the ag positive feedback loop , a gene regulatory network model was used to simulate a network lacking the loop for ag . here , the whole set of data ( including many classical epistasis analyses ) helped to build a dynamic network and to predict a gene regulatory subgraph that had been overlooked and that was later verified by independent experiments in another laboratory ( gmez - mena et al . as its name suggests , the latter relies on the self - activation of the gene wer ( see recent review in bentez et al . a network - based study of this patterning system suggests that these two models act in a partially redundant manner during root development , conferring robustness to the overall system when both are considered ( see a more detailed discussion in alvarez - buylla et al . , 2010 ; they show that boolean network models are useful tools in integrating the reported experimental molecular data , as well as to detect missing interactions , postulate novel ones and design new crosses and experiments . in this article we have focused on the assumptions of hierarchical and single - path notions of gene regulation , as well as on the importance of considering temporal dynamics of gene regulation when performing a classical epistasis analysis . with the use of simple examples , we have shown how if we assume non - synchronous dynamics and complex non - hierarchical , multi - path gene interactions , more precise inferences of gene interactions can be reached . a network - based perspective not only complements classical epistasis analysis , but it also challenges the notion of a blueprint contained in genes , a linear relationship between genotype and phenotype , and the atomization of an organism s traits and cell types based on the premise that genes are particulate , stable , and separable and hence can be studied in isolation of other regulatory elements ( greenspan , 2001 ; newman et al . besides the use of network modeling to address how genes map onto phenotypical traits and such developmental processes evolve ( albert and othmer , 2003 ; espinosa - soto et al . we specifically argued that boolean network approaches , which like classical epistasis analysis use a logical approach , naturally complement it and provide more accurate inferences of gene interactions . network approaches are contributing to the integration of complex interactions at the genetic and other levels of organization , creating a formal language to build up rigorous databases , and the creation of a novel set of terms and concepts for understanding biological research . we think that the use of network - based approaches is a promising field and its application in order to understand a wide range of biological systems is underway .

the cerebral cortex , including the hippocampal formation , is composed of two main classes of neurons : principal and non - principal cells . the first and the more numerous class ( around 80% of the neurons ) corresponds mainly to pyramidal cells , which are excitatory glutamatergic neurons ( for reviews see defelipe and jones , 1988 ; spruston , 2008 ) . the second class is formed for its vast majority by gamma - aminobutyric acid ( gaba ) neurons ( freund and buzsaki , 1996 ; huang et al . gaba is the major inhibitory neurotransmitter in adult brain ( krnjevic and schwartz , 1967 ) . these gabaergic non - principal neurons have been also referred as interneurons because most of them have an axon that remains restricted to the local structure . however , several types of such so - called interneurons have an axon that project to distant brain regions ( ribak et al . , 1986 ; , 2007 ; tomioka and rockland , 2007 ; for review see jinno , 2009 ) . these gabaergic neurons form a very heterogenous population that plays a crucial role in regulating the activity of neuronal networks and complex interactions among principal cells ( somogyi and klausberger , 2005 ; skaggs et al . , 2007 ) . therefore , the coordination and integration of cortical functions depends on the number and diversity of gabaergic neurons . in fact , dysfunction or cell death of several specific types of gabaergic neurons has been described as a hallmark of various psychiatric and neurological disorders , such as schizophrenia ( for reviews see lewis et al . , 2005 ; gonzalez - burgos and lewis , 2008 ; maldonado - aviles et al . , 2009 ) and epilepsy ( houser , 1999 ; , 2003 ; poluch et al . , 2008 ; for reviews see levitt et al . , 2004 ; kostovic et al . , 2007 ; leviton and gressens , 2007 ; metin et al . , 2008 ) . understanding mechanisms that regulate the development of gabaergic neurons is a crucial pre - requisite step to assess pathological processes that can take place during development and lead to such neurological and psychiatric diseases . up to date most of our knowledge regarding gabaergic neuron development are based on studies performed in rodents . although many types of cortical interneurons appear to be common to all species , some types in primates , like the double bouquet cells ( defelipe and jones , 1988 ; yanez et al . , 2005 ; defelipe et al . , 2006 ; melchitzky and lewis , 2008 ; povysheva et al . , 2008 ; cruz et al . , 2009 ; jones , 2009 ; zaitsev et al . , 2009 ) , or interneurons of cortical layer i ( rakic and zecevic , 2003 ; kostovic et al . , 2005 ; in addition , several studies suggest an increased proportion of cortical gabaergic neurons between rodents and primates ( hendry et al . , 1987 ; gabbott and bacon , 1996 ; such increases in the number and diversity of gabaergic neurons have been suggested to be closely related to the tremendous increased brain complexity that occurs during mammalian evolution ( jones , 2009 ; pierani and wassef , 2009 ; rakic , 2009 ; rakic et al . whereas there is a consensus for all species examined that principal glutamatergic neurons originate in the local ventricular ( vz ) and subventricular zones ( svz ) of the dorsal telencephalon ( pallium ) and migrate along radial glia to their target cortical layer ( rakic , 1971 ; noctor et al . , 2001 ; for reviews see rakic , 2006 2009 ) , this seems not to be the case for gabaergic neurons ( molnar et al . , 2006 ; wonders and anderson , 2006 ; metin et al . , 2008 ; petanjek et al . , there are compelling evidences that cortical gabaergic neurons ( figure 1 ) are not generated in these cortical ( pallial ) proliferative zones . they are produced in proliferative zones of the ventral ( basal ) telencephalon ( subpallium ) , the ganglionic eminence ( ge ) . from this region , newly born gabaergic neurons migrate tangentially into the cortex ( for review see wonders and anderson , 2006 ) . surprisingly , data obtained in the human ( letinic et al . , 2002 ; rakic and zecevic , 2003 ; fertuzinhos et al . , 2009 ) and monkey cortex ( petanjek et al . , 2009 ) point out significant evolutionary changes with respect to the origin of cortical gabaergic neurons . therefore , the aim of this paper is to update the literature in this field ( see petanjek et al . , 2008 ) , giving an overview of the main data about the origin and migration of cortical gabaergic neurons in rodents and primates . ( a ) shematic drawing of migratory routes for cortical neurons in the dorsal telencephalon ( pallium ) . principal ( pyramidal ) neurons ( excitatory glutamatergic neurons ) originate locally in the proliferative ventricular ( vz ) and subventricular ( svz ) zones of the dorsal telencephalon . early - generated neurons display a bipolar morphology and migrate orthogonal to the surface of the brain along radially oriented glial fibres ( radial migration ) . according to data obtained in rodents , hippocampal and cortical gabaergic neurons originate in the ventral proliferative zones , i.e. ganglionic eminence ( ge ) and migrate tangentially to their target cortical regions ( red arrowed lines ) . the largest stream follows the lower intermediate zone ( iz)/upper subventricular zone ( svz ) . in the marginal zone ( mz ) numerous tangentially oriented neurons are present below the cortical plate ( cp ) , in the subplate . migratory routes are shown on schematized frontal section of human fetus at postconceptual week 15 ( th thalamus ; c caudate nucleus ; p putamen ; cc corpus callosum ) . ( b ) microphotography of a golgi impregnated section through the whole thickness of dorsal telencephalon in a human fetus at postconceptual week 10 . numerous prospective pyramidal neurons at the beginning of differentiation are present in the cortical plate . red arrows indicate major locations and direction of tangential migration . modified by permission from kostovic et al . golgi - impregnation studies have first described tangentially migrating cells in the fetal telencephalon ( stensaas , 1968 ) . increased attention started when it was established that these cells indeed correspond to gabaergic neurons ( van eden et al . , 1989 ; dediego et al . , 1994 ) . although the stream of those cells was observed all along the intermediate zone ( iz ) of the dorsal telencephalon , from the ge to the most medial part of dorsal hippocampus , it was difficult at this time to conceive that cortical gabaergic neurons could originate from outside the cortical proliferative zones ( dediego et al . , 1994 ) . lineage experiments later established that clones of gabaergic neurons are tangentially dispersed , whereas radially arranged clones are formed primarily of glutamatergic neurons ( tan et al . , 1998 ; noctor et al . , 2001 ; for reviews see molnar et al . , 2006 ; dehay and kennedy , 2007 ; metin et al . , 2008 ; ( 2002 ) further demonstrated that cortical glutamatergic neurons are produced in proliferative zones of the dorsal telencephalon ( pallium ) , but not gabaergic cells . they suggested that gabaergic neurons originate from progenitors located outside the pallium . in vitro studies directly established that in rodents gabaergic neurons migrate tangentially from the ventral telencephalon ( i.e. ge ) into the cortex . these migrating neurons follow several routes to reach their target regions ( figure 1 ) . the major stream of tangentially migrating gabaergic neurons is present at the border of iz and svz ( lower part of iz and upper part of svz ) . in addition , smaller streams of migrating cells are present in the subplate ( sp ) and the upper part of marginal zone ( mz ) ( anderson et al . , 1997 ; pleasure et al . , 2000 ; heng et al . , 2007 ; metin et al . , 2007 ; for reviews see molnar et al . , 2006 ; wonders and anderson , 2006 ; metin et al . , 2008 evidence that gabaergic neurons originate mainly from progenitors in the ge was provided by studies using several lines of knockout mice for genes expressed specifically in the ventral telencephalon . a marked reductions in the number of neocortical gabaergic neurons is observed at birth in all these lines including mutants for the transcription factors dlx1 and dlx2 ( anderson et al . , 1997 ; pleasure et al . , 2000 ; wonders and anderson , 2005 ; long et al . , 2009 ) , nkx2.1 ( sussel et al . , 1999 ; pleasure et al . , 2000 ; butt et al . , 2008 ; nobrega - pereira et al . , together these studies have clearly established that in rodents almost all cortical gabaergic neurons originate from the ge and migrate tangentially to their target cortical layers . interestingly , there are compelling evidences that different subdomains within the ge generate different populations of cortical gabaergic neurons . the ge displays two major divisions : the medial and lateral ge ( sidman and rakic , 1973 ; o'rahilly and mller , 2006 ; petanjek et al . , 2008 ) . recent findings demonstrated the existence of two additional components , the caudal and septal divisions ( xu et al . , 2004 ; small , defined regions of the ge , showed that neurons from the medial part migrate laterally and spread widely throughout the cortex , whereas most neurons from the caudal part migrate toward the most caudal end of the telencephalon ( ang et al . , 2003 ; liodis et al . , 2007 ; kanatani et al . , 2008 ; zhao et al . , 2008 ; for reviews see wonders and anderson , 2006 ; metin et al . the vast majority of cortical gabaergic neurons are provided by the medial ge and at a lower extent by the caudal ge . however , significant , although smaller numbers of cortical gabaergic neurons originate from the lateral ge , especially at later stages ( xu et al . , 2004 ; wonders and anderson , 2006 ; wonders et al . , 2008 ) . among the different subpopulations of gabaergic neurons , most of somatostatin - expressing neurons and all parvalbumin - containing cells derive from the medial ge whereas the majority of calretinin - containing cells and half of the npy - expressing neurons arise from the caudal ge . even within the medial ge , somatostatin cells are primarily generated within the dorsal part of the medial ge whereas parvalbumin neurons are provided by the ventral part of the medial ge ( wonders et al . , 2008 ) . therefore , the ge is organized in molecularly different subdomains that produce different subpopulations of cortical gabaergic neurons ( nery et al . , 2002 ; bellion and metin , 2005 ; bielle et al . , 2005 ; butt et al . , 2005 , 2007 ; , 2006 ; wonders and anderson , 2006 ; flames et al . , 2007 ; , 2007 ; ghanem et al . , 2007 ; guillemot , 2007 ; kohwi et al . , 2007 ; liodis et al . , 2007 ; molyneaux et al . , 2007 ; batista - brito et al . , 2008 ; bayatti et al . , 2008 ; du et al . , 2008 ; garcia - moreno et al . , 2008 ; kanatani et al . , 2008 ; in addition to spatial diversity in the origin of gabaergic neuron subpopulations , the generation of these different subgroups may occur at specific time windows ( butt et al . , 2005 , 2008 ; ghanem et al . , 2007 ; batista - brito et al . , 2008 ; cai et al . however , the precise spatio - temporal pattern of production , for the different gabaergic subtypes , has yet to be established . several studies have provided evidence that mammalian evolution uses different developmental rules to afford for cortical gabaergic neurons to an expanding neocortex . in human and non - human primates , cortical gabaergic neurons are produced not only by the ge ( as in rodents ) , but also massively by the proliferative zones of the dorsal telencephalon ( letinic et al . , 2002 ; rakic and zecevic , 2003 ; fertuzinhos et al . , 2009 ; ( 2002 ) were first to demonstrate the existence of two distinct lineages of cortical gabaergic neurons using retroviral labelling in organotypic slice cultures of the fetal human forebrain . one lineage originates from the ge as in rodents but give rise to only 35% of the cortical gabaergic neurons in human . the second that represents 65% of cortical gabaergic neurons originates from progenitors within the proliferative vz and svz of the dorsal telencephalon . in this study , the authors suggested that the dorsal telencephalic origin of cortical gabaergic neurons might be human - specific ( letinic et al . , 2002 ) , but we demonstrated more recently that this occurs also in the macaque monkey ( macaca rhesus and macaca fascicularis ) ( petanjek et al . , 2009 ) . both in human and macaque , neurogenesis of gabaergic neurons within the ventral and dorsal telencephalon occurs with distinct temporal profiles . whereas at early stages of primate fetal development , ge is an exclusive site of origin for cortical gabaergic neurons , later on the dorsal telencephalic proliferative layers are the major source for these neurons ( letinic et al . , 2002 ; petanjek et al . , 2009 ) . in the human fetal brain mash1 , a transcription factor known to be involved in the early specification of gabaergic neurons ( meredith and johnson , 2000 ; letinic et al . , 2002 ; britz et al . , 2006 ; guillemot , 2007 ; long et al . , 2009 ) , was expressed only by the progenitors in the ge from postconceptional week ( pcw ) 10 to pcw13 ( letinic et al . , 2002 ) . in the macaque fetal brain ( figure 2 ) , progenitors labeled for the gaba synthesizing enzyme , glutamic acid decarboxylase 65 ( gad65 ) and mash1 were present almost exclusively in the ge and the septal vz / svz ( septal eminence ) from embryonic day ( e ) 47 to e55 ( petanjek et al . , 2009 ) . neurogenesis of gabaergic neurons in the ventral telencephalon of a monkey fetus at embryonic day ( e ) 55 . ( a , b ) adjacent sections labeled for mash1 ( a ) and gad65 ( b ) from a e55 fetal brain at a rostral level in which the lateral ventricle is dilated ( paleocortical ventricle indicated by a star ) . ( a ) progenitors labeled for mash1 were present mainly in the ganglionic eminence ( ge ) [ box magnified in panel ( c ) ] and septal ( sept ) proliferative zone [ box magnified in panel ( d ) ] . ( b ) gad65-containing cells were observed in the same regions of the ventral telencephalon as mash1 , the ge and the sept [ boxes enlarged in panels ( e ) and ( f ) ] . ( c f ) figures correspond to magnified regions indicated by boxes in ( a , b ) . ( c ) in the ge many cells were labeled for mash1 in the ventricular ( vz ) and the subventricular zone ( svz ) . ( d ) in the prospective septal region ( sept ) , numerous cells highly labeled for mash1 were observed in the vz whereas the cells in the svz displayed a moderate level of labeling . these cells were mainly located in the svz , and only a few cells were present in the vz . ( f ) many cells labeled for gad65 were present in the svz of the sept . ( g ) confocal micrographs of an adjacent coronal section double - labeled for mash1 ( red , nuclear ) and gad65 ( green , cytoplasmic ) showing that in the svz of ge numerous mash1 containing progenitors were labeled for gad65 . scale bars : 1 mm ( a , b ) , 50 m ( c g ) , 10 m [ box in ( f ) ] . abbreviation : cc corpus callosum . modified by permission from petanjek et al . large contingent of gad65-containing cells with morphological features of tangentially oriented migrating neurons were present in the upper svz and lower iz as well as in the sp and mz of the dorsal telencephalon ( figure 3 ) . such migratory pathways of gabaergic neurons from the ge to the prospective cortical region were observed also in human from pcw10 to pcw13 ( letinic et al . , 2002 ) . so , during the early fetal period both in monkey ( e47e55 ) and in human ( pcw10 pcw13 ) , migratory cortical gabaergic neurons arise from the ge . tangential migration of gabaergic neurons in coronal sections of a monkey fetus at embryonic day ( e ) 55 . ( a ) mash1 containing progenitors were present in the ganglionic eminence ( ge ) but not in the neighboring structures including the caudate nucleus ( caud ) , capsula interna ( ci ) , and adjacent zones of the dorsal telencephalon : ventricular zone ( vz ) , subventricular zone ( svz ) and intermediate zone ( iz ) . in addition a stream of post - mitotic - like , gad65-containing neurons extending from the ge to dorsal telencephalon was observed in the lower iz / upper svz . ( c ) gad65-containing cells with a characteristic morphology of post - mitotic migrating neurons , including a leading process ( arrows ) leaving the ge . ( d ) in the iz and upper svz of dorsal telencephalon , the majority of gad65 migrating neurons were tangentially oriented ( arrows ) . ( e ) tangentially oriented gad65 migrating neurons are also observed in the subplate ( sp ) ( arrows ) , cortical plate ( cp ) and marginal zone ( mz ) of the dorsal telencephalon . ( f , g ) confocal micrographs of an adjacent coronal section double - labeled for ki67 ( f ) or mash1 ( g ) ( red , nuclear ) and gad65 ( green , cytoplasmic ) . at e55 , in the svz of the ge many gad65-labeled cells were labeled for ki67 ( f ) and therefore corresponded to proliferating cells . in contrast none of the migrating gad65-containing cells in the iz displayed detectable level of ki67 or mash1 ( g ) and therefore likely corresponded to early post - mitotic neurons . scale bars : 350 m ( a , b ) , 50 m ( f , g ) , 20 m , ( c e ) . modified by permission from petanjek et al . later on during fetal development ( e64e75 in monkey ; pcw15pcw20 in human ) , cortical gabaergic neurons are , in addition to the ge , massively generated by the proliferative regions of the dorsal telencephalon . in macaque , numerous mash1-containing progenitors coexpressing gad65 were present in the svz and vz of the entire dorsal telencephalon ( figure 4 ) . this is in contrast to earlier stage ( e47e55 ) , when almost all migrating neurons were tangentially oriented ( figure 3 ) . similar patterns of gabaergic neuron progenitors and migrating gabaergic neurons were described in human fetuses ( letinic et al . , 2002 ) . neurogenesis of gabaergic neurons in the ventral and dorsal telencephalon of monkey fetuses at embryonic day ( e ) 68 , e75 and e88 . ( a d ) mash1 expression . ( a ) at e75 , mash1 containing progenitors were present in the ganglionic eminence ( ge ) as well as in ventricular ( vz ) and subventricular ( svz ) zone of the entire dorsal telencephalon . in both areas , ( b ) at e68 , the same pattern of labeling for mash1 is already present in the vz and svz of the dorsal telencephalon . no labeling is observed in the upper part of intermediate zone ( iz ) , the subplate ( sp ) and the cortical plate ( cp ) . the numerous mash1 highly labeled cells located in the vz and svz of the dorsal telencephalon . ( d ) at e88 , end of neurogenesis , the intensity of labeling for mash1 was strongly decreased as compared to e68 ( b ) and e75 ( a , c ) . ( e ) at e75 , gad65-containing progenitors were present in the ge , but also in the proliferative vz and svz of the dorsal telencephalon in contrast to earlier stages . the stream of migrating gad65-containing neurons observed at e55 [ compare with panel ( b ) in figure 3 ] in the lower part of iz and upper part of svz was still present ( arrows ) . in contrast to previous stages many migrating - like post - mitotic neurons with one leading process were now observed in the vz and svz of the dorsal telencephalon . ( g ) at e75 , in addition to migrating - like post - mitotic neurons , many round - shaped cells without any process were labeled for gad65 ( arrows ) . these cells displayed a similar morphology to that observed in the ge and septal region . ( h ) at e75 , many migrating - like gad65-neurons in the svz of the dorsal telencephalon displayed multidirectional orientations . ( i j ) confocal micrographs of an adjacent coronal section double - labeled for mash1 ( i ) or ki67 ( j , k ) ( red , nuclear ) and gad65 ( green , cytoplasmic ) . ( i ) at e75 , in the dorsal telencephalon , most mash1-containing cells of svz also contained gad65 , whereas many mash1-labeled cells in the vz did not . insert : aggregates of cells double - labeled for mash1 and gad65 were frequently observed in the svz of the dorsal telencephalon , suggesting cells undergoing division . ( j ) at e75 in the svz of the dorsal telencephalon [ compare with panel ( g ) ] most gad65-containing cells were labeled for ki67 and therefore corresponded to progenitors of gabaergic neurons . ( k ) migrating - like gad65-containing cells in the iz of the dorsal telencephalon were not labeled for ki67 . scale bars : 550 m ( a , e ) , 250 m ( b ) , 60 m ( c , d ) and 30 m ( f h ) ; 50 m ( i , j ) , 10 m [ insert ( i , k ) ] . modified by permission from petanjek et al . furthermore , our results strongly support the view that the progenitors of gabaergic neurons in the cortical ( dorsal ) proliferative zones are generated locally , rather than arriving from the ge . our main argument is that at e47e55 , no mash1-containing cells were observed in the dorsal telencephalic wall close to the ge whereas onset of mash1 expression was already observed in the vz / svz of the most dorsal part of the telencephalon ( figures 5 and 6b ) . this pattern of mash1 expression in dorsal progenitors indicates that these progenitors did not migrate from the ge . however , at earlier stage ( e47e55 ) , the mash1-containing cells in the dorsal telencephalon did not contain gad65 and thus may not have a gabaergic phenotype . we can not exclude the possibility that these mash1-containing progenitors generate other types of cells such as oligodendrocytes ( jakovcevski and zecevic , 2005 ; guillemot , 2007 ; parras et al . , 2007 ) or reflect a regulation of the progenitor division mode not related with specification of gabaergic neurons ( britz et al . , 2006 ) . however , at e64 only few mash1-containing cells express pdgfr- , a marker of oligodendrocyte progenitors , and the vast majority of mash1-containing cells in the vz / svz of the entire dorsal telencephalon expresses gad65 ( petanjek et al . , 2009 ) . thus , the expression of mash1 in the dorsal telencephalon at e47e55 corresponds likely to symmetrically dividing prospective gabaergic precursors ( rakic , 2009 ; rakic et al . , 2009 ) and marks the onset of gabaergic neurogenesis in this region . mash1 and gad65 labeling patterns in coronal sections of monkey fetal telencephalon at embryonic day ( e ) 47 suggest that neurogenesis of gabaergic neurons is induced locally . ( a ) at e47 mash1 was highly expressed in the proliferative zone of the ventral telencephalon , the ganglionic eminence ( ge ) . it followed a clear dorso - ventral decreasing gradient in the labeling intensity within the entire cortical wall . moderate level of labeling was observed in the most dorsal part of the telencephalon [ indicated by square and magnified in panel ( c ) ] . no mash1 immunolabeling was observed close to the ge , nor in the most ventral part of the medial telencephalic wall , that corresponds at this level to dorsal hippocampus ( dhipp ) . ( c ) some cells moderately labeled for mash1 were observed in the proliferative zones of the most dorsal part of the dorsal telencephalon . most of these mash1 labeled cells were present in the upper part of subventricular zone ( svz ) , on the border with the intermediate zone ( iz ) . some cells were also observed in the deepest part of ventricular zone ( vz ) . ( d ) in contrast to mash1 no gad65-containing cells were observed in the vz and svz of the most dorsal part of the dorsal telencephalon at e47 . however in this region , some gad65-containing cells with a characteristic morphology of early post - mitotic migrating neurons , including a leading process , and tangentially oriented , were observed in the iz and the cortical plate ( cp ) ( arrows ) . scale bars : 1 mm ( a ) , 50 m ( b d ) . sources of cortical gabaergic neuron in a mouse fetus and in primate fetuses at early developmental stage ( embryonic day ( e ) 47 to e55 in macaque / postconceptional week ( pcw ) 10 to pcw13 in human ) . nissl staining of frontal brain sections through telencephalic vesicle at the level of septum and paleocortical ventricle ( asterix ) in a mouse fetus at embryonic day ( e ) 15 [ panel ( a ) and in a human fetus at postconceptional week 10 panel ( b ) ] . ( a ) in the rodent , the vast majority of cortical and hippocampal gabaergic neurons are produced by ganglionic eminence ( ge ) ( large star ) . a very small percentage of the gabaergic neurons is supposed to be also produced in dorsal proliferative zones ( small stars ( b ) in the monkey and human brain , the ge is also an important source of cortical gabaergic neurons ( large star ) . data from macaque monkey ( petanjek et al . , 2009 ) suggest that already at this ages ( see below ) a production of gabaergic neurons started in the proliferative zones of most dorsal part of the pallium ( small stars full red arrows ) . small figure [ left down in panel ( b ) ] is a mouse section at e15 ( a ) reduced in size to be comparable with human fetus at pcw10 ( b ) . therefore , in human and monkey , cortical gabaergic neurons are generated by two distinct sources : the ge and the proliferative zone of the dorsal telencephalon . in addition , the dorsal proliferative zone is not only an additional but also a major pool of gabaergic progenitors in primates ( figure 7 ) . nissl staining of frontal brain sections through rostral part of the telencephalic vesicle in a human fetus at postconceptional week ( pcw ) 15 ( at a level of the anterior horn ) . in human ( pcw15 pcw20 ) as in macaque monkey [ embryonic day ( e ) 64 to e75 ] later during gestation , gabaergic neurons are produced massively by the proliferative zones of the dorsal telencephalon ( smaller stars full red arrows ) . according to letinic et al . vz ventricular and subventricular zone ; cp cortical plate ; cc corpus callosum . in the macaque , the dorsal and ventral lineage of gabaergic neurons displayed different cortical distributions . early - generated gabaergic neurons from the ge populated mainly the mz and the sp . indeed , as early as e47 , morphologically differentiated gad - containing neurons were observed first in the mz and the sp just below the cortical plate ( cp ) . their number increased in the sp with the development of this layer at a period when progenitors of gabaergic neurons are mainly observed in the ventral telencephalon ( petanjek et al . , 2009 ) . a similar pattern of early - generated gabaergic neurons was described in human fetuses ( zecevic and milosevic , 1997 ; meyer et al . , 2000 ; it was shown that sp neurons as well as early - generated neurons in the hippocampal mz are functional and play a crucial role in early - generated network activity ( khazipov et al . , 2001 ; hanganu et al . , 2002 ; moore et al . , 2009 ) . in human and non - human primates gabaergic neurons in the cp are generated in both ge and dorsal proliferative zones . in macaque , from e64e75 onwards , gabaergic neurons penetrate into the cp following an inside - out gradient . their number increases during a period when progenitors of gabaergic neurons are massively present in proliferative regions of the entire dorsal telencephalon . in human , two thirds of cp interneurons are generated in the dorsal telencephalon ( letinic et al . , 2002 ) . ( 2009 ) investigates the proportion of different subpopulations of gabaergic neurons in brains of human fetuses or infants affected by holoprosencephaly . this syndrome is characterized by severe striatal hypoplasia and atrophy of the ge ( judas et al . , 2003 ) . interestingly , in holoprosencephaly , the numbers of cortical gabaergic neurons expressing nitric oxide synthase , neuropeptide y or somatostatin were significantly reduced in comparison to healthy infants . in contrast , calretinin - containing neurons were present in normal numbers as well as principal neurons . these findings show that , in human , nitric oxide synthase- , neuropeptide y- and somatostatin - containing neurons originate from the ge whereas calretinin neurons are generated by the dorsal telencephalon . it is interesting to note that gabaergic neurons identified as missing or significantly reduced ( fertuzinhos et al . , 2009 ) are those normally found in greatest numbers in derivatives of the mz and sp ( kostovic and rakic , 1990 ; delalle et al . , 1997 ; rakic and zecevic , 2003 ; meyer , 2007 ; kostovic et al . , 2008 ; petanjek et al . , in addition , the population of calretinin neurons , generated mainly in the dorsal telencephalon in human , corresponds to the population of gabaergic neurons which proportion increases dramatically in primates ( hendry et al . , 1987 ; gabbott and bacon , 1996 ; zaitsev et al . , 2009 ) and displays primate - specific subtypes such as double bouquet cells ( defelipe and jones , 1988 ; defelipe , 2002 ; yanez et al . , 2005 ; defelipe et al . , , these studies favor the hypothesis that dorsal production occurs principally as an answer to an increased evolutionary need for specific classes of cortical gabaergic neurons . whereas in primates a large majority of cortical gabaergic neurons are produced in the proliferative regions of the dorsal telencephalon ( figure 7 ) , in rodents cortical gabaergic neurons are generated almost exclusively in the ge ( figure 6a ) . a puzzling question is whether this dorsal telencephalic origin of cortical gabaergic neurons is primate - specific or reflects an enhancement of preexisting developmental mechanisms . we have to stress that mammalian species examined until now included only mouse , rat , ferret , monkey and human . however , in favor of the later hypothesis , a production of gabaergic neurons in dorsal proliferative zones has been reported in mouse ( figure 6a ) , but it accounts for a very small fraction of neurons present at maturity ( xu et al . , 2004 ; molnar et al . , 2006 ; wonders and anderson , 2006 ) . in this species , the progenitor cells of the dorsal telencephalon are able to generate gaba - expressing neurons in vitro ( he et al . , 2001 ) . however , in the ferret , that has a convoluted neocortex and displays a 23 times longer neurogenesis compared to mouse and rat , very few ( less than 5% ) cortical gabaergic neurons originated from proliferative zones of the dorsal telencephalon ( anderson et al . , 2002 ) . 2004 ; molnar et al . , 2006 ; wonders and anderson , 2006 ) . however , the massive neurogenesis of cortical gabaergic neurons from dorsal telencephalon is most likely primate - specific . during tangential migration they pass by a complex route between numerous growing axon bundles and cross regional boundaries . the length of their trajectory is greater than that of radially migrating neurons ( figure 1 ) . therefore , the mechanisms of cellular interactions enabling proper positioning and specification of gabaergic neurons are more complex than for radially migrating neurons ( metin et al . , 2006 , 2008 ) . one can suggests that dorsal production of cortical gabaergic neurons in primates might occur in order to facilitate migration routes through an expanding neocortex ( figures 6 and 7 ) . an exclusive ventral telencephalic origin of cortical gabaergic neurons in primates would imply extremely long and complex migratory routes for such neurons ( figure 6b ; compare the real size between embryonic mouse and human fetus ) . in keeping with this hypothesis , it might be expected that the percentage of gabaergic neurons produced dorsally will increase in larger brains in order to keep migratory routes shorter and simpler . however , the data obtained in ferret ( anderson et al . , 2002 ) , which displays a convoluted neocortex significantly larger compared to rodents but shows limited cortical gabaergic neurons generated by the dorsal telencephalon such as described in mouse , do not support this hypothesis . extensive dorsal production of gabaergic neurons in primates can be related to an increased need in number and/or specific types for gabaergic neurons in brains with more complex cortical circuitries ( fertuzinhos et al . , 2009 ; jones , 2009 ) . the massive neurogenesis of cortical gabaergic neurons from dorsal telencephalon suggests distinct properties of dorsal telencephalic progenitors in primates compared to rodents . interestingly , it was demonstrated that the proliferative behavior of cortical neuronal precursors during neurogenesis differs between rodents and primates . this leads to significant differences in morphology and function of their dorsal proliferative zones ( smart et al . , 2002 ; kriegstein et al . , 2006 ; dehay and kennedy , 2007 ; noctor et al . , 2007 , 2008 ; , 2008 ; fish et al . , 2008 ; javaherian and kriegstein , 2009 ; tabata et al . , 2009 ) . in the human embryo , in comparison to the rodent embryo , there is a dramatic increase in surface area and thickness of the vz at the earliest stage of proliferation ( carney et al . , 2007 ) . it displays a different type of cellular organization ( smart et al . , 2002 ; zecevic et al . , 2005 ; kriegstein et al . , there is a new , outer compartment within the svz , which displays a number of unique features , and exists much longer during development ( molnar et al . , 2006 ; dehay and kennedy , 2007 ; bystron et al . , 2008 ; kostovic and vasung 2009 ; jovanov - milosevic et al . , 2009 ) . so , it is not unreasonable to suggest that the increase in complexity , or even the specific cellular and laminar organization of dorsal proliferative zones in primates are significantly connected to the massive production of gabaergic neurons . there has been an impressive amount of research over past years in the field of gabaergic neuron development . the vast majority of this work has been done in rodents , birds and reptiles . these studies give us insight into molecular and cellular mechanisms of gabaergic neuron specification , proper positioning and differentiation . in higher mammals , especially primates , primate data have pointed out significant changes in the origin ( figures 6b and 7 ) , specification and migration of cortical gabaergic neurons compared to lower mammals ( figure 6a ) . they strongly support the view that our understanding of gabaergic neuron development in the human brain can not be assessed only on studies performed in rodents . 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 .

gamma - aminobutyric - acidergic ( gabaergic ) cells form a very heterogeneous population of neurons that play a crucial role in the coordination and integration of cortical functions . their number and diversity increase through mammalian brain evolution . does evolution use the same or different developmental rules to provide the increased population of cortical gabaergic neurons ? in rodents , these neurons are not generated in the pallial proliferative zones as glutamatergic principal neurons . they are produced almost exclusively by the subpallial proliferative zones , the ganglionic eminence ( ge ) and migrate tangentially to reach their target cortical layers . the ge is organized in molecularly different subdomains that produce different subpopulations of cortical gabaergic neurons . in humans and non - human primates , in addition to the ge , cortical gabaergic neurons are also abundantly generated by the proliferative zones of the dorsal telencephalon . neurogenesis in ventral and dorsal telencephalon occurs with distinct temporal profiles . these dorsal and ventral lineages give rise to different populations of gabaergic neurons . early - generated gabaergic neurons originate from the ge and mostly migrate to the marginal zone and the subplate . later - generated gabaergic neurons , originating from both proliferative sites , populate the cortical plate . interestingly , the pool of gabaergic progenitors in dorsal telencephalon produces mainly calretinin neurons , a population known to be significantly increased and to display specific features in primates . we conclude that the development of cortical gabaergic neurons have exclusive features in primates that need to be considered in order to understand pathological mechanisms leading to some neurological and psychiatric diseases .

Introduction In Rodents GABAergic Neurons Originate Exclusively from the Ganglionic Eminence and Migrate Tangentially to the Cortex In Primates the Dorsal Telencephalon is a Major Source for Cortical GABAergic Neurons The Dorsal And Ventral Sites of Neurogenesis Produce Different Populations of Cortical GABAergic Neurons Massive Dorsal Origin of Cortical GABAergic Neurons is Primate-Specific Concluding Remarks Conflict of Interest Statement

these gabaergic non - principal neurons have been also referred as interneurons because most of them have an axon that remains restricted to the local structure . these gabaergic neurons form a very heterogenous population that plays a crucial role in regulating the activity of neuronal networks and complex interactions among principal cells ( somogyi and klausberger , 2005 ; skaggs et al . therefore , the coordination and integration of cortical functions depends on the number and diversity of gabaergic neurons . understanding mechanisms that regulate the development of gabaergic neurons is a crucial pre - requisite step to assess pathological processes that can take place during development and lead to such neurological and psychiatric diseases . although many types of cortical interneurons appear to be common to all species , some types in primates , like the double bouquet cells ( defelipe and jones , 1988 ; yanez et al . , 2005 ; in addition , several studies suggest an increased proportion of cortical gabaergic neurons between rodents and primates ( hendry et al . , 1987 ; gabbott and bacon , 1996 ; such increases in the number and diversity of gabaergic neurons have been suggested to be closely related to the tremendous increased brain complexity that occurs during mammalian evolution ( jones , 2009 ; pierani and wassef , 2009 ; rakic , 2009 ; rakic et al . whereas there is a consensus for all species examined that principal glutamatergic neurons originate in the local ventricular ( vz ) and subventricular zones ( svz ) of the dorsal telencephalon ( pallium ) and migrate along radial glia to their target cortical layer ( rakic , 1971 ; noctor et al . , there are compelling evidences that cortical gabaergic neurons ( figure 1 ) are not generated in these cortical ( pallial ) proliferative zones . they are produced in proliferative zones of the ventral ( basal ) telencephalon ( subpallium ) , the ganglionic eminence ( ge ) . , 2009 ) point out significant evolutionary changes with respect to the origin of cortical gabaergic neurons . , 2008 ) , giving an overview of the main data about the origin and migration of cortical gabaergic neurons in rodents and primates . ( a ) shematic drawing of migratory routes for cortical neurons in the dorsal telencephalon ( pallium ) . principal ( pyramidal ) neurons ( excitatory glutamatergic neurons ) originate locally in the proliferative ventricular ( vz ) and subventricular ( svz ) zones of the dorsal telencephalon . early - generated neurons display a bipolar morphology and migrate orthogonal to the surface of the brain along radially oriented glial fibres ( radial migration ) . according to data obtained in rodents , hippocampal and cortical gabaergic neurons originate in the ventral proliferative zones , i.e. ganglionic eminence ( ge ) and migrate tangentially to their target cortical regions ( red arrowed lines ) . in the marginal zone ( mz ) numerous tangentially oriented neurons are present below the cortical plate ( cp ) , in the subplate . although the stream of those cells was observed all along the intermediate zone ( iz ) of the dorsal telencephalon , from the ge to the most medial part of dorsal hippocampus , it was difficult at this time to conceive that cortical gabaergic neurons could originate from outside the cortical proliferative zones ( dediego et al . , 2008 ; ( 2002 ) further demonstrated that cortical glutamatergic neurons are produced in proliferative zones of the dorsal telencephalon ( pallium ) , but not gabaergic cells . in vitro studies directly established that in rodents gabaergic neurons migrate tangentially from the ventral telencephalon ( i.e. in addition , smaller streams of migrating cells are present in the subplate ( sp ) and the upper part of marginal zone ( mz ) ( anderson et al . , 2008 evidence that gabaergic neurons originate mainly from progenitors in the ge was provided by studies using several lines of knockout mice for genes expressed specifically in the ventral telencephalon . , together these studies have clearly established that in rodents almost all cortical gabaergic neurons originate from the ge and migrate tangentially to their target cortical layers . interestingly , there are compelling evidences that different subdomains within the ge generate different populations of cortical gabaergic neurons . the vast majority of cortical gabaergic neurons are provided by the medial ge and at a lower extent by the caudal ge . however , significant , although smaller numbers of cortical gabaergic neurons originate from the lateral ge , especially at later stages ( xu et al . among the different subpopulations of gabaergic neurons , most of somatostatin - expressing neurons and all parvalbumin - containing cells derive from the medial ge whereas the majority of calretinin - containing cells and half of the npy - expressing neurons arise from the caudal ge . even within the medial ge , somatostatin cells are primarily generated within the dorsal part of the medial ge whereas parvalbumin neurons are provided by the ventral part of the medial ge ( wonders et al . therefore , the ge is organized in molecularly different subdomains that produce different subpopulations of cortical gabaergic neurons ( nery et al . , 2008 ; in addition to spatial diversity in the origin of gabaergic neuron subpopulations , the generation of these different subgroups may occur at specific time windows ( butt et al . several studies have provided evidence that mammalian evolution uses different developmental rules to afford for cortical gabaergic neurons to an expanding neocortex . in human and non - human primates , cortical gabaergic neurons are produced not only by the ge ( as in rodents ) , but also massively by the proliferative zones of the dorsal telencephalon ( letinic et al . , 2009 ; ( 2002 ) were first to demonstrate the existence of two distinct lineages of cortical gabaergic neurons using retroviral labelling in organotypic slice cultures of the fetal human forebrain . one lineage originates from the ge as in rodents but give rise to only 35% of the cortical gabaergic neurons in human . the second that represents 65% of cortical gabaergic neurons originates from progenitors within the proliferative vz and svz of the dorsal telencephalon . in this study , the authors suggested that the dorsal telencephalic origin of cortical gabaergic neurons might be human - specific ( letinic et al . both in human and macaque , neurogenesis of gabaergic neurons within the ventral and dorsal telencephalon occurs with distinct temporal profiles . whereas at early stages of primate fetal development , ge is an exclusive site of origin for cortical gabaergic neurons , later on the dorsal telencephalic proliferative layers are the major source for these neurons ( letinic et al . in the human fetal brain mash1 , a transcription factor known to be involved in the early specification of gabaergic neurons ( meredith and johnson , 2000 ; letinic et al . in the macaque fetal brain ( figure 2 ) , progenitors labeled for the gaba synthesizing enzyme , glutamic acid decarboxylase 65 ( gad65 ) and mash1 were present almost exclusively in the ge and the septal vz / svz ( septal eminence ) from embryonic day ( e ) 47 to e55 ( petanjek et al . ( a ) progenitors labeled for mash1 were present mainly in the ganglionic eminence ( ge ) [ box magnified in panel ( c ) ] and septal ( sept ) proliferative zone [ box magnified in panel ( d ) ] . ( b ) gad65-containing cells were observed in the same regions of the ventral telencephalon as mash1 , the ge and the sept [ boxes enlarged in panels ( e ) and ( f ) ] . ( c ) in the ge many cells were labeled for mash1 in the ventricular ( vz ) and the subventricular zone ( svz ) . such migratory pathways of gabaergic neurons from the ge to the prospective cortical region were observed also in human from pcw10 to pcw13 ( letinic et al . so , during the early fetal period both in monkey ( e47e55 ) and in human ( pcw10 pcw13 ) , migratory cortical gabaergic neurons arise from the ge . ( a ) mash1 containing progenitors were present in the ganglionic eminence ( ge ) but not in the neighboring structures including the caudate nucleus ( caud ) , capsula interna ( ci ) , and adjacent zones of the dorsal telencephalon : ventricular zone ( vz ) , subventricular zone ( svz ) and intermediate zone ( iz ) . in addition a stream of post - mitotic - like , gad65-containing neurons extending from the ge to dorsal telencephalon was observed in the lower iz / upper svz . ( e ) tangentially oriented gad65 migrating neurons are also observed in the subplate ( sp ) ( arrows ) , cortical plate ( cp ) and marginal zone ( mz ) of the dorsal telencephalon . at e55 , in the svz of the ge many gad65-labeled cells were labeled for ki67 ( f ) and therefore corresponded to proliferating cells . later on during fetal development ( e64e75 in monkey ; pcw15pcw20 in human ) , cortical gabaergic neurons are , in addition to the ge , massively generated by the proliferative regions of the dorsal telencephalon . neurogenesis of gabaergic neurons in the ventral and dorsal telencephalon of monkey fetuses at embryonic day ( e ) 68 , e75 and e88 . ( a ) at e75 , mash1 containing progenitors were present in the ganglionic eminence ( ge ) as well as in ventricular ( vz ) and subventricular ( svz ) zone of the entire dorsal telencephalon . in both areas , ( b ) at e68 , the same pattern of labeling for mash1 is already present in the vz and svz of the dorsal telencephalon . no labeling is observed in the upper part of intermediate zone ( iz ) , the subplate ( sp ) and the cortical plate ( cp ) . the numerous mash1 highly labeled cells located in the vz and svz of the dorsal telencephalon . ( e ) at e75 , gad65-containing progenitors were present in the ge , but also in the proliferative vz and svz of the dorsal telencephalon in contrast to earlier stages . in contrast to previous stages many migrating - like post - mitotic neurons with one leading process were now observed in the vz and svz of the dorsal telencephalon . ( g ) at e75 , in addition to migrating - like post - mitotic neurons , many round - shaped cells without any process were labeled for gad65 ( arrows ) . ( i ) at e75 , in the dorsal telencephalon , most mash1-containing cells of svz also contained gad65 , whereas many mash1-labeled cells in the vz did not . ( j ) at e75 in the svz of the dorsal telencephalon [ compare with panel ( g ) ] most gad65-containing cells were labeled for ki67 and therefore corresponded to progenitors of gabaergic neurons . furthermore , our results strongly support the view that the progenitors of gabaergic neurons in the cortical ( dorsal ) proliferative zones are generated locally , rather than arriving from the ge . our main argument is that at e47e55 , no mash1-containing cells were observed in the dorsal telencephalic wall close to the ge whereas onset of mash1 expression was already observed in the vz / svz of the most dorsal part of the telencephalon ( figures 5 and 6b ) . however , at e64 only few mash1-containing cells express pdgfr- , a marker of oligodendrocyte progenitors , and the vast majority of mash1-containing cells in the vz / svz of the entire dorsal telencephalon expresses gad65 ( petanjek et al . ( a ) at e47 mash1 was highly expressed in the proliferative zone of the ventral telencephalon , the ganglionic eminence ( ge ) . no mash1 immunolabeling was observed close to the ge , nor in the most ventral part of the medial telencephalic wall , that corresponds at this level to dorsal hippocampus ( dhipp ) . ( c ) some cells moderately labeled for mash1 were observed in the proliferative zones of the most dorsal part of the dorsal telencephalon . ( d ) in contrast to mash1 no gad65-containing cells were observed in the vz and svz of the most dorsal part of the dorsal telencephalon at e47 . however in this region , some gad65-containing cells with a characteristic morphology of early post - mitotic migrating neurons , including a leading process , and tangentially oriented , were observed in the iz and the cortical plate ( cp ) ( arrows ) . ( a ) in the rodent , the vast majority of cortical and hippocampal gabaergic neurons are produced by ganglionic eminence ( ge ) ( large star ) . a very small percentage of the gabaergic neurons is supposed to be also produced in dorsal proliferative zones ( small stars ( b ) in the monkey and human brain , the ge is also an important source of cortical gabaergic neurons ( large star ) . , 2009 ) suggest that already at this ages ( see below ) a production of gabaergic neurons started in the proliferative zones of most dorsal part of the pallium ( small stars full red arrows ) . therefore , in human and monkey , cortical gabaergic neurons are generated by two distinct sources : the ge and the proliferative zone of the dorsal telencephalon . in addition , the dorsal proliferative zone is not only an additional but also a major pool of gabaergic progenitors in primates ( figure 7 ) . in human ( pcw15 pcw20 ) as in macaque monkey [ embryonic day ( e ) 64 to e75 ] later during gestation , gabaergic neurons are produced massively by the proliferative zones of the dorsal telencephalon ( smaller stars full red arrows ) . in the macaque , the dorsal and ventral lineage of gabaergic neurons displayed different cortical distributions . early - generated gabaergic neurons from the ge populated mainly the mz and the sp . indeed , as early as e47 , morphologically differentiated gad - containing neurons were observed first in the mz and the sp just below the cortical plate ( cp ) . their number increased in the sp with the development of this layer at a period when progenitors of gabaergic neurons are mainly observed in the ventral telencephalon ( petanjek et al . a similar pattern of early - generated gabaergic neurons was described in human fetuses ( zecevic and milosevic , 1997 ; meyer et al . , 2000 ; it was shown that sp neurons as well as early - generated neurons in the hippocampal mz are functional and play a crucial role in early - generated network activity ( khazipov et al . in human and non - human primates gabaergic neurons in the cp are generated in both ge and dorsal proliferative zones . their number increases during a period when progenitors of gabaergic neurons are massively present in proliferative regions of the entire dorsal telencephalon . in human , two thirds of cp interneurons are generated in the dorsal telencephalon ( letinic et al . ( 2009 ) investigates the proportion of different subpopulations of gabaergic neurons in brains of human fetuses or infants affected by holoprosencephaly . interestingly , in holoprosencephaly , the numbers of cortical gabaergic neurons expressing nitric oxide synthase , neuropeptide y or somatostatin were significantly reduced in comparison to healthy infants . these findings show that , in human , nitric oxide synthase- , neuropeptide y- and somatostatin - containing neurons originate from the ge whereas calretinin neurons are generated by the dorsal telencephalon . , in addition , the population of calretinin neurons , generated mainly in the dorsal telencephalon in human , corresponds to the population of gabaergic neurons which proportion increases dramatically in primates ( hendry et al . , , these studies favor the hypothesis that dorsal production occurs principally as an answer to an increased evolutionary need for specific classes of cortical gabaergic neurons . whereas in primates a large majority of cortical gabaergic neurons are produced in the proliferative regions of the dorsal telencephalon ( figure 7 ) , in rodents cortical gabaergic neurons are generated almost exclusively in the ge ( figure 6a ) . however , in favor of the later hypothesis , a production of gabaergic neurons in dorsal proliferative zones has been reported in mouse ( figure 6a ) , but it accounts for a very small fraction of neurons present at maturity ( xu et al . in this species , the progenitor cells of the dorsal telencephalon are able to generate gaba - expressing neurons in vitro ( he et al . however , in the ferret , that has a convoluted neocortex and displays a 23 times longer neurogenesis compared to mouse and rat , very few ( less than 5% ) cortical gabaergic neurons originated from proliferative zones of the dorsal telencephalon ( anderson et al . however , the massive neurogenesis of cortical gabaergic neurons from dorsal telencephalon is most likely primate - specific . therefore , the mechanisms of cellular interactions enabling proper positioning and specification of gabaergic neurons are more complex than for radially migrating neurons ( metin et al . one can suggests that dorsal production of cortical gabaergic neurons in primates might occur in order to facilitate migration routes through an expanding neocortex ( figures 6 and 7 ) . an exclusive ventral telencephalic origin of cortical gabaergic neurons in primates would imply extremely long and complex migratory routes for such neurons ( figure 6b ; compare the real size between embryonic mouse and human fetus ) . in keeping with this hypothesis , it might be expected that the percentage of gabaergic neurons produced dorsally will increase in larger brains in order to keep migratory routes shorter and simpler . , 2002 ) , which displays a convoluted neocortex significantly larger compared to rodents but shows limited cortical gabaergic neurons generated by the dorsal telencephalon such as described in mouse , do not support this hypothesis . the massive neurogenesis of cortical gabaergic neurons from dorsal telencephalon suggests distinct properties of dorsal telencephalic progenitors in primates compared to rodents . interestingly , it was demonstrated that the proliferative behavior of cortical neuronal precursors during neurogenesis differs between rodents and primates . so , it is not unreasonable to suggest that the increase in complexity , or even the specific cellular and laminar organization of dorsal proliferative zones in primates are significantly connected to the massive production of gabaergic neurons . in higher mammals , especially primates , primate data have pointed out significant changes in the origin ( figures 6b and 7 ) , specification and migration of cortical gabaergic neurons compared to lower mammals ( figure 6a ) .

there is great interest in understanding the composition of the tissue microenvironment and its consequence for immune responses . one of the most important microenvironmental factors is the oxygenation status of tissues , since oxygen affects a plethora of cellular processes including innate and adaptive cellular immune defence mechanisms . cells in different tissues are exposed to a wide range of oxygen concentrations , and local oxygen concentrations between 1 and 12% o2 are physiologic . in particular the liver has a unique anatomical structure that creates an oxygen gradient between 8% o2 and 4% o2 within the liver compartments and the po2 is even more reduced during infection to 1% o2 [ 2 , 3 ] . human tryptophan 2,3-dioxygenase ( tdo ) is a liver enzyme with a well - described function in tryptophan homeostasis and crucial immunoregulatory features . nicely demonstrated that tdo mice exhibit an increased sensitivity to endotoxin - induced shock , indicating the potential relevance of tdo in anti - inflammatory reactions . further hints towards an immunoregulatory function of tdo derive from the facts that tdo is expressed in hepatocarcinomas and other malignancies and tdo - mediated production of tryptophan metabolites protects tumor cells against immune rejection [ 69 ] . interestingly , hypoxia is frequently observed in tumoral tissue and influences host defence [ 1012 ] . hence , hypoxia is a microenvironmental factor that might have an impact also on tdo - mediated functions . it was shown by us that recombinantly expressed human tdo is able to inhibit the growth of bacteria , parasites , and viruses and also infected tissue displays low oxygen levels or hypoxia . several factors contribute to these infection- or inflammation - associated hypoxic states , for example , increased oxygen consumption by inflamed resident cells , infiltrating inflammatory cells , and proliferating pathogens as well as a decreased oxygen supply due to vascular pathology and microthrombosis . here we were interested in tdo functions under hypoxia . using stably transfected hela t - rex cells expressing recombinant human tdo and liver homogenates from wt and tdo mice we found that the tdo - mediated degradation of tryptophan to kynurenine was inhibited under low oxygen concentrations . consequently the antimicrobial functions of tdo against tryptophan - auxotroph bacteria and parasites were abrogated in vitro . in summary our studies revealed that low oxygen levels might be detrimental for antimicrobial effector molecules ( ido , tdo ) , inhibiting appropriate immune reactions during infections , which possibly leads to inadequate microbial clearance and subsequent overwhelming or chronic infections . hela t - rex cells were purchased from invitrogen ( karlsruhe , germany ) and stably transfected with pcdna4-htdo vector ( invitrogen , karlsruhe , germany ) containing human liver tdo cdna to produce hela - htdo cells as described by us . the expression of recombinant htdo in hela - htdo cells was induced by stimulation with tetracycline . the cells were cultured in iscove 's modified dulbecco 's medium ( imdm ) ( gibco , grand island , usa ) , supplied with 5% heat - inactivated fetal calf serum ( fcs ) in culture flasks ( costar , cambridge , usa ) and split weekly in 1 : 10 ratios by using trypsin / edta ( gibco , grand island , usa ) . hypoxia growth experiments were carried out using alternatively a heracell 150 i co2 incubator ( thermo fisher scientific , langenselbold , germany ) or the anoxomat system ( mart microbiology b.v . , the imdm was buffered with sodium bicarbonate and therefore had an optimal buffering capacity at the 10% co2 environment to maintain the physiological ph . consequently the ph values of the cell culture medium with or without cells under normoxia and hypoxia were in the range of 7.157.48 . 1 10 hela - htdo cells were seeded per cm in 25 cm cell culture flask ( corning , ny , usa ) , h - thymidine was added at day 0 , and the cells were incubated under normoxia ( 20% o2 ) or hypoxia ( 1% o2 ) for 17 days . the incorporation of h - thymidine was detected using liquid scintillation spectrometry ( 1205 betaplate , perkinelmer , rodgau jgesheim , germany ) . 3 10 hela - htdo cells per well were incubated in 96-well costar microtiter plates ( corning , ny , usa ) for 24 h under normoxia ( 20% o2 ) or hypoxia ( 1% o2 ) . then the cells were washed with pbs and stained with calcein am ( 1 : 2000 ) or ethidium - homodimer-1 ( eth - d-1 ) ( 1 : 500 ) ( live / dead viability / cytotoxicity kit , invitrogen , karlsruhe , germany ) . after incubation time of 45 minutes the fluorescence was detected using the plate reader synergy mx ( winooski , vt , usa ) . calcein was excited using a fluorescein optical filter ( 485 10 nm ) and eth - d-1 using a rhodamine optical filter ( 530 12 nm ) . the fluorescence emissions were acquired separately as well , calcein at 530 12 nm and eth - d-1 at 645 12 nm . tdo activity was determined by analysing kynurenine concentration in supernatants of stimulated or unstimulated hela - htdo , using ehrlich 's reagent as described before . assessment of mtdo activity in liver homogenates was done according to the method of moreau et al . , with some modifications : livers were homogenized in three times the weight using a tissue homogenizer ( precellys , vwr international , erlangen , germany ) . 400 l liver homogenisate was added to 1 ml tdo - buffer ( 200 mm potassium phosphate buffer ( ph 7.0 ) , 10 mm ascorbic acid , 5 m hematin , and 10 mm l - tryptophan ) in 48-well microtiter plates and was incubated without lid under normoxia ( 20% o2 ) or hypoxia ( 1% o2 ) for 4 h at 37c . then the reaction was stopped using 1/10 v / v 30% trichloroacetic acid , again incubated for 30 min at 60c , and centrifuged . supernatants were mixed with an equal volume of ehrlich 's reagent and absorbance was detected at 492 nm with a microplate reader ( slt lab instruments , crailsheim , germany ) . louis , usa ) was diluted in culture medium or buffer and used as standard . for the calculation of the kynurenine content , linear regression and graphpad prism software were used . for in vitro infection studies 3 10 hela - htdo were incubated under normoxia ( 20% o2 ) or hypoxia ( 1% o2 ) in the presence or absence of tetracycline for 72 h. thereafter they were infected with different bacteria or parasites . for bacterial infections enterococcus faecalis ( atcc 29212 ) was used . tryptophan auxotrophy was tested before starting infection experiments . bacteria were grown on brain heart infusion agar ( difco , hamburg , germany ) , containing 5% sheep blood and incubated at 37c in 5% co2-enriched atmosphere or in cell culture medium in the absence or presence of cells . tetracycline - sensitivity was tested and a half - maximal bacterial growth was observed in the presence of 40 g / ml tetracycline under normoxia and under hypoxia , which is thousandfold more than the concentration we used in the experiments . for use in experiments , a 24 h old single bacterial colony was picked , resuspended in tryptophan - free rpmi 1640 ( gibco , life technologies , darmstadt , germany ) , and diluted serially . ten l of the bacterial suspension corresponding to 10100 cfu was added to each well of the 96-well microtiter plates containing preincubated hela - htdo cells . after 18 h bacterial growth was monitored using a microplate photometer ( slt lab instruments , crailsheim , germany ) by measuring the optical density at 620 nm . in some experiments , the bacterial population present in the cultures was enumerated by counting colony forming units ( cfu ) after plating 10 l aliquots of serially diluted culture supernatants on blood agar . toxoplasma gondii ( rh strain , atcc , wesel , germany ) or neospora caninum ( nc-1 strain , kind gift of g. schares , greifswald - insel riems , germany ) tachyzoites were maintained in human foreskin fibroblasts ( atcc , wesel , germany ) in imdm containing 5% fcs . tachyzoites were harvested after 5 days of incubation , resuspended in pbs , and counted . preincubated hela - htdo cells were infected with 3 10 toxoplasma or 4 10 neospora per well . parasite growth was determined by the h - uracil incorporation method as described before . in brief 48 h after infection 0.33 ci h - uracil was added and after additional 24 h host cells were lysed by freeze and thaw cycles . the incorporation of h - uracil was detected using liquid scintillation spectrometry ( 1205 betaplate , perkinelmer , rodgau jgesheim , germany ) . 3 10 hela - tdo cells were left unstimulated or stimulated with tetracycline ( 10 ng / ml ) for 72 h under normoxia ( 20% o2 ) or hypoxia ( 1% o2 ) . then the cells were harvested and lysed by three freeze and thaw cycles in a protease inhibitor cocktail ( roche diagnostics gmbh , mannheim , germany ) . proteins were separated by electrophoresis using 10% nupage novex bis - tris mini gels in the appropriate electrophoresis system ( invitrogen , karlsruhe , germany ) and semidry blotted on nitrocellulose membranes ( carboglas , schleicher & schuell , dassel , germany ) . after blocking of the membranes with 3% ( w / v ) skim milk powder in tbs for 1 h at room temperature , they were incubated in the respective primary antibodies overnight at 4c . louis , usa ) or anti - human - tdo2 antibody ( gtx 40401 , genetex , irvine , usa ) was diluted in 3% ( w / v ) skim milk powder in tbs . after washing the membranes were incubated with goat - anti - mouse hrp - conjugated or goat - anti - rabbit hrp - conjugated igg ( 1 : 10000 , jackson immunoresearch lab . , dianova , hamburg , germany ) , diluted in 3% ( w / v ) skim milk powder in tbs , for 2 h at room temperature . after additional washing steps proteins were detected by enhanced chemiluminescence ( amersham pharmacia biotech , freiburg , germany ) . 3 10 hela - htdo cells were incubated with or without tetracycline ( 10 ng / ml ) for 72 h under normoxia ( 20% o2 ) or hypoxia ( 1% o2 ) in 20 ml cell culture medium in culture flasks . then the supernatants were harvested and used as cell culture medium for freshly isolated 1.5 10 ficoll - separated peripheral blood lymphocytes ( pbl)/well . pbl were activated using the monoclonal anti - cd3 antibody okt3 ; unstimulated pbl and tryptophan - supplemented pbl served as control group . t cell proliferation was determined after three days by adding h - thymidine for 24 h. the incorporation of h - thymidine was detected using liquid scintillation spectrometry ( 1205 betaplate , perkinelmer , rodgau jgesheim , germany ) . this study was carried out in strict accordance with the german animal welfare act and a protocol approved by the local authorities . mice were housed under spf conditions in the animal facility and were 812 weeks old . all experiments were done in triplicate and data are given as mean standard deviation ( sd ; figures 1(a ) , 4(a ) , 4(c ) , 5(a ) , and 6(a ) ) of a representative experiment or as mean standard error of the mean ( sem ; figures 1(b ) , 2(a)2(c ) , 3 , 4(b ) , 4(d ) , 5(b ) , 6(b ) , and 7 ) of three to eight independent experiments . for statistical analysis the two - tailed paired t - test ( figure 3 ) or the two - tailed unpaired t - test ( all other data ) was used and significant differences were marked with asterisks ( p 0.05 ; p 0.01 ; p 0.001 ; p 0.0001 ) . the analysis was performed with graphpad prism software ( graphpad software inc . , san diego , ca ) . we have shown before that the tryptophan - degrading enzyme human tryptophan 2,3-dioxygenase ( htdo ) , expressed in a tetracycline - inducible hela - cell based system , has antibacterial , antiparasitic , and antiviral capacities in vitro . these antimicrobial effects are the result of the htdo - induced degradation of tryptophan , which is an essential amino acid for tryptophan - auxotroph organisms , since the supplementation with additional tryptophan abrogated the effects . in order to analyse htdo - mediated antimicrobial effects under hypoxic conditions , we first monitored hela - htdo cell survival under hypoxia . proliferation studies revealed no significant differences in cell growth , when hela - htdo cells were cultured under normoxia or hypoxia ( 1% o2 ) for 7 days ( figure 1(a ) ) . additionally we observed no significant alterations in cell proliferation under normoxia or hypoxia when the cells were stimulated with tetracycline ( data not shown ) . therefore we concluded that the hela - htdo cells thoroughly survive the hypoxic environment , at least beyond the three - day incubation phases in the subsequent analyses . this cell survival was also confirmed using a fluorescence - based method of assessing cell viability and cell death ( figure 1(b ) ) . for these experiments hela - htdo cells were incubated for 24 h under normoxia ( 20% o2 ) or hypoxia ( 1% o2 ) and afterwards stained with calcein am to detect intracellular esterase activity as indication for living cells or ethidium - homodimer-1 ( eth - d-1 ) that enters nonintact plasma membranes of dead cells . figure 1(b ) shows that the relative fluorescence signal intensity after staining with calcein am does not significantly differ in normoxia- or hypoxia - treated hela - htdo cells ( white bars ) . furthermore eth - d-1 treatment revealed no enhanced cell death under hypoxia ( plaid bars ) . the enzymatic activity of htdo under hypoxia was analysed by determination of the tetracycline - induced htdo - mediated conversion of tryptophan to kynurenine in cell culture supernatants after 72 h of incubation . tetracycline stimulated hela - htdo cells produced kynurenine dose - dependently , when they were incubated under normoxia in the presence of tryptophan ( figure 2(a ) ) . interestingly the cells generated significantly lower amounts of kynurenine under hypoxic conditions ( 1% o2 ) ( figure 2(a ) ) . when tetracycline stimulated cells were incubated under different oxygen concentrations , the kynurenine amounts positively correlated with the amounts of oxygen present ( figure 2(b ) ) . reoxygenation studies confirmed survival of hela - htdo cells and preservation of enzymatic function within these cells under hypoxic conditions ( figure 2(c ) ) . in these experiments the tetracycline - induced tdo - mediated kynurenine production was determined after 72 h of incubation under normoxia ( 20% o2 ) or hypoxia ( 1% o2 ) and compared to the kynurenine production within cell supernatants after subsequent 48 h incubation under normoxia . the tdo - mediated conversion of tryptophan to kynurenine was drastically inhibited by hypoxia , but the cells were able to produce high amounts of kynurenine in the following normoxic phase , demonstrating cell survival and preservation of enzymatic activity . interestingly , normoxia - pretreated and hypoxia - pretreated cells showed no significant differences in their tryptophan - degrading capacity in the reoxygenation phase ab initio ( figure 2(d ) ) . next , western blot analyses were performed to get quantitative information about htdo protein amounts in hela - htdo cells that were stimulated under normoxia ( 20% o2 ) and hypoxia ( 1% o2 ) . figure 3(a ) the protein amount of htdo , induced by tetracycline stimulation of hela - htdo cells , was not altered upon hypoxia as compared to the normoxia control . the stimulation of the cells with the proinflammatory cytokine ifn- did not induce a tdo expression in the cells , as expected . the summary of eight densitometric evaluations of independent western blot analyses is shown in figure 3(b ) . since -actin protein amounts are also reduced upon hypoxia , the ratio of htdo protein to -actin protein was calculated . there were no significant differences detectable in htdo protein amounts under normoxic and hypoxic conditions detectable . although htdo protein levels were unaltered under hypoxia ( figure 3 ) , the enzymatic activity of htdo was reduced up to nearly 90% , as determined by measurement of kynurenine in hela - htdo cell supernatants after 72 h of incubation under normoxia ( 20% o2 ) or hypoxia ( 1% o2 ) ( figure 2 ) . in order to analyse htdo - mediated antimicrobial functions under hypoxia , we infected unstimulated or tetracycline prestimulated hela - htdo cells with different tryptophan - auxotroph pathogens . figure 4(a ) shows the result of infection experiments with the facultative anaerobe , gram - positive bacterium enterococcus faecalis . enterococci grew in the presence of unstimulated hela - htdo cells , whereas bacterial growth was inhibited by tdo - positive cells , which have been stimulated with 5 ng / ml tetracycline ( white bars ) under normoxic conditions . interestingly , this tdo - mediated antibacterial effect was lost under hypoxic conditions ( grey bars ) . the same result was observed in infection experiments using other tryptophan - auxotroph bacteria , such as group b streptococci and staphylococci ( data not shown ) . figure 4(b ) shows that moreover the growth of the obligate intracellular apicomplexan parasite neospora caninum ( nc-1 strain ) was inhibited within activated hela - htdo cells under normoxia and that this antiparasitic effect was abolished under hypoxic conditions . a more detailed analysis of the htdo - mediated antibacterial effect under different low oxygen conditions ( 110% o2 ) in comparison to normoxia is shown in figure 5 . the antibacterial efficiency of htdo correlated with the presence of oxygen and low oxygen conditions significantly inhibited the antibacterial effect as determined by the optical density ( figure 5(a ) ) or by counting the colony forming units ( figure 5(b ) ) . we have mentioned before that tdo regulates immune reactions in vitro and in vivo , for example , by creating tolerance towards the rejection of tumor cells . furthermore tumoral tissues are often poorly vascularized and inefficiently supplied with blood and contain only low oxygen amounts [ 10 , 11 ] . hence , we tested the immunoregulatory property of htdo in hela - htdo cells under hypoxia in vitro . in order to avoid ifn- production by allogeneic t cells which would result in ido induction in hela cells by coculture , supernatants of tetracycline - activated or unstimulated and hypoxia- or normoxia - treated hela - htdo served as culture medium for freshly isolated peripheral blood lymphocytes ( pbl ) . t cell growth was triggered by the addition of the monoclonal anti - cd3 antibody okt3 and monitored by the h - thymidine incorporation method . this okt3 stimulation induced strong t cell proliferation , which is illustrated in a single experiment in figure 6(a ) . the t cell proliferation was inhibited by the tdo - mediated depletion of tryptophan , since it could be restored by the supplementation of tryptophan . however , such t cell inhibition did not occur in conditioned medium that has been harvested from hypoxia - treated hela - htdo cells , which demonstrates the loss of immunoregulatory htdo functions under hypoxia . although stably transfected hela cells are a useful tool to efficiently examine htdo functions in vitro , we extended our studies and confirmed the data by more physiological ex vivo liver homogenate experiments . in these experiments freshly isolated liver tissue of wt or tdo - deficient mice was homogenized in pbs and incubated in the presence of 20% o2 or 19% o2 in a buffer that allows the determination of tdo protein activity by the production of kynurenine . since the normoxic and the hypoxic groups contained the same amounts of murine tdo protein , the direct effect of normoxia and hypoxia on tdo enzyme activity could be revealed ( figure 7 ) . under hypoxia murine tdo produced significantly lower kynurenine amounts as compared to the normoxia control group . furthermore , no kynurenine was detectable in liver homogenates of tdo - deficient mice , as expected . in this study we investigated the influence of hypoxia on the activity of the tryptophan - degrading enzyme human tryptophan 2,3-dioxygenase ( htdo ) by using a the tetracycline - inducible hela t - rex system together with ex vivo studies analysing murine tdo . under normoxic conditions ( 20% o2 ) htdo activity reduced tryptophan amounts in tetracycline stimulated hela - htdo cells and cell culture supernatants . the liver has a unique anatomical structure that creates an oxygen gradient within the liver compartments . incoming highly oxygenated blood via the hepatic artery is subsequently mixed with oxygen - depleted blood in the hepatic portal vein . then the blood flows towards the central vein of the lobule and is depleted of oxygen , resulting in an oxygen pressure ( po2 ) of about 8% o2 in the periportal area and of about 4% o2 in parenchymatic perivenous areas . for example , po2 of ~1.3% was detected within the liver tissue of schistosoma mansoni - infected mice . furthermore a high expression rate of the hypoxia inducible factor-1 ( hif-1 ) was detected in the infiltrative belt surrounding hepatic alveolar echinococcosis lesions within rat livers . overexpression of hif-1 in the actively multiplying infiltrative region of these lesions was closely related to angiogenesis and microvasculature . similar po2 levels of approximately 1 to 3% o2 were detected in other inflamed and infected tissues in the skin , the lungs , and the gut . for example , leishmania major infected mice display low oxygen levels of about 2.8% o2 in pronounced skin lesions , while the resolution of the wound was accompanied by an increase of lesional oxygen levels . hence there is a correlation between infected and inflamed tissues and low oxygen amounts within these tissues . given the fact that htdo has antimicrobial properties under normoxic conditions in vitro and that a microbial - induced hypoxic state is detected within liver tissue , we checked whether putative htdo - mediated antimicrobial effects might persist under low oxygen conditions . therefore the expression and activity of recombinant human tdo in transfected hela cells as well as ex vivo murine tdo were analysed under normoxic and hypoxic conditions . in first step the survival of hela - htdo cells within a hypoxic microenvironment of 1% o2 was confirmed in cell proliferation tests , in fluorescence - based cell viability / cytotoxicity assays and in reoxygenation - based enzyme activity studies . all of these tests provided no indication for an enhanced hypoxia - induced cell death of hela - htdo cells . then the enzymatic activity of htdo was determined by measurement of kynurenine , which is the product of the tdo - mediated tryptophan degradation . human tdo efficiently catalysed the formation of kynurenine under normoxic conditions ( 20% o2 ) , whereas significantly lower levels of kynurenine were generated under oxygen concentrations detected in liver tissue physiologically ( 110% o2 ) . our ex vivo studies using liver homogenates from wildtype and tdo - deficient mice clearly show that also the mtdo - dependent degradation of tryptophan is significantly reduced under low oxygen conditions ( < 9% o2 ) . in particular the low po2 of 13% o2 , which has been detected in infected liver tissue significantly restricted the enzymatic activity of human and murine tdo enzymes . the reason for the lower tryptophan conversion rate under hypoxic conditions in vitro could be general downregulation of tdo protein levels or a reduced enzymatic function . the analysis of human tdo protein amounts within normoxia- and hypoxia - treated hela - htdo cells via western blot analyses showed no decrease of htdo protein amounts under hypoxia . htdo protein levels were correlated with the respective -actin band , since it is known that hypoxia caused general alterations in protein expression levels . therefore a decrease in htdo expression could not account for the decrease of htdo activity under hypoxia . hence the enzymatic activity of htdo must be altered , matching the fact that tdo is a protoheme - containing enzyme that catalyses the insertion of o2 into the pyrrole ring of l - tryptophan and is therefore dependent on cellular oxygen portions . in line with that we observed that normoxia - pretreated and hypoxia - pretreated cells showed no significant differences in their tryptophan - degrading capacity in the reoxygenation phase ab initio . a second dioxygenase that likewise catalyses the degradation of tryptophan is the enzyme indoleamine 2,3-dioxygenase ( ido ) . already in the 1980s it was shown that the intracellular degradation of tryptophan , induced by ifn- , restricted the growth of the intracellular parasite toxoplasma gondii in human fibroblasts . since then ido crystallized as broad - spectrum antimicrobial effector molecule that is effective against a variety of tryptophan - auxotroph pathogens in vitro . interestingly , ido - mediated tryptophan depletion is also inhibited under low oxygen concentrations ( 3% o2 ) which results in a loss of ido - mediated antimicrobial effects in vitro [ 19 , 25 , 26 ] . this was first shown in infection experiments using the intracellular bacterium chlamydia trachomatis in human fallopian tube cells and pinpointed to the hypoxia - dependent inhibition of ifn- signalling . this loss of ido - mediated antimicrobial effects under hypoxia raised the question to what extent tdo might lose its antimicrobial properties upon hypoxic circumstances . here we show that the antibacterial and antiparasitic effects of htdo were lost in hela - htdo cells under hypoxia . human tdo was no longer able to inhibit the growth of enterococcus faecalis and other tryptophan - auxotroph bacteria such as staphylococcus aureus or group b streptococci ( data not shown ) . furthermore the htdo - mediated defence against the intracellular parasite neospora caninum was lost under hypoxic conditions . since htdo is expressed within hela - htdo cells by the addition of tetracycline and not by ifn- stimulation , perturbations in the ifn- signalling pathway can not account for these observations , but only the lack of molecular oxygen . unfortunately it is impossible to analyse potential antimicrobial functions of mtdo or htdo in isolated primary hepatocytes , since these cells readily undergo dedifferentiation and lose hepatocyte function . therefore further studies need to be performed analysing such tdo functions in ex vivo induced stem cells ( e.g. , embryonic stem cells , pluripotent stem cells , and hepatic progenitor cells ) that are differentiated into hepatocyte - like cells with potential tdo function . since there are also no data claiming for an antimicrobial function of mtdo in vivo up to now , the immunoregulatory function of tdo is in the focus of research . here we show that also the t cell inhibitory function of htdo was lost in hela - htdo cells upon hypoxic conditions . again hypoxia prevented htdo - mediated tryptophan depletion and kynurenine production which led to unhindered okt3-driven t cell proliferation in supernatants of htdo - positive hela - htdo cells . in consistence with findings from other groups we detected reduced overall t cell proliferation under hypoxia . for example , atkuri et al . clearly demonstrated that the influence of oxygen levels on the t cell proliferation depends on the stimulus used to activate t cells . while the proliferation in response to phytohemagglutinin was not altered under different oxygen conditions , the cd3/cd28 crosslinking and the stimulation with con a lead to significant higher proliferation under atmospheric oxygen levels than under physiologic oxygen levels ( 5% and 10% o2 ) , with the latter being comparable to our experimental setting . this observation might be of relevance in vivo , since hypoxia is frequently observed in tumoural tissue [ 10 , 11 ] . tumor cells , as well as healthy cells , adapt to hypoxia by various appropriate physiologic responses , for example , by altered expression of genes that switch from oxidative to glycolytic metabolism [ 29 , 30 ] . these cellular responses are caused by the induction of the hypoxia - inducible factor ( hif ) protein complex due to hypoxia . the hif complex regulates the expression of more than 100 genes included in metabolism , angiogenesis , vascular tone , cell differentiation , and apoptosis , among them various enzymes . tdo mrna is frequently expressed within human hepatocarcinomas , but information about tdo activity is missing and the cellular source is still unknown [ 6 , 7 ] . tdo - mediated production of tryptophan metabolites protects tdo - transfected tumour cells against immune rejection [ 8 , 9 ] . our data indicate hypoxia as an environmental factor which is present in tumoural tissue that strongly impacts tdo activities and might therefore be beneficial for tumour growth . the strong influence of low oxygen amounts on innate and adaptive immunity in both inflamed resident cells and infiltrating immune cells was described before [ 3234 ] . since several antimicrobial and immunoregulatory effector molecules in addition to tdo and ido , as , for example , the phagocyte nadph oxidase ( phox ) , type 2 nitric oxide synthase ( nos2 ) , and mitochondria rely on molecular oxygen as a substrate , hypoxia impairs their activity , which might promote the survival of pathogens . therefore low oxygen levels might lead to an inadequate control of microorganisms and to subsequent overwhelming or chronic infections .

tryptophan is an essential amino acid for hosts and pathogens . the liver enzyme tryptophan 2,3-dioxygenase ( tdo ) provokes , by its ability to degrade tryptophan to n - formylkynurenine , the precursor of the immune - relevant kynurenines , direct and indirect antimicrobial and immunoregulatory states . up to now these tdo - mediated broad - spectrum effector functions have never been observed under hypoxia in vitro , although physiologic oxygen concentrations in liver tissue are low , especially in case of infection . here we analysed recombinant expressed human tdo and ex vivo murine tdo functions under different oxygen conditions and show that tdo - induced restrictions of clinically relevant pathogens ( bacteria , parasites ) and of t cell proliferation are abrogated under hypoxic conditions . we pinpointed the loss of tdo efficiency to the reduction of tdo activity , since cell survival and tdo protein levels were unaffected . in conclusion , the potent antimicrobial as well as immunoregulatory effects of tdo were substantially impaired under hypoxic conditions that pathophysiologically occur in vivo . this might be detrimental for the appropriate host immune response towards relevant pathogens .

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

one of the most important microenvironmental factors is the oxygenation status of tissues , since oxygen affects a plethora of cellular processes including innate and adaptive cellular immune defence mechanisms . cells in different tissues are exposed to a wide range of oxygen concentrations , and local oxygen concentrations between 1 and 12% o2 are physiologic . human tryptophan 2,3-dioxygenase ( tdo ) is a liver enzyme with a well - described function in tryptophan homeostasis and crucial immunoregulatory features . nicely demonstrated that tdo mice exhibit an increased sensitivity to endotoxin - induced shock , indicating the potential relevance of tdo in anti - inflammatory reactions . further hints towards an immunoregulatory function of tdo derive from the facts that tdo is expressed in hepatocarcinomas and other malignancies and tdo - mediated production of tryptophan metabolites protects tumor cells against immune rejection [ 69 ] . hence , hypoxia is a microenvironmental factor that might have an impact also on tdo - mediated functions . it was shown by us that recombinantly expressed human tdo is able to inhibit the growth of bacteria , parasites , and viruses and also infected tissue displays low oxygen levels or hypoxia . several factors contribute to these infection- or inflammation - associated hypoxic states , for example , increased oxygen consumption by inflamed resident cells , infiltrating inflammatory cells , and proliferating pathogens as well as a decreased oxygen supply due to vascular pathology and microthrombosis . here we were interested in tdo functions under hypoxia . using stably transfected hela t - rex cells expressing recombinant human tdo and liver homogenates from wt and tdo mice we found that the tdo - mediated degradation of tryptophan to kynurenine was inhibited under low oxygen concentrations . consequently the antimicrobial functions of tdo against tryptophan - auxotroph bacteria and parasites were abrogated in vitro . in summary our studies revealed that low oxygen levels might be detrimental for antimicrobial effector molecules ( ido , tdo ) , inhibiting appropriate immune reactions during infections , which possibly leads to inadequate microbial clearance and subsequent overwhelming or chronic infections . hela t - rex cells were purchased from invitrogen ( karlsruhe , germany ) and stably transfected with pcdna4-htdo vector ( invitrogen , karlsruhe , germany ) containing human liver tdo cdna to produce hela - htdo cells as described by us . , the imdm was buffered with sodium bicarbonate and therefore had an optimal buffering capacity at the 10% co2 environment to maintain the physiological ph . consequently the ph values of the cell culture medium with or without cells under normoxia and hypoxia were in the range of 7.157.48 . calcein was excited using a fluorescein optical filter ( 485 10 nm ) and eth - d-1 using a rhodamine optical filter ( 530 12 nm ) . the fluorescence emissions were acquired separately as well , calcein at 530 12 nm and eth - d-1 at 645 12 nm . tdo activity was determined by analysing kynurenine concentration in supernatants of stimulated or unstimulated hela - htdo , using ehrlich 's reagent as described before . assessment of mtdo activity in liver homogenates was done according to the method of moreau et al . for the calculation of the kynurenine content , linear regression and graphpad prism software were used . for in vitro infection studies 3 10 hela - htdo were incubated under normoxia ( 20% o2 ) or hypoxia ( 1% o2 ) in the presence or absence of tetracycline for 72 h. thereafter they were infected with different bacteria or parasites . tetracycline - sensitivity was tested and a half - maximal bacterial growth was observed in the presence of 40 g / ml tetracycline under normoxia and under hypoxia , which is thousandfold more than the concentration we used in the experiments . ten l of the bacterial suspension corresponding to 10100 cfu was added to each well of the 96-well microtiter plates containing preincubated hela - htdo cells . in some experiments , the bacterial population present in the cultures was enumerated by counting colony forming units ( cfu ) after plating 10 l aliquots of serially diluted culture supernatants on blood agar . proteins were separated by electrophoresis using 10% nupage novex bis - tris mini gels in the appropriate electrophoresis system ( invitrogen , karlsruhe , germany ) and semidry blotted on nitrocellulose membranes ( carboglas , schleicher & schuell , dassel , germany ) . after blocking of the membranes with 3% ( w / v ) skim milk powder in tbs for 1 h at room temperature , they were incubated in the respective primary antibodies overnight at 4c . t cell proliferation was determined after three days by adding h - thymidine for 24 h. the incorporation of h - thymidine was detected using liquid scintillation spectrometry ( 1205 betaplate , perkinelmer , rodgau jgesheim , germany ) . we have shown before that the tryptophan - degrading enzyme human tryptophan 2,3-dioxygenase ( htdo ) , expressed in a tetracycline - inducible hela - cell based system , has antibacterial , antiparasitic , and antiviral capacities in vitro . these antimicrobial effects are the result of the htdo - induced degradation of tryptophan , which is an essential amino acid for tryptophan - auxotroph organisms , since the supplementation with additional tryptophan abrogated the effects . in order to analyse htdo - mediated antimicrobial effects under hypoxic conditions , we first monitored hela - htdo cell survival under hypoxia . this cell survival was also confirmed using a fluorescence - based method of assessing cell viability and cell death ( figure 1(b ) ) . for these experiments hela - htdo cells were incubated for 24 h under normoxia ( 20% o2 ) or hypoxia ( 1% o2 ) and afterwards stained with calcein am to detect intracellular esterase activity as indication for living cells or ethidium - homodimer-1 ( eth - d-1 ) that enters nonintact plasma membranes of dead cells . furthermore eth - d-1 treatment revealed no enhanced cell death under hypoxia ( plaid bars ) . the enzymatic activity of htdo under hypoxia was analysed by determination of the tetracycline - induced htdo - mediated conversion of tryptophan to kynurenine in cell culture supernatants after 72 h of incubation . interestingly the cells generated significantly lower amounts of kynurenine under hypoxic conditions ( 1% o2 ) ( figure 2(a ) ) . when tetracycline stimulated cells were incubated under different oxygen concentrations , the kynurenine amounts positively correlated with the amounts of oxygen present ( figure 2(b ) ) . reoxygenation studies confirmed survival of hela - htdo cells and preservation of enzymatic function within these cells under hypoxic conditions ( figure 2(c ) ) . in these experiments the tetracycline - induced tdo - mediated kynurenine production was determined after 72 h of incubation under normoxia ( 20% o2 ) or hypoxia ( 1% o2 ) and compared to the kynurenine production within cell supernatants after subsequent 48 h incubation under normoxia . the tdo - mediated conversion of tryptophan to kynurenine was drastically inhibited by hypoxia , but the cells were able to produce high amounts of kynurenine in the following normoxic phase , demonstrating cell survival and preservation of enzymatic activity . next , western blot analyses were performed to get quantitative information about htdo protein amounts in hela - htdo cells that were stimulated under normoxia ( 20% o2 ) and hypoxia ( 1% o2 ) . although htdo protein levels were unaltered under hypoxia ( figure 3 ) , the enzymatic activity of htdo was reduced up to nearly 90% , as determined by measurement of kynurenine in hela - htdo cell supernatants after 72 h of incubation under normoxia ( 20% o2 ) or hypoxia ( 1% o2 ) ( figure 2 ) . in order to analyse htdo - mediated antimicrobial functions under hypoxia , we infected unstimulated or tetracycline prestimulated hela - htdo cells with different tryptophan - auxotroph pathogens . figure 4(a ) shows the result of infection experiments with the facultative anaerobe , gram - positive bacterium enterococcus faecalis . enterococci grew in the presence of unstimulated hela - htdo cells , whereas bacterial growth was inhibited by tdo - positive cells , which have been stimulated with 5 ng / ml tetracycline ( white bars ) under normoxic conditions . interestingly , this tdo - mediated antibacterial effect was lost under hypoxic conditions ( grey bars ) . the same result was observed in infection experiments using other tryptophan - auxotroph bacteria , such as group b streptococci and staphylococci ( data not shown ) . figure 4(b ) shows that moreover the growth of the obligate intracellular apicomplexan parasite neospora caninum ( nc-1 strain ) was inhibited within activated hela - htdo cells under normoxia and that this antiparasitic effect was abolished under hypoxic conditions . a more detailed analysis of the htdo - mediated antibacterial effect under different low oxygen conditions ( 110% o2 ) in comparison to normoxia is shown in figure 5 . we have mentioned before that tdo regulates immune reactions in vitro and in vivo , for example , by creating tolerance towards the rejection of tumor cells . hence , we tested the immunoregulatory property of htdo in hela - htdo cells under hypoxia in vitro . t cell growth was triggered by the addition of the monoclonal anti - cd3 antibody okt3 and monitored by the h - thymidine incorporation method . this okt3 stimulation induced strong t cell proliferation , which is illustrated in a single experiment in figure 6(a ) . the t cell proliferation was inhibited by the tdo - mediated depletion of tryptophan , since it could be restored by the supplementation of tryptophan . however , such t cell inhibition did not occur in conditioned medium that has been harvested from hypoxia - treated hela - htdo cells , which demonstrates the loss of immunoregulatory htdo functions under hypoxia . although stably transfected hela cells are a useful tool to efficiently examine htdo functions in vitro , we extended our studies and confirmed the data by more physiological ex vivo liver homogenate experiments . in these experiments freshly isolated liver tissue of wt or tdo - deficient mice was homogenized in pbs and incubated in the presence of 20% o2 or 19% o2 in a buffer that allows the determination of tdo protein activity by the production of kynurenine . since the normoxic and the hypoxic groups contained the same amounts of murine tdo protein , the direct effect of normoxia and hypoxia on tdo enzyme activity could be revealed ( figure 7 ) . under hypoxia murine tdo produced significantly lower kynurenine amounts as compared to the normoxia control group . furthermore , no kynurenine was detectable in liver homogenates of tdo - deficient mice , as expected . in this study we investigated the influence of hypoxia on the activity of the tryptophan - degrading enzyme human tryptophan 2,3-dioxygenase ( htdo ) by using a the tetracycline - inducible hela t - rex system together with ex vivo studies analysing murine tdo . then the blood flows towards the central vein of the lobule and is depleted of oxygen , resulting in an oxygen pressure ( po2 ) of about 8% o2 in the periportal area and of about 4% o2 in parenchymatic perivenous areas . for example , po2 of ~1.3% was detected within the liver tissue of schistosoma mansoni - infected mice . furthermore a high expression rate of the hypoxia inducible factor-1 ( hif-1 ) was detected in the infiltrative belt surrounding hepatic alveolar echinococcosis lesions within rat livers . similar po2 levels of approximately 1 to 3% o2 were detected in other inflamed and infected tissues in the skin , the lungs , and the gut . for example , leishmania major infected mice display low oxygen levels of about 2.8% o2 in pronounced skin lesions , while the resolution of the wound was accompanied by an increase of lesional oxygen levels . given the fact that htdo has antimicrobial properties under normoxic conditions in vitro and that a microbial - induced hypoxic state is detected within liver tissue , we checked whether putative htdo - mediated antimicrobial effects might persist under low oxygen conditions . therefore the expression and activity of recombinant human tdo in transfected hela cells as well as ex vivo murine tdo were analysed under normoxic and hypoxic conditions . then the enzymatic activity of htdo was determined by measurement of kynurenine , which is the product of the tdo - mediated tryptophan degradation . human tdo efficiently catalysed the formation of kynurenine under normoxic conditions ( 20% o2 ) , whereas significantly lower levels of kynurenine were generated under oxygen concentrations detected in liver tissue physiologically ( 110% o2 ) . our ex vivo studies using liver homogenates from wildtype and tdo - deficient mice clearly show that also the mtdo - dependent degradation of tryptophan is significantly reduced under low oxygen conditions ( < 9% o2 ) . in particular the low po2 of 13% o2 , which has been detected in infected liver tissue significantly restricted the enzymatic activity of human and murine tdo enzymes . the reason for the lower tryptophan conversion rate under hypoxic conditions in vitro could be general downregulation of tdo protein levels or a reduced enzymatic function . the analysis of human tdo protein amounts within normoxia- and hypoxia - treated hela - htdo cells via western blot analyses showed no decrease of htdo protein amounts under hypoxia . htdo protein levels were correlated with the respective -actin band , since it is known that hypoxia caused general alterations in protein expression levels . therefore a decrease in htdo expression could not account for the decrease of htdo activity under hypoxia . a second dioxygenase that likewise catalyses the degradation of tryptophan is the enzyme indoleamine 2,3-dioxygenase ( ido ) . already in the 1980s it was shown that the intracellular degradation of tryptophan , induced by ifn- , restricted the growth of the intracellular parasite toxoplasma gondii in human fibroblasts . since then ido crystallized as broad - spectrum antimicrobial effector molecule that is effective against a variety of tryptophan - auxotroph pathogens in vitro . interestingly , ido - mediated tryptophan depletion is also inhibited under low oxygen concentrations ( 3% o2 ) which results in a loss of ido - mediated antimicrobial effects in vitro [ 19 , 25 , 26 ] . this was first shown in infection experiments using the intracellular bacterium chlamydia trachomatis in human fallopian tube cells and pinpointed to the hypoxia - dependent inhibition of ifn- signalling . this loss of ido - mediated antimicrobial effects under hypoxia raised the question to what extent tdo might lose its antimicrobial properties upon hypoxic circumstances . here we show that the antibacterial and antiparasitic effects of htdo were lost in hela - htdo cells under hypoxia . furthermore the htdo - mediated defence against the intracellular parasite neospora caninum was lost under hypoxic conditions . unfortunately it is impossible to analyse potential antimicrobial functions of mtdo or htdo in isolated primary hepatocytes , since these cells readily undergo dedifferentiation and lose hepatocyte function . therefore further studies need to be performed analysing such tdo functions in ex vivo induced stem cells ( e.g. since there are also no data claiming for an antimicrobial function of mtdo in vivo up to now , the immunoregulatory function of tdo is in the focus of research . here we show that also the t cell inhibitory function of htdo was lost in hela - htdo cells upon hypoxic conditions . again hypoxia prevented htdo - mediated tryptophan depletion and kynurenine production which led to unhindered okt3-driven t cell proliferation in supernatants of htdo - positive hela - htdo cells . in consistence with findings from other groups we detected reduced overall t cell proliferation under hypoxia . clearly demonstrated that the influence of oxygen levels on the t cell proliferation depends on the stimulus used to activate t cells . while the proliferation in response to phytohemagglutinin was not altered under different oxygen conditions , the cd3/cd28 crosslinking and the stimulation with con a lead to significant higher proliferation under atmospheric oxygen levels than under physiologic oxygen levels ( 5% and 10% o2 ) , with the latter being comparable to our experimental setting . this observation might be of relevance in vivo , since hypoxia is frequently observed in tumoural tissue [ 10 , 11 ] . tumor cells , as well as healthy cells , adapt to hypoxia by various appropriate physiologic responses , for example , by altered expression of genes that switch from oxidative to glycolytic metabolism [ 29 , 30 ] . these cellular responses are caused by the induction of the hypoxia - inducible factor ( hif ) protein complex due to hypoxia . tdo mrna is frequently expressed within human hepatocarcinomas , but information about tdo activity is missing and the cellular source is still unknown [ 6 , 7 ] . tdo - mediated production of tryptophan metabolites protects tdo - transfected tumour cells against immune rejection [ 8 , 9 ] . since several antimicrobial and immunoregulatory effector molecules in addition to tdo and ido , as , for example , the phagocyte nadph oxidase ( phox ) , type 2 nitric oxide synthase ( nos2 ) , and mitochondria rely on molecular oxygen as a substrate , hypoxia impairs their activity , which might promote the survival of pathogens .

nuclear factor b ( nf-b)/rel transcription factors exert important effects in diverse tissues , including epithelia . nf-b subunits are inhibited by inhibitor of b ( ib ) proteins and are activated by an upstream cascade involving ib kinases , which is controlled by a number of cell surface receptors , such as tnfr1 ( baldwin , 2001 ; dixit and mak , 2002 ) . in skin , epidermis lacking the rela nf-b subunit displays hyperproliferation that is epithelial cell autonomous ( zhang et al . , 2004 ) . rela inhibits epidermal growth by opposing the action of another group of tnfr1 effectors , namely the c - jun nh2-terminal kinase ( jnk ) cascade ( zhang et al . , 2004 ) . nf-b epidermal effects are not confined to physiologic growth restraint , but have also been implicated recently in epidermal squamous cell carcinoma ( scc ) , which is the second most common cancer in the united states . a majority of human sccs display evidence of nf-b hypofunction , and experimentally induced nf-b blockade with ib promotes scc in both murine and human epidermal tissue ( van hogerlinden et al . , 1999 therefore , nf-b affects epithelial homeostasis as well as carcinogenesis , although the nf-b targets altering cellular growth in these settings are unknown . the critical transition through the g1 phase of the cell cycle into dna replication is regulated by cdks , including cdk4/6 and their d - type cyclin partners , which act in concert to remove the mid - g1 rb block ( murray , 2004 ) . although redundancy in cdk mediated g1 progression is clear ( for review see su and stumpff , 2004 ) , the functional importance of g1 cdks and cyclins is supported by the overlapping defects resulting from their ablation as well as by their frequent amplification in human cancers ( cheung et al . , 2001 ; yasmeen et al . , 2003 ; additionally , in human epidermal cells , cdk4 down - regulation has been identified as a safeguard against neoplastic transformation by oncogenic ras ( lazarov et al . , 2002 ) . in this context , cdk4 protein degradation is triggered by ras in a process that can be inhibited by ib ( dajee et al . , 2003 ) , suggesting that nf-b opposes epidermal tumorigenesis by altering levels of a core cell cycle regulator . in agreement with this , nf-b caused selective cdk4 down - regulation , which led to g1 arrest ( dajee et al . however , these experiments relied on overexpression of active nf-b subunits , and it is uncertain whether these findings point to a physiologic role for cdk4 regulation by nf-b in epidermal growth control . here , we demonstrate that interfering with nf-b function in epidermis by multiple distinct genetic approaches increases expression and tissue distribution of cdk4 . this cdk4 up - regulation is dependent on both tnfr1 and jnk cascade function , and is consistent with a model in which tnfr1 originates antagonistic effects on the core cell cycle machinery through opposing actions by two of its major downstream effectors , nf-b and jnk . cdk4 ablation abolished epidermal growth impacts of conditional nf-b blockade , confirming the functional importance of cdk4 in this setting . antagonist regulation of cdk4 expression by nf-b and tnfr1/jnk therefore mediates homeostatic growth control in epidermis . to study the basis for the growth deregulation that occurs with nf-b hypofunction in the nontransformed epidermis , we examined the levels of cell cycle regulators . we focused initially on cdk4 because of prior findings in neoplasia overexpression studies ( dajee et al . , 2003 ) . to define effects on cdk4 in the context of nf-b subunit loss of function by genetic deletion this tissue , generated by an embryo - grafting approach to immunodeficient mice that circumvents the mid - gestational embryonic lethality of rela knockout mice ( zhang et al . , 2004 ) , displayed a markedly more widespread distribution of cdk4 protein ; in contrast , distribution of another g1 cdk , cdk2 , was unaltered ( fig . although normally confined to the proliferative basal layer of epithelium that is adherent to the underlying basement membrane zone ( bmz ) , cdk4 protein was found many cell layers above the bmz in rela - deficient epidermis , which is consistent with the recently demonstrated expansion of the proliferative cell compartment that occurs in this context ( zhang et al . , 2004 ) . quantitatively , cdk4 protein levels were more than fourfold above the normal level seen in epidermal extracts from rela - null tissue ( fig . therefore , nf-b rela deficiency leads to cdk4 up - regulation in nonneoplastic epidermis , indicating that rela is required to maintain physiologic cdk4 expression levels in this setting . cdk4 , cdk2 ( orange ) , the nidogen bmz marker ( green ) , and hoechst 33342 ( blue ) are shown . note the increase in cdk4 protein in rela epidermis that extends many cell layers above the bmz compared with the lack of altered cdk2 distribution . the addition of cdk4 peptide immunogen ( peptide ) abolished detection , and verified cdk4 antibody specificity . e14.5 keratinocytes from rela , rela , and rela mice embryos were immunoblotted for cdk4 , rela , and k14 ; optical densitometric quantitation for cdk4 protein is shown below each cdk4 band , normalized to keratin 14 ( k14 ) tnfr1 activates nf-b as well as other effector pathways , including the jnk kinase cascade , which has emerged as a major oppositional force to nf-b action in a variety of cell types ( de smaele et al . , 2001 ; tang et al . , 2001 ; reuther - madrid et al . , 2002 ) . in light of recent findings indicating that nf-b restrains epidermal proliferation by antagonizing tnfr1-jnk action ( zhang et al . , 2004 ) , we wished to determine if the cdk4 up - regulation that occurs with nf-b impairment requires intact tnfr1 and jnk function . to examine tnfr1 , tnfr1rela double knockout mice were generated ; these mice are viable through the postnatal period ( alcamo et al . , 2001 ) . epidermal extracts demonstrated no increase in cdk4 levels with rela deficiency on a tnfr1-null background ( fig . 2 a ) , confirming that tnfr1 is required for the cdk4 up - regulation that occurs with nf-b loss of function . tnf , the major tnfr1 ligand , down - regulated cdk4 protein levels in primary human epidermal cells , indicating that cdk4 represents a downstream target of tnf action ( fig . cdk4 down - regulation by tnf was blocked by expression of an nf-b inhibitory ib super - repressor mutant ( van antwerp et al . , 2 b ) . although neither ib nor mkk7 altered levels of tnfr1 protein ( fig . 2 c ) , antibodies to tnfr1 blocked the cdk4 increases seen in epidermal cells expressing ib ( fig . jnk cascade inhibition , via both genetic and pharmacologic means , also blocked ib-driven cdk4 up - regulation in human epidermal cells in vitro ( fig . 2003 ) using overexpression of nf-b subunits , altered nf-b function failed to change cdk4 mrna levels significantly ( fig . 2 f ) , indicating that cdk4 regulation by nf-b occurs at the posttranscriptional level . expression of ib in genetically engineered human epidermal tissue regenerated on immunodeficient mice recapitulated the expansion of cdk4 expression seen in rela null tissue ; this expansion was largely reversed by either coexpression of dominant - negative jnk or topical application of a jnk inhibitor ( fig . epidermal cdk4 up - regulation caused by nf-b hypofunction , therefore , requires intact function of both tnfr1 and the jnk cascade . cdk4 up - regulation caused by nf-b hypofunction requires intact function of tnfr1 and jnk . ( a ) a lack of cdk4 up - regulation with rela deficiency in the absence of tnfr1 in epidermal extract immunoblots from rela tnfr1 , relatnfr1 , and relatnfr1 mice . primary human keratinocytes expressing ib super - repressor mutant ( ibm ) , active mkk7 , or lacz control were treated with ( + ) or without ( ) tnf. ( c ) nf-b blockade fails to increase tnfr1 expression in keratinocytes that express ibm as well as mkk7 and lacz controls . ( d ) blockade of ib-induced cdk4 up - regulation by antibodies to tnfr1 in keratinocytes expressing ibm or lacz treated with blocking antibodies to tnfr1 or tnfr2 . keratinocytes expressing either lacz control or ibm were either cotransduced with a retroviral expression vector for dominant - negative jnk1-apf ( apf ) or treated with the jnk inhibitor sp600125 . ( f ) northern blot of human keratinocytes harvested 24 and 48 h after expression of ibm or lacz control . ( g ) interfering with jnk cascade function blocks cdk4 up - regulation in response to nf-b blockade in vivo . jnk function was impaired in human epidermis engineered to express ib via either coexpression of the dominant - negative jnk - apf mutant or topical application of sp600125 . note the presence of the cdk4 protein ( orange ) confined to the basal layer in control tissue ( small bracket ) and its spread upward in epidermis expressing ibm ( large bracket ) compared with the diminished cdk4 distribution seen in ib-expressing epidermis subjected to jnk inhibition . cdk4 up - regulation in rela epidermis could reflect either a requirement for cdk4 in the hyperproliferation that occurs with nf-b impairment or , in light of recent studies ( for review see su and stumpff , 2004 ) , it could represent a nonessential secondary effect . distinguishing between these two possibilities requires the ability to examine the consequences of nf-b impairment in the absence of cdk4 . generation of relacdk4 double knockout mice , however , is a formidable challenge because of the death of rela embryos by e14.5 ( beg et al . , 1995 ) and the decreased viability and infertility of cdk4 mice ( rane et al . , 1999 ) . to circumvent this , we established a conditional approach to inhibit nf-b by fusing a 4-hydoxytamoxifen ( 4oht)-responsive mutant estrogen receptor ( er ) domain ( littlewood et al . , 1995 ) to the cooh terminus of sp , which is a p50 mutant that dominantly inhibits nf-b function ( logeat et al . , 1991 ) . expression of sp : er in epidermal cells selectively blocked nf-b directed reporter gene activity and down - regulated expression of the nf-b target , ib , in a 4oht - responsive manner ( fig . ( a ) sp : er inducibly inhibits nf-b reporter gene expression in response to 4oht . reporter gene activity sd from triplicate independent experiments is shown in cells transfected with luciferase reporter plasmids for nf-b as well as ef-1 specificity control after 48 h with or without 4oht ( 50 nm ) . extracts from primary human keratinocytes transduced with retrovectors encoding either lacz or sp : er and grown for 48 h in the absence ( ) or in the presence ( + ) of 4oht were immunoblotted with ib antibodies . to determine the effects of nf-b hypofunction in the setting of cdk4 deficiency in tissue , we first generated multiple lines of epidermis - targeted k14-sp : er transgenic mice ; all three expressing lines demonstrated comparable findings ( unpublished data ) . k14-sp : er mice displayed increased epidermal cdk4 protein levels in response to topical 4oht ( fig . 4 a ) , confirming that nf-b inhibition in mature epidermis also produces cdk4 up - regulation . k14-sp : er mice were then bred with cdk4 mice to generate k14-sp : er cdk4 mice . conditional nf-b blockade via 4oht increased epidermal proliferation in cdk4 epidermis , resulting in marked hyperplasia in 5 d ; in contrast , nf-b inhibition in cdk4-deficient tissue produced no changes in either epidermal mitotic index or morphology ( fig . therefore , cdk4 is required for the epidermal hyperproliferation that occurs with nf-b loss of function . epidermal extracts from wild - type ( wt ) and k14-sp : er transgenic mice ( tg ) treated with 4oht or vehicle control for 5 d were immunoblotted for sp : er , cdk4 , and actin . histology of k14-sp : er / cdk4 and k14-sp : er / cdk4 murine skin treated with either 4oht or vehicle alone for 1 wk . note 4oht - induced hyperplasia in k14-sp : ercdk4 epidermis and its complete absence in k14-sp : ercdk4 skin . note the increase in green ki-67(+ ) cells ( arrows ) that occurs with nf-b blockade only in the presence of cdk4 . ( d ) mitotic index quantitation of ki-67(+ ) epidermal cells/100 m skin of k14-sp : er / cdk4 and k14-sp : er / cdk4 murine skin in response to 4oht . the present work indicates that suppressing cdk4 up - regulation and confining cdk4 within the epidermal basal layer represents a nonredundant role for nf-b rela that serves as a physiologic restraint on epidermal growth . moreover , these findings indicate that cdk4 is essential for the increased epidermal proliferation that accompanies nf-b impairment . this does not reflect a global deficit in proliferation in cdk4-deficient epidermis , as shown recently by normal hyperproliferation of cdk4 mice in response to other stimuli , including phorbol ester and wounding ( rodriguez - puebla et al . , 2002 ) . although recent neoplasia - focused work suggests that active nf-b subunits regulate cdk4 expression at the posttranscriptional level in a proteasome inhibitor sensitive manner ( dajee et al . , 2003 ) , further studies are needed to determine the mechanistic basis for cdk4 responsiveness to altered nf-b function . the current data are consistent with recent studies directed at epidermal neoplasia , where cdk4 levels were established to be the critical determinant of whether an epidermal cell undergoes permanent growth arrest or malignant transformation ( lazarov et al . , 2002 ; dajee et al . , 2003 ) . this appears to contradict the orthodox view that regulation of cyclin levels is the vital protein expression control mechanism of cellular proliferation ( murray , 2004 ) and points to cdk4 protein as a limiting factor in core cell cycle machinery in normal and neoplastic epidermis . in agreement with this possibility , epidermal overexpression of cdk4 causes greater hyperproliferation and malignant transformation than does expression of cyclin d1 from the same promoter ( robles et al . , 1996 ; miliani de marval et al . , 2004 ) . the well - characterized positive regulation of d - type cyclins by nf-b ( guttridge et al . , 1999 ) may thus be balanced by active suppression of cdk4 , indicating that additional signals , such as jnk induction , are necessary to overcome cdk4 repression by nf-b to support g1 progression in epidermis . we observed that intact tnfr1 and jnk function was required for cdk4 up - regulation caused by nf-b impairment . while nf-b - jnk antagonism is increasingly recognized as a potent regulator of signaling outcomes in a variety of settings , this work identifies the first core cell cycle target regulated in an opposing fashion by these pathways . tnfr1 activates both nf-b as well as jnk ( aggarwal , 2000 ) , and our findings suggest that cdk4 levels , and their resultant effects on epidermal growth control , may depend on the relative balance between these two competing pathways . although pro - growth functions by the jnk cascade , aside from cdk4 effects noted here , have not been systematically assessed in normal epidermis , the data suggest that jnk and its ap1 targets facilitate epidermal neoplasia . ap1 function , via deletion of either jnk2 ( chen et al . , 2001 ) or c - jun ( zenz et al . , 2003 ) as well as by overexpression of dominant - negative c - jun ( thompson et al . , 2002 ) increases resistance to topical chemical carcinogenesis , but oncogenic ap1 subunits promote epidermal tumor formation ( wang et al . , 1995 ) . although further studies are needed to define the role of jnk cascade action in epidermal growth control , these data are consistent with a potential role for this nf-b - opposed pathway in promoting epidermal proliferation . our findings suggest that the cdk4-dependent epidermal growth deregulation that occurs with nf-b inhibition is intrinsic to epidermal cells and does not require significant inflammation . pure cultures of both murine and human keratinocytes with impaired nf-b function caused by rela deficiency and ib overexpression , respectively , display both cdk4 up - regulation and hyperproliferation , underscoring the cell - autonomous nature of this process . in tissue , we detected neither an influx of inflammatory cells nor an up - regulation of inflammatory cytokines associated with the cdk4 induction that occurs with conditional nf-b blockade ( fig . this lack of inflammation is consistent with prior findings with constitutively rela - deficient epidermis ( zhang et al . , 2004 ) . although inflammatory cell infiltration is readily excluded in these experiments by immunohistochemical analysis , such analyses do not exclude action by basal levels of local proinflammatory cytokines , such as tnf , which are constitutively expressed by epidermal cells . tnf represents a likely ligand for tnfr1 in the epidermal growth deregulation caused by nf-b loss of function , as demonstrated recently by relac - rel tnf mice , which display epidermal hypoplasia ( gugasyan et al . , 2004 ) . moreover , the blockade of cdk4 up - regulation by antibodies to tnfr1 in pure cultures of keratinocytes observed here suggests that tnfr1 operates in an epithelial cell - intrinsic manner rather than by a more indirect route , such as via inflammatory cells . this premise is supported by the recent demonstration that tnfr1 deletion abolishes hyperplasia and tumorigenesis in epidermis overexpressing ib in a manner that can not be restored by administration of tnfr1 wild - type hematopoietic cells ( lind et al . , 2004 ) 2004 ) demonstrated that significant cdk4 up - regulation was observed only in tumorigenesis , and further study of differences between the k5-ib mice and the three distinct approaches pursued here is merited . in which locally produced tnf activates epidermal cell tnfr1 , which then alters cdk4 protein levels in a nf-b and jnk - regulated manner to control cell cycle progression in epidermis . primary human keratinocytes were grown as described previously ( robbins et al . , 2001 ) . we grew murine keratinocytes from rela , rela , and rela e14.5-old embryos for 3 d before performing protein extraction . for gene transfer , 4 10 keratinocytes were plated on 35-mm wells 1 d before infection with retroviruses . no drug selection was used at any point , and > 99% efficiency gene transfer was verified by immunofluorescence staining ( robbins et al . , 2001 ) . retroviruses were generated encoding lacz , ibm ( seitz et al . , 1998 ) , and dominant - negative jnk1-apf ( derijard et al . , 1994 ) in the plzrs backbone . keratinocytes infected with retroviruses encoding lacz , ibm , or mkk7 were treated with either neutralizing antibodies against tnfr1 ( 50 g / ml ; mab 225 ; r&d systems ) , tnfr2 control ( 50 g / ml ; mab 226 ; r&d systems ) , the jnk inhibitor sp600125 ( 10 m ; biomol research laboratories , inc . ) , or tnf ( 15 ng / ml ; sigma - aldrich ) for 48 h before immunoblotting . rela , rela , and rela skin tissues were generated through embryonic skin grafting on immunodeficient cb.17 scid / scid mice ( oro et al . , 1997 ) and analyzed at 6 wk . inducible nf-b blockade was achieved through in - frame cooh - terminal fusion of the p50 mutant ( sp ) dominant negative for nf-b function ( provided by a. israel , cnrs ura 2582 , paris , france ; logeat et al . , 1991 ) with the 4oht - responsive mutant er ( littlewood et al . , 1995 ) . sp : er fusion was subcloned into the pentr1a plasmid ( invitrogen ) and transferred to plzrs retrovector and pbskii keratin 14 promoter plasmids modified to contain gateway destination sequences ( gibco brl ) . we confirmed expression of sp : er protein in three independent lines of k14-sp : er transgenic mice by immunoblotting epidermal extracts . k14-sp : er mice were crossbred with cdk4 mice ( provided by e. reddy and m. barbacid , temple university school of medicine , philadelphia , pa ; rane et al . , 1999 ) to generate k14sp : er , cdk4 mice . to induce nf-b blockade , we applied 1 mg / d 4oht ( in 95% ethanol ) for 5 d on back skin of 4-wk - old mice ; five mice / group . for human skin studies , primary human keratinocytes were engineered to express lacz control , ib , or ib + dominant - negative jnk1 ( jnk - apf ) and grafted on immunodeficient cb.17 scid / scid mice ( n = 5 independent grafts per analyzed group ) . for jnk inhibition , we applied 1 mg / d sp600125 topically to human skin grafts for 5 d before analysis . for immunostaining , 5-m cryosections were incubated either with primary rabbit antibodies against cdk4 or cdk2 ( santa cruz biotechnology , inc . ) along with rat anti mouse nidogen ( chemicon ) , or rat anti ki-67 ( dakocytomation ) along with rabbit anti mouse laminin 5 ( gift of m.p . marinkovich , stanford university , stanford , ca ) followed by cy2 or cy3-conjugated secondary antibodies ( jackson immunoresearch laboratories ) . regenerated human skin cryosections were incubated with primary mouse antibodies against cdk4 ( labvision ) along with rabbit anti , cells were transfected with plasmids encoding sp : er and either nf-b or ef1 luciferase reporter plasmids along with cmv - driven renilla - luciferase internal controls for transfection efficiency using fugene reagents ( roche ) . 8 h after transfection , cells were treated with ethanol solvent or 50 nm 4oht for 48 h before analysis with dual luciferase reagents ( promega ) . primary human keratinocytes were grown as described previously ( robbins et al . , 2001 ) . we grew murine keratinocytes from rela , rela , and rela e14.5-old embryos for 3 d before performing protein extraction . for gene transfer , 4 10 keratinocytes were plated on 35-mm wells 1 d before infection with retroviruses . no drug selection was used at any point , and > 99% efficiency gene transfer was verified by immunofluorescence staining ( robbins et al . , 2001 ) . retroviruses were generated encoding lacz , ibm ( seitz et al . , 1998 ) , and dominant - negative jnk1-apf ( derijard et al . , 1994 ) in the plzrs backbone . keratinocytes infected with retroviruses encoding lacz , ibm , or mkk7 were treated with either neutralizing antibodies against tnfr1 ( 50 g / ml ; mab 225 ; r&d systems ) , tnfr2 control ( 50 g / ml ; mab 226 ; r&d systems ) , the jnk inhibitor sp600125 ( 10 m ; biomol research laboratories , inc . ) , or tnf ( 15 ng / ml ; sigma - aldrich ) for 48 h before immunoblotting . rela , rela , and rela skin tissues were generated through embryonic skin grafting on immunodeficient cb.17 scid / scid mice ( oro et al . , 1997 ) and analyzed at 6 wk . inducible nf-b blockade was achieved through in - frame cooh - terminal fusion of the p50 mutant ( sp ) dominant negative for nf-b function ( provided by a. israel , cnrs ura 2582 , paris , france ; logeat et al . , 1991 ) with the 4oht - responsive mutant er ( littlewood et al . , 1995 ) . sp : er fusion was subcloned into the pentr1a plasmid ( invitrogen ) and transferred to plzrs retrovector and pbskii keratin 14 promoter plasmids modified to contain gateway destination sequences ( gibco brl ) . we confirmed expression of sp : er protein in three independent lines of k14-sp : er transgenic mice by immunoblotting epidermal extracts . k14-sp : er mice were crossbred with cdk4 mice ( provided by e. reddy and m. barbacid , temple university school of medicine , philadelphia , pa ; rane et al . , 1999 ) to generate k14sp : er , cdk4 mice . to induce nf-b blockade , we applied 1 mg / d 4oht ( in 95% ethanol ) for 5 d on back skin of 4-wk - old mice ; five mice / group . for human skin studies , primary human keratinocytes were engineered to express lacz control , ib , or ib + dominant - negative jnk1 ( jnk - apf ) and grafted on immunodeficient cb.17 scid / scid mice ( n = 5 independent grafts per analyzed group ) . for jnk inhibition , we applied 1 mg / d sp600125 topically to human skin grafts for 5 d before analysis . for immunostaining , 5-m cryosections were incubated either with primary rabbit antibodies against cdk4 or cdk2 ( santa cruz biotechnology , inc . ) along with rat anti mouse nidogen ( chemicon ) , or rat anti ki-67 ( dakocytomation ) along with rabbit anti mouse laminin 5 ( gift of m.p . marinkovich , stanford university , stanford , ca ) followed by cy2 or cy3-conjugated secondary antibodies ( jackson immunoresearch laboratories ) . regenerated human skin cryosections were incubated with primary mouse antibodies against cdk4 ( labvision ) along with rabbit anti for reporter gene assays , cells were transfected with plasmids encoding sp : er and either nf-b or ef1 luciferase reporter plasmids along with cmv - driven renilla - luciferase internal controls for transfection efficiency using fugene reagents ( roche ) . 8 h after transfection , cells were treated with ethanol solvent or 50 nm 4oht for 48 h before analysis with dual luciferase reagents ( promega ) .

nuclear factor b ( nf-b ) mediates homeostatic growth inhibition in the epidermis , and a loss of nf-b function promotes proliferation and oncogenesis . to identify mechanisms responsible for these effects , we impaired nf-b action in the epidermis by three different genetic approaches , including conditional nf-b blockade . in each case , epidermal hyperplasia was accompanied by an increase in both protein levels and tissue distribution of the g1 cell cycle kinase , cdk4 . cdk4 up - regulation required intact tnfr1 and c - jun nh2-terminal kinase ( jnk ) function . cdk4 gene deletion concomitant with conditional nf-b blockade demonstrated that cdk4 is required for growth deregulation . therefore , epidermal homeostasis depends on antagonist regulation of cdk4 expression by nf-b and tnfr1/jnk .

Introduction Results and discussion Materials and methods Cell culture and gene transfer Animal studies Protein expression analysis and microscopy Reporter gene assays

nuclear factor b ( nf-b)/rel transcription factors exert important effects in diverse tissues , including epithelia . nf-b subunits are inhibited by inhibitor of b ( ib ) proteins and are activated by an upstream cascade involving ib kinases , which is controlled by a number of cell surface receptors , such as tnfr1 ( baldwin , 2001 ; dixit and mak , 2002 ) . rela inhibits epidermal growth by opposing the action of another group of tnfr1 effectors , namely the c - jun nh2-terminal kinase ( jnk ) cascade ( zhang et al . nf-b epidermal effects are not confined to physiologic growth restraint , but have also been implicated recently in epidermal squamous cell carcinoma ( scc ) , which is the second most common cancer in the united states . a majority of human sccs display evidence of nf-b hypofunction , and experimentally induced nf-b blockade with ib promotes scc in both murine and human epidermal tissue ( van hogerlinden et al . , 1999 therefore , nf-b affects epithelial homeostasis as well as carcinogenesis , although the nf-b targets altering cellular growth in these settings are unknown . the critical transition through the g1 phase of the cell cycle into dna replication is regulated by cdks , including cdk4/6 and their d - type cyclin partners , which act in concert to remove the mid - g1 rb block ( murray , 2004 ) . , 2003 ; additionally , in human epidermal cells , cdk4 down - regulation has been identified as a safeguard against neoplastic transformation by oncogenic ras ( lazarov et al . in this context , cdk4 protein degradation is triggered by ras in a process that can be inhibited by ib ( dajee et al . , 2003 ) , suggesting that nf-b opposes epidermal tumorigenesis by altering levels of a core cell cycle regulator . in agreement with this , nf-b caused selective cdk4 down - regulation , which led to g1 arrest ( dajee et al . however , these experiments relied on overexpression of active nf-b subunits , and it is uncertain whether these findings point to a physiologic role for cdk4 regulation by nf-b in epidermal growth control . here , we demonstrate that interfering with nf-b function in epidermis by multiple distinct genetic approaches increases expression and tissue distribution of cdk4 . this cdk4 up - regulation is dependent on both tnfr1 and jnk cascade function , and is consistent with a model in which tnfr1 originates antagonistic effects on the core cell cycle machinery through opposing actions by two of its major downstream effectors , nf-b and jnk . cdk4 ablation abolished epidermal growth impacts of conditional nf-b blockade , confirming the functional importance of cdk4 in this setting . antagonist regulation of cdk4 expression by nf-b and tnfr1/jnk therefore mediates homeostatic growth control in epidermis . to study the basis for the growth deregulation that occurs with nf-b hypofunction in the nontransformed epidermis , we examined the levels of cell cycle regulators . to define effects on cdk4 in the context of nf-b subunit loss of function by genetic deletion this tissue , generated by an embryo - grafting approach to immunodeficient mice that circumvents the mid - gestational embryonic lethality of rela knockout mice ( zhang et al . , 2004 ) , displayed a markedly more widespread distribution of cdk4 protein ; in contrast , distribution of another g1 cdk , cdk2 , was unaltered ( fig . although normally confined to the proliferative basal layer of epithelium that is adherent to the underlying basement membrane zone ( bmz ) , cdk4 protein was found many cell layers above the bmz in rela - deficient epidermis , which is consistent with the recently demonstrated expansion of the proliferative cell compartment that occurs in this context ( zhang et al . quantitatively , cdk4 protein levels were more than fourfold above the normal level seen in epidermal extracts from rela - null tissue ( fig . therefore , nf-b rela deficiency leads to cdk4 up - regulation in nonneoplastic epidermis , indicating that rela is required to maintain physiologic cdk4 expression levels in this setting . cdk4 , cdk2 ( orange ) , the nidogen bmz marker ( green ) , and hoechst 33342 ( blue ) are shown . note the increase in cdk4 protein in rela epidermis that extends many cell layers above the bmz compared with the lack of altered cdk2 distribution . the addition of cdk4 peptide immunogen ( peptide ) abolished detection , and verified cdk4 antibody specificity . e14.5 keratinocytes from rela , rela , and rela mice embryos were immunoblotted for cdk4 , rela , and k14 ; optical densitometric quantitation for cdk4 protein is shown below each cdk4 band , normalized to keratin 14 ( k14 ) tnfr1 activates nf-b as well as other effector pathways , including the jnk kinase cascade , which has emerged as a major oppositional force to nf-b action in a variety of cell types ( de smaele et al . , 2004 ) , we wished to determine if the cdk4 up - regulation that occurs with nf-b impairment requires intact tnfr1 and jnk function . epidermal extracts demonstrated no increase in cdk4 levels with rela deficiency on a tnfr1-null background ( fig . 2 a ) , confirming that tnfr1 is required for the cdk4 up - regulation that occurs with nf-b loss of function . tnf , the major tnfr1 ligand , down - regulated cdk4 protein levels in primary human epidermal cells , indicating that cdk4 represents a downstream target of tnf action ( fig . cdk4 down - regulation by tnf was blocked by expression of an nf-b inhibitory ib super - repressor mutant ( van antwerp et al . jnk cascade inhibition , via both genetic and pharmacologic means , also blocked ib-driven cdk4 up - regulation in human epidermal cells in vitro ( fig . 2003 ) using overexpression of nf-b subunits , altered nf-b function failed to change cdk4 mrna levels significantly ( fig . 2 f ) , indicating that cdk4 regulation by nf-b occurs at the posttranscriptional level . expression of ib in genetically engineered human epidermal tissue regenerated on immunodeficient mice recapitulated the expansion of cdk4 expression seen in rela null tissue ; this expansion was largely reversed by either coexpression of dominant - negative jnk or topical application of a jnk inhibitor ( fig . epidermal cdk4 up - regulation caused by nf-b hypofunction , therefore , requires intact function of both tnfr1 and the jnk cascade . cdk4 up - regulation caused by nf-b hypofunction requires intact function of tnfr1 and jnk . ( a ) a lack of cdk4 up - regulation with rela deficiency in the absence of tnfr1 in epidermal extract immunoblots from rela tnfr1 , relatnfr1 , and relatnfr1 mice . ( c ) nf-b blockade fails to increase tnfr1 expression in keratinocytes that express ibm as well as mkk7 and lacz controls . ( d ) blockade of ib-induced cdk4 up - regulation by antibodies to tnfr1 in keratinocytes expressing ibm or lacz treated with blocking antibodies to tnfr1 or tnfr2 . ( g ) interfering with jnk cascade function blocks cdk4 up - regulation in response to nf-b blockade in vivo . jnk function was impaired in human epidermis engineered to express ib via either coexpression of the dominant - negative jnk - apf mutant or topical application of sp600125 . note the presence of the cdk4 protein ( orange ) confined to the basal layer in control tissue ( small bracket ) and its spread upward in epidermis expressing ibm ( large bracket ) compared with the diminished cdk4 distribution seen in ib-expressing epidermis subjected to jnk inhibition . cdk4 up - regulation in rela epidermis could reflect either a requirement for cdk4 in the hyperproliferation that occurs with nf-b impairment or , in light of recent studies ( for review see su and stumpff , 2004 ) , it could represent a nonessential secondary effect . distinguishing between these two possibilities requires the ability to examine the consequences of nf-b impairment in the absence of cdk4 . generation of relacdk4 double knockout mice , however , is a formidable challenge because of the death of rela embryos by e14.5 ( beg et al . to circumvent this , we established a conditional approach to inhibit nf-b by fusing a 4-hydoxytamoxifen ( 4oht)-responsive mutant estrogen receptor ( er ) domain ( littlewood et al . , 1995 ) to the cooh terminus of sp , which is a p50 mutant that dominantly inhibits nf-b function ( logeat et al . expression of sp : er in epidermal cells selectively blocked nf-b directed reporter gene activity and down - regulated expression of the nf-b target , ib , in a 4oht - responsive manner ( fig . extracts from primary human keratinocytes transduced with retrovectors encoding either lacz or sp : er and grown for 48 h in the absence ( ) or in the presence ( + ) of 4oht were immunoblotted with ib antibodies . to determine the effects of nf-b hypofunction in the setting of cdk4 deficiency in tissue , we first generated multiple lines of epidermis - targeted k14-sp : er transgenic mice ; all three expressing lines demonstrated comparable findings ( unpublished data ) . k14-sp : er mice displayed increased epidermal cdk4 protein levels in response to topical 4oht ( fig . 4 a ) , confirming that nf-b inhibition in mature epidermis also produces cdk4 up - regulation . conditional nf-b blockade via 4oht increased epidermal proliferation in cdk4 epidermis , resulting in marked hyperplasia in 5 d ; in contrast , nf-b inhibition in cdk4-deficient tissue produced no changes in either epidermal mitotic index or morphology ( fig . therefore , cdk4 is required for the epidermal hyperproliferation that occurs with nf-b loss of function . epidermal extracts from wild - type ( wt ) and k14-sp : er transgenic mice ( tg ) treated with 4oht or vehicle control for 5 d were immunoblotted for sp : er , cdk4 , and actin . note the increase in green ki-67(+ ) cells ( arrows ) that occurs with nf-b blockade only in the presence of cdk4 . the present work indicates that suppressing cdk4 up - regulation and confining cdk4 within the epidermal basal layer represents a nonredundant role for nf-b rela that serves as a physiologic restraint on epidermal growth . moreover , these findings indicate that cdk4 is essential for the increased epidermal proliferation that accompanies nf-b impairment . this does not reflect a global deficit in proliferation in cdk4-deficient epidermis , as shown recently by normal hyperproliferation of cdk4 mice in response to other stimuli , including phorbol ester and wounding ( rodriguez - puebla et al . although recent neoplasia - focused work suggests that active nf-b subunits regulate cdk4 expression at the posttranscriptional level in a proteasome inhibitor sensitive manner ( dajee et al . , 2003 ) , further studies are needed to determine the mechanistic basis for cdk4 responsiveness to altered nf-b function . this appears to contradict the orthodox view that regulation of cyclin levels is the vital protein expression control mechanism of cellular proliferation ( murray , 2004 ) and points to cdk4 protein as a limiting factor in core cell cycle machinery in normal and neoplastic epidermis . in agreement with this possibility , epidermal overexpression of cdk4 causes greater hyperproliferation and malignant transformation than does expression of cyclin d1 from the same promoter ( robles et al . the well - characterized positive regulation of d - type cyclins by nf-b ( guttridge et al . , 1999 ) may thus be balanced by active suppression of cdk4 , indicating that additional signals , such as jnk induction , are necessary to overcome cdk4 repression by nf-b to support g1 progression in epidermis . we observed that intact tnfr1 and jnk function was required for cdk4 up - regulation caused by nf-b impairment . while nf-b - jnk antagonism is increasingly recognized as a potent regulator of signaling outcomes in a variety of settings , this work identifies the first core cell cycle target regulated in an opposing fashion by these pathways . tnfr1 activates both nf-b as well as jnk ( aggarwal , 2000 ) , and our findings suggest that cdk4 levels , and their resultant effects on epidermal growth control , may depend on the relative balance between these two competing pathways . although pro - growth functions by the jnk cascade , aside from cdk4 effects noted here , have not been systematically assessed in normal epidermis , the data suggest that jnk and its ap1 targets facilitate epidermal neoplasia . , 2001 ) or c - jun ( zenz et al . , 2003 ) as well as by overexpression of dominant - negative c - jun ( thompson et al . although further studies are needed to define the role of jnk cascade action in epidermal growth control , these data are consistent with a potential role for this nf-b - opposed pathway in promoting epidermal proliferation . our findings suggest that the cdk4-dependent epidermal growth deregulation that occurs with nf-b inhibition is intrinsic to epidermal cells and does not require significant inflammation . pure cultures of both murine and human keratinocytes with impaired nf-b function caused by rela deficiency and ib overexpression , respectively , display both cdk4 up - regulation and hyperproliferation , underscoring the cell - autonomous nature of this process . in tissue , we detected neither an influx of inflammatory cells nor an up - regulation of inflammatory cytokines associated with the cdk4 induction that occurs with conditional nf-b blockade ( fig . tnf represents a likely ligand for tnfr1 in the epidermal growth deregulation caused by nf-b loss of function , as demonstrated recently by relac - rel tnf mice , which display epidermal hypoplasia ( gugasyan et al . moreover , the blockade of cdk4 up - regulation by antibodies to tnfr1 in pure cultures of keratinocytes observed here suggests that tnfr1 operates in an epithelial cell - intrinsic manner rather than by a more indirect route , such as via inflammatory cells . , 2004 ) 2004 ) demonstrated that significant cdk4 up - regulation was observed only in tumorigenesis , and further study of differences between the k5-ib mice and the three distinct approaches pursued here is merited . in which locally produced tnf activates epidermal cell tnfr1 , which then alters cdk4 protein levels in a nf-b and jnk - regulated manner to control cell cycle progression in epidermis . we grew murine keratinocytes from rela , rela , and rela e14.5-old embryos for 3 d before performing protein extraction . no drug selection was used at any point , and > 99% efficiency gene transfer was verified by immunofluorescence staining ( robbins et al . , 1994 ) in the plzrs backbone . rela , rela , and rela skin tissues were generated through embryonic skin grafting on immunodeficient cb.17 scid / scid mice ( oro et al . inducible nf-b blockade was achieved through in - frame cooh - terminal fusion of the p50 mutant ( sp ) dominant negative for nf-b function ( provided by a. israel , cnrs ura 2582 , paris , france ; logeat et al . , 1999 ) to generate k14sp : er , cdk4 mice . to induce nf-b blockade , we applied 1 mg / d 4oht ( in 95% ethanol ) for 5 d on back skin of 4-wk - old mice ; five mice / group . for human skin studies , primary human keratinocytes were engineered to express lacz control , ib , or ib + dominant - negative jnk1 ( jnk - apf ) and grafted on immunodeficient cb.17 scid / scid mice ( n = 5 independent grafts per analyzed group ) . for jnk inhibition , we applied 1 mg / d sp600125 topically to human skin grafts for 5 d before analysis . we grew murine keratinocytes from rela , rela , and rela e14.5-old embryos for 3 d before performing protein extraction . no drug selection was used at any point , and > 99% efficiency gene transfer was verified by immunofluorescence staining ( robbins et al . , 1998 ) , and dominant - negative jnk1-apf ( derijard et al . , 1994 ) in the plzrs backbone . rela , rela , and rela skin tissues were generated through embryonic skin grafting on immunodeficient cb.17 scid / scid mice ( oro et al . inducible nf-b blockade was achieved through in - frame cooh - terminal fusion of the p50 mutant ( sp ) dominant negative for nf-b function ( provided by a. israel , cnrs ura 2582 , paris , france ; logeat et al . to induce nf-b blockade , we applied 1 mg / d 4oht ( in 95% ethanol ) for 5 d on back skin of 4-wk - old mice ; five mice / group . for human skin studies , primary human keratinocytes were engineered to express lacz control , ib , or ib + dominant - negative jnk1 ( jnk - apf ) and grafted on immunodeficient cb.17 scid / scid mice ( n = 5 independent grafts per analyzed group ) . for jnk inhibition , we applied 1 mg / d sp600125 topically to human skin grafts for 5 d before analysis .

diabetes status and medications were evaluated by a questionnaires completed with the aid of trained nurses . participants who had fpg values > 126 mg / dl ( 7 mmol / l ) or who received medications for diabetes were excluded . the questionnaire , along with anthropometric measurement and risk - factor assessment , were used to assess participants medical and metabolic status . abdominal computed tomography ( ct ) all study participants were contacted by telephone , e - mail , or postal mail 13 years after the initial visit , and follow - up visits were scheduled according to the respondent s availability . written informed consent was obtained from each individual . anthropometric measurement of each subject was performed by trained nurses in the morning after fasting for at least 8 h. body height was recorded to the nearest 0.5 cm and body weight to the nearest 0.1 kg . bmi was defined as body weight ( kilograms ) divided by the square of body height ( meters ) . wc - ic was measured in the horizontal plane at the superior border of the right iliac crest . wc - mid was measured in the horizontal plane midway between lowest rib and the iliac crest . both wc - ic and wc - mid were measured to the nearest 0.1 cm at the end of a normal expiration . before recording the measurement , the nurse would ensure that the tape was snug but did not compress the skin and was parallel to the floor . wc - ic and wc - mid were measured repeatedly in 10 men and 10 women by 3 trained nurses on 3 consecutive days . the coefficients of variation for wc - ic were 0.8% ( range 0.51.7% ) for women and 0.6% ( range 0.31.4% ) for men . the coefficients of variation for wc - mid were 0.4% ( range 00.7% ) for men and 0.9% ( range 0.51.9% ) for women . blood pressure was recorded to the nearest 2 mmhg by a mercury sphygmomanometer with the arm supported at heart level after sitting quietly for 10 min . fpg was measured after fasting for at least 8 h. a standard oral 75-g glucose tolerance test was performed to measure 2-h postprandial plasma glucose ( 2hpg ) . plasma glucose and fasting serum total cholesterol , triglycerides ( tg ) , hdl cholesterol ( hdl - c ) , ldl cholesterol ( ldl - c ) , and high - sensitivity c - reactive protein ( hscrp ) concentrations were measured with an automatic analyzer ( toshiba tba 120fr ; toshiba medical systems co. , ltd . , hba1c was measured by automatic analyzers ( hlc-723 g7 hplc systems ; tosoh corporation , tokyo , japan ) . the hba1c assay was certified by the national glycohemoglobin standardization program ( 23 ) and standardized to the diabetes control and complications trial reference assay . imaging of each subject in a supine position was performed on a 16-mdct scanner ( lightspeed 16 ; ge healthcare , milwaukee , wi ) ( 120 kvp , 400 mas , slice thickness 5 mm ) . image analysis software ( imagej , version 1.44 ; national institutes of health , bethesda , md ) was used with an attenuation range of 50 to 250 hounsfield units to quantify the abdominal subcutaneous fat area ( sfa ) and vfa , expressed in centimeters squared , on a single cross - sectional image obtained at the level of the umbilicus . hypertension was present if blood pressure was 140/90 mmhg or if the subject was taking medication for hypertension . diabetes was diagnosed when fpg was 126 mg / dl ( 7 mmol / l ) , 2hpg 200 mg / dl ( 11.1 mmol / l ) , and hba1c 6.5% or if the subject was taking medication for diabetes ( 24 ) . ms was defined in accordance with the updated ncep atp iii guideline ( 7 ) . data are presented as means and sds for continuous variables and as a percentage for categorical variables . pearson correlation coefficients and partial correlation coefficients were used to assess the relationship among wc , abdominal fat areas , and metabolic variables . the association of the different diagnostic criteria for central obesity with high vfa and with metabolic disease was analyzed by receiver operating characteristic ( roc ) curve analysis . roc statistics were calculated by using percentile values of disease case measures relative to the corresponding marker distribution among controls ( 25,26 ) . optimal cutoffs were derived from the roc curve with the shortest distance to sensitivity = 1 and 1 specificity = 0 . kaplan - meier failure curves were used to estimate the cumulative incidence of hypertension , diabetes , and ms in individuals with and without central obesity defined by wc - ic or wc - mid cutoff values . the statistical analyses were performed with stat / se 11 for windows ( statacorp lp , college station , tx ) . from 2006 to 2012 , individuals aged 18 years who had received health examinations at the national taiwan university hospital yun - lin branch during the previous year and had fasting plasma glucose ( fpg ) levels < 126 mg / dl ( 7 mmol / l ) were invited to participate in the taiwan lifestyle study ( 1922 ) . diabetes status and medications were evaluated by a questionnaires completed with the aid of trained nurses . participants who had fpg values > 126 mg / dl ( 7 mmol / l ) or who received medications for diabetes were excluded . the questionnaire , along with anthropometric measurement and risk - factor assessment , were used to assess participants medical and metabolic status . abdominal computed tomography ( ct ) all study participants were contacted by telephone , e - mail , or postal mail 13 years after the initial visit , and follow - up visits were scheduled according to the respondent s availability . written informed consent was obtained from each individual . anthropometric measurement of each subject was performed by trained nurses in the morning after fasting for at least 8 h. body height was recorded to the nearest 0.5 cm and body weight to the nearest 0.1 kg . bmi was defined as body weight ( kilograms ) divided by the square of body height ( meters ) . wc - ic was measured in the horizontal plane at the superior border of the right iliac crest . wc - mid was measured in the horizontal plane midway between lowest rib and the iliac crest . both wc - ic and wc - mid were measured to the nearest 0.1 cm at the end of a normal expiration . before recording the measurement , the nurse would ensure that the tape was snug but did not compress the skin and was parallel to the floor . wc - ic and wc - mid were measured repeatedly in 10 men and 10 women by 3 trained nurses on 3 consecutive days . the coefficients of variation for wc - ic were 0.8% ( range 0.51.7% ) for women and 0.6% ( range 0.31.4% ) for men . the coefficients of variation for wc - mid were 0.4% ( range 00.7% ) for men and 0.9% ( range 0.51.9% ) for women . blood pressure was recorded to the nearest 2 mmhg by a mercury sphygmomanometer with the arm supported at heart level after sitting quietly for 10 min . fpg was measured after fasting for at least 8 h. a standard oral 75-g glucose tolerance test was performed to measure 2-h postprandial plasma glucose ( 2hpg ) . plasma glucose and fasting serum total cholesterol , triglycerides ( tg ) , hdl cholesterol ( hdl - c ) , ldl cholesterol ( ldl - c ) , and high - sensitivity c - reactive protein ( hscrp ) concentrations were measured with an automatic analyzer ( toshiba tba 120fr ; toshiba medical systems co. , ltd . , hba1c was measured by automatic analyzers ( hlc-723 g7 hplc systems ; tosoh corporation , tokyo , japan ) . the hba1c assay was certified by the national glycohemoglobin standardization program ( 23 ) and standardized to the diabetes control and complications trial reference assay . imaging of each subject in a supine position was performed on a 16-mdct scanner ( lightspeed 16 ; ge healthcare , milwaukee , wi ) ( 120 kvp , 400 mas , slice thickness 5 mm ) . image analysis software ( imagej , version 1.44 ; national institutes of health , bethesda , md ) was used with an attenuation range of 50 to 250 hounsfield units to quantify the abdominal subcutaneous fat area ( sfa ) and vfa , expressed in centimeters squared , on a single cross - sectional image obtained at the level of the umbilicus . hypertension was present if blood pressure was 140/90 mmhg or if the subject was taking medication for hypertension . diabetes was diagnosed when fpg was 126 mg / dl ( 7 mmol / l ) , 2hpg 200 mg / dl ( 11.1 mmol / l ) , and hba1c 6.5% or if the subject was taking medication for diabetes ( 24 ) . ms was defined in accordance with the updated ncep atp iii guideline ( 7 ) . data are presented as means and sds for continuous variables and as a percentage for categorical variables . pearson correlation coefficients and partial correlation coefficients were used to assess the relationship among wc , abdominal fat areas , and metabolic variables . the association of the different diagnostic criteria for central obesity with high vfa and with metabolic disease was analyzed by receiver operating characteristic ( roc ) curve analysis . roc statistics were calculated by using percentile values of disease case measures relative to the corresponding marker distribution among controls ( 25,26 ) . optimal cutoffs were derived from the roc curve with the shortest distance to sensitivity = 1 and 1 specificity = 0 . kaplan - meier failure curves were used to estimate the cumulative incidence of hypertension , diabetes , and ms in individuals with and without central obesity defined by wc - ic or wc - mid cutoff values . the statistical analyses were performed with stat / se 11 for windows ( statacorp lp , college station , tx ) . the clinical characteristics of the participants ( n = 1,898 ) are summarized in table 1 . wc - ic values were significantly higher than wc - mid in both sexes , and the differences between wc - ic and wc - mid were greater in women than in men ( p < 0.001 ) . the partial correlation coefficients adjusted for age in men and women were 0.91 and 0.834 , respectively . clinical characteristics of the study subjects there were 425 participants , including 150 males and 275 females , who underwent abdominal ct for assessment of abdominal fat areas . comparing participants with and without ct measurements , those who had ct evaluations showed slightly higher hdl - c ( 52 12 vs. 50 13 mg / dl ; p < 0.05 ) and ldl - c values ( 121 34 vs. 117 32 mg / dl ; p < 0.05 ) . women with ct measurement had higher wc - ic than women without ct measurement ( 85 9 vs. 83 9 cm ; p < 0.05 ) . as shown in table 2 , both wc - ic and wc - mid correlated significantly with bmi , total abdominal fat area , vfa , and sfa . wc - mid predicted high vfa ( vfa 50th percentile in the corresponding sex ) more often than wc - ic in women ( area under the roc [ auc ] 0.825 for wc - ic , 0.860 for wc - mid ; p = 0.0142 ) , but not in men ( auc 0.855 wc - ic , 0.865 wc - mid ; p = 0.454 ) . associations between wc - ic and wc - mid , bmi , and abdominal fat areas the data presented in supplementary table 1 show that both wc - ic and wc - mid correlated significantly with systolic and diastolic blood pressure , fpg , 2hpg , hba1c , tg , hdl - c , and hscrp in both sexes , and wc - mid was better correlated than wc - ic with these metabolic variables in both sexes . results in table 3 show that the identification of individuals with hypertension , diabetes , and ms by wc - ic and wc - mid was fair ( auc 0.680.7 for wc - ic and 0.70.75 for wc - mid ) . wc - mid had slightly better association with hypertension , diabetes , and ms than wc - ic ( p < 0.05 comparing auc ) . the optimal cutoffs for wc - ic and wc - mid varied , depending on which disease to identify . generally , wc - ic was more sensitive , whereas wc - mid was more specific . wc - mid had a higher age - adjusted auc than wc - ic for diabetes in men , hypertension in women , and ms in both sexes ( supplementary table 2 ) . the data in supplementary table 3 show that wc - mid at its optimal cutoffs had the highest auc for hypertension in females and diabetes and ms in both sexes . using the cutoffs of 90 and 80 cm ( males and females , respectively ) , wc - mid had significantly higher auc for hypertension and diabetes in women and for ms in both sexes ( all age - adjusted p < 0.05 ) ( supplementary table 3 ) . the differences in auc for hypertension , diabetes , and ms among four criteria were larger in women ( 0.040.07 ) than in men ( 0.020.05 ) . different definitions of central obesity to identify or predict hypertension , diabetes , or ms there were 1,503 subjects who stayed in the study for at least 12 months . among them , the data in table 3 indicate that the performance of wc - ic and wc - mid to predict incident hypertension , diabetes , and ms was fair ( auc 0.620.68 for wc - ic , 0.650.68 for wc - mid ) . the aucs for wc - ic and wc - mid for hypertension , diabetes , and ms were not statistically different ( age - adjusted p > 0.05 ) . the optimal cutoff values for wc - ic and wc - mid to predict different diseases varied , and wc - ic was more sensitive , whereas wc - mid was more specific . as demonstrated by the data in supplementary table 2 , there was no significant difference between wc - ic and wc - mid to predict hypertension , diabetes , or ms . however , wc - mid had slightly higher aucs than wc - ic for diabetes and for hypertension in women ( both age - adjusted p values 0.050.1 ) . data in supplementary table 3 show that the best criteria for highest auc depended on the disease to be predicted and the sex to be considered . using the cutoffs of 90/80 cm ( male / female ) , wc - ic and wc - mid showed similar auc for hypertension , diabetes , and ms in both sexes ( all age - adjusted p > 0.05 ; supplementary table 3 ) . there were 639 subjects , including 206 men and 433 women , who did not have hypertension at baseline . during the follow - up period ( median 31.7 months , interquartile range 16.145.6 ) , 87 subjects developed hypertension . as shown in fig . 1a and b , there was no difference in the incidence of hypertension in subjects with or without central obesity , neither by wc - ic or wc - mid criteria ( both p > 0.05 ) . there were 801 subjects , including 292 men and 509 women , who did not have diabetes at baseline . during follow - up ( median 30.8 months , interquartile range 16.046.3 ) , 60 developed diabetes . as shown in fig . 1c and d , the cumulative incidence of diabetes was significantly higher in the individuals who met wc - mid criteria for central obesity ( p = 0.003 ) and not for those with wc - ic criteria ( p = 0.112 ) . there were 587 subjects , including 179 men and 408 women , who had less than two components of ms at baseline . during follow - up ( median 31.4 months , interquartile range 16.149.6 ) , 162 subjects clustered three or more components of ms . figure 1e and f shows that the cumulative incidence of ms was not significantly different in subjects who had central obesity by wc - ic criteria ( p = 0.988 ) or wc - mid criteria ( p = 0.223 ) . kaplan - meier curves for the cumulative incidence of developing hypertension ( a and b ) , diabetes ( c and d ) , or ms ( e and f ) by wc - ic ( a , c , and e ) or wc - mid ( b , d , and f ) to define central obesity . to the best of our knowledge , this is the first comprehensive study to compare different measurements of wc to define central obesity . we showed that wc - mid predicts high vfa better than wc - ic in women . correlation , as compared by auc , with hypertension , diabetes , and ms was better by wc - mid criteria than wc - ic criteria . however , the performance of wc - ic and wc - mid to predict hypertension , diabetes , and ms was similar , although only central obesity by wc - mid criteria , and not wc - ic criteria , predicted future diabetes incidence . overall , our findings suggest that wc - mid is a better measurement of central obesity than wc - ic . it is practical to keep the current cutoffs for central obesity ( i.e. , 90 [ males]/80 [ females ] cm ) . using these cutoffs , in the current study , we found that the sensitivity of wc - ic measurement values for identifying or predicting hypertension , diabetes , and ms was greater than that of wc - mid measurements . since central obesity is a screening tool for metabolic diseases , higher sensitivity of wc - ic values may be a desirable attribute . furthermore , wc - ic can be more precisely located than wc - mid , which may make it more consistent during follow - up . therefore , in a recent scientific statement of the american heart association , wc - ic with cutoffs at 90/80 ( male / female ) cm for asians has been recommended to define central obesity ( 2 ) . in contrast , wc - mid correlated better to vfa and metabolic variables and worked better to identify and predict metabolic diseases in the current study . indeed , when optimal cutoffs were used , wc - mid showed better performance than wc - ic did , with more balanced sensitivity and specificity . furthermore , although measurement of wc - mid is a slightly more complex procedure , findings from the current study and from mason and katzmarzyk ( 27 ) have shown that the reproducibility of wc - mid measurement is also high . thus , if modification of the cutoffs for central obesity is to be considered , wc - mid is a better location of measurement than wc - ic . the impact of the location of wc measurement varies by sex . in the current study , the difference between wc - ic and wc - mid was larger in women ( 5.6 cm ) than in men ( 1.7 cm ) , which is in concordance with a report in caucasians ( 28 ) . this may explain why the differences between the correlation coefficients of vfa to wc - ic and wc - mid were larger in women than in men in present study ( table 2 ) . it could also explain why wc - mid in women , but not in men , showed significantly higher aucs than wc - ic for hypertension , diabetes , and ms when cutoffs of 90/80 ( male / female ) cm were used ( supplementary table 3 ) . also , the differences in auc among the four wc criteria used to identify these diseases were larger in women than in men ( supplementary table 3 ) , and similar findings were also reported in the previous study in caucasians ( 29 ) . in that study , the differences between wc - ic and wc - mid were 0.4 cm in men and 1.1 cm in women , and the differences in auc for components of ms among wc - ic , wc - mid , wc at umbilicus , and minimal wc were larger in women ( 0.0530.088 ) than in men ( 0.0030.029 ) . all of these findings suggest that the location of wc measurement has greater impact in women than in men . the optimal cutoffs depend on the diseases to be identified or predicted . in the current study , the optimal cutoffs for both wc - ic and wc - mid were all different ( table 3 ) . supporting our findings , the optimal cutoffs for wc were also different for different metabolic diseases in another large cross - sectional study in taiwan that included 55,563 people ( 11 ) . in that study , optimal cutoffs were determined based on the performance for identifying at least one disease , including hypertension , diabetes , and dyslipidemia . moreover , there have been two chinese studies investigating the optimal cutoffs based on the relationship of wc to vfa ( 18,30 ) . ( 30 ) reported that individuals in a cross - sectional study with vfa > 80 cm had higher risk of ms . ye et al . ( 18 ) showed that subjects with vfa > 90 cm have a higher risk of future incidence of diabetes . , it seems essential to have a consensus for the use of wc before optimal cutoffs can be determined . questions that should be addressed include the following : for which diseases are associations with wc most important ? are the cutoffs based on disease identification or disease prediction ? should vfa be considered ? in the current study , wc - mid was better correlated to vfa than wc - ic . various reports have shown that visceral adipose tissue ( vat ) produces and releases adipokines , which are linked to the development of metabolic abnormalities ( 3133 ) . fatty acids from vat drain to the liver , and the increased fat influx may increase hepatic tg content , resulting in increased hepatic glucose output and vldl tg production ( 34,35 ) . vat also secretes higher levels of proinflammatory cytokines than subcutaneous adipose tissue ( 36,37 ) . all of these mechanisms provide for a potentially pathogenic role of vat in the development of metabolic abnormalities , and indeed , increased vfa has been associated with increased risk of hypertension , diabetes , and dyslipidemia in humans ( 38,39 ) . however , wc has been shown to have a stronger correlation to sfa than vfa in caucasians , indicating that wc is a better index of sfa ( 28 ) . in present study , although wc also showed stronger correlation with sfa than vfa , the differences in the correlation coefficients were small , especially for wc - mid ( 0.010.04 in men and 00.03 in women ; table 2 ) . these findings suggest that wc , particularly wc - mid , can be viewed as an index of both sfa and abdominal vfa in asians . the strength of this study is in the completeness of its comparison of wc - ic and wc - mid . we compared the biologic roles of wc - mid and wc - ic through their relationships to vfa and metabolic variables . in contrast , this study was limited in that only wc - ic and wc - mid were compared and evaluated . from the literature , there are at least eight different measurement locations for wc ( 40 ) . however , since wc - ic or wc - mid have been the recommended locations by world health organization , ncep atp iii , and idf ( 68 ) , the findings of the current study are practical . moreover , the study subjects were not a random sample because the incidence of hypertension and diabetes were higher ( 5.1%/person - year and 2.8%/person - year , respectively ) . this may be due to a higher percentage of subjects who had prehypertension and prediabetes in the cohort ( 24 and 39% , respectively ) , since people at risk are more willing to be followed . in conclusion , wc - mid proved in this study to be a better measurement to define central obesity than wc - ic in asians , since wc - mid was more closely related to abdominal vfa and metabolic variables and had better results for identifying and predicting metabolic diseases . our data further indicate that there is a need to re - evaluate the location of wc measurement and cutoffs for central obesity in different ethnic groups .

objectivewaist circumference ( wc ) is used to define central obesity . this study aimed to compare the performance of two recommended locations of wc measurement.research design and methodsa cohort of 1,898 subjects who were without diabetes from 2006 to 2012 were followed for a median of 31 months ( taiwan lifestyle study ) . the wc - ic , recommended by the national cholesterol education program third adult treatment panel , was measured at the superior border of the iliac crest , and the wc - mid , recommended by world health organization and international diabetes federation , was measured midway between the lowest ribs and the iliac crest . the abdominal subcutaneous fat area ( sfa ) and visceral fat area ( vfa ) were assessed by computed tomography.resultsthere was greater difference between wc - ic and wc - mid measurements in women than in men ( p < 0.001 ) . both wc - ic and wc - mid correlated significantly with bmi , vfa , and sfa ( all p < 0.001 ) . wc - mid was better correlated to vfa than wc - ic , particularly in women , and it correlated more strongly to blood pressure , plasma glucose , hemoglobin a1c , triglyceride levels , hdl cholesterol , and c - reactive protein ( all p < 0.05 ) . the association of wc - mid with hypertension , diabetes , and metabolic syndrome was slightly better than that of wc - ic ( area under the receiver operator curve 0.7 vs. 0.69 , 0.71 vs. 0.68 , and 0.75 vs. 0.7 , respectively ; all age - adjusted p < 0.05 ) . with 90 cm ( male)/80 cm ( female ) as criteria for central obesity , wc - mid , but not wc - ic , predicted the incidence of diabetes development ( age - adjusted p = 0.003).conclusionswc - mid is a better measurement to define central obesity than wc - ic , particularly in women .

RESEARCH DESIGN AND METHODS Participants Anthropometry Risk factor measurements Quantification of abdominal adipose tissue by CT Definitions Statistical analysis RESULTS CONCLUSIONS

the questionnaire , along with anthropometric measurement and risk - factor assessment , were used to assess participants medical and metabolic status . abdominal computed tomography ( ct ) all study participants were contacted by telephone , e - mail , or postal mail 13 years after the initial visit , and follow - up visits were scheduled according to the respondent s availability . bmi was defined as body weight ( kilograms ) divided by the square of body height ( meters ) . wc - ic was measured in the horizontal plane at the superior border of the right iliac crest . wc - mid was measured in the horizontal plane midway between lowest rib and the iliac crest . both wc - ic and wc - mid were measured to the nearest 0.1 cm at the end of a normal expiration . wc - ic and wc - mid were measured repeatedly in 10 men and 10 women by 3 trained nurses on 3 consecutive days . the coefficients of variation for wc - ic were 0.8% ( range 0.51.7% ) for women and 0.6% ( range 0.31.4% ) for men . the coefficients of variation for wc - mid were 0.4% ( range 00.7% ) for men and 0.9% ( range 0.51.9% ) for women . blood pressure was recorded to the nearest 2 mmhg by a mercury sphygmomanometer with the arm supported at heart level after sitting quietly for 10 min . fpg was measured after fasting for at least 8 h. a standard oral 75-g glucose tolerance test was performed to measure 2-h postprandial plasma glucose ( 2hpg ) . plasma glucose and fasting serum total cholesterol , triglycerides ( tg ) , hdl cholesterol ( hdl - c ) , ldl cholesterol ( ldl - c ) , and high - sensitivity c - reactive protein ( hscrp ) concentrations were measured with an automatic analyzer ( toshiba tba 120fr ; toshiba medical systems co. , ltd . the hba1c assay was certified by the national glycohemoglobin standardization program ( 23 ) and standardized to the diabetes control and complications trial reference assay . image analysis software ( imagej , version 1.44 ; national institutes of health , bethesda , md ) was used with an attenuation range of 50 to 250 hounsfield units to quantify the abdominal subcutaneous fat area ( sfa ) and vfa , expressed in centimeters squared , on a single cross - sectional image obtained at the level of the umbilicus . hypertension was present if blood pressure was 140/90 mmhg or if the subject was taking medication for hypertension . pearson correlation coefficients and partial correlation coefficients were used to assess the relationship among wc , abdominal fat areas , and metabolic variables . the association of the different diagnostic criteria for central obesity with high vfa and with metabolic disease was analyzed by receiver operating characteristic ( roc ) curve analysis . optimal cutoffs were derived from the roc curve with the shortest distance to sensitivity = 1 and 1 specificity = 0 . kaplan - meier failure curves were used to estimate the cumulative incidence of hypertension , diabetes , and ms in individuals with and without central obesity defined by wc - ic or wc - mid cutoff values . from 2006 to 2012 , individuals aged 18 years who had received health examinations at the national taiwan university hospital yun - lin branch during the previous year and had fasting plasma glucose ( fpg ) levels < 126 mg / dl ( 7 mmol / l ) were invited to participate in the taiwan lifestyle study ( 1922 ) . the questionnaire , along with anthropometric measurement and risk - factor assessment , were used to assess participants medical and metabolic status . abdominal computed tomography ( ct ) all study participants were contacted by telephone , e - mail , or postal mail 13 years after the initial visit , and follow - up visits were scheduled according to the respondent s availability . bmi was defined as body weight ( kilograms ) divided by the square of body height ( meters ) . wc - ic was measured in the horizontal plane at the superior border of the right iliac crest . wc - mid was measured in the horizontal plane midway between lowest rib and the iliac crest . both wc - ic and wc - mid were measured to the nearest 0.1 cm at the end of a normal expiration . wc - ic and wc - mid were measured repeatedly in 10 men and 10 women by 3 trained nurses on 3 consecutive days . the coefficients of variation for wc - ic were 0.8% ( range 0.51.7% ) for women and 0.6% ( range 0.31.4% ) for men . the coefficients of variation for wc - mid were 0.4% ( range 00.7% ) for men and 0.9% ( range 0.51.9% ) for women . blood pressure was recorded to the nearest 2 mmhg by a mercury sphygmomanometer with the arm supported at heart level after sitting quietly for 10 min . fpg was measured after fasting for at least 8 h. a standard oral 75-g glucose tolerance test was performed to measure 2-h postprandial plasma glucose ( 2hpg ) . plasma glucose and fasting serum total cholesterol , triglycerides ( tg ) , hdl cholesterol ( hdl - c ) , ldl cholesterol ( ldl - c ) , and high - sensitivity c - reactive protein ( hscrp ) concentrations were measured with an automatic analyzer ( toshiba tba 120fr ; toshiba medical systems co. , ltd . , hba1c was measured by automatic analyzers ( hlc-723 g7 hplc systems ; tosoh corporation , tokyo , japan ) . the hba1c assay was certified by the national glycohemoglobin standardization program ( 23 ) and standardized to the diabetes control and complications trial reference assay . image analysis software ( imagej , version 1.44 ; national institutes of health , bethesda , md ) was used with an attenuation range of 50 to 250 hounsfield units to quantify the abdominal subcutaneous fat area ( sfa ) and vfa , expressed in centimeters squared , on a single cross - sectional image obtained at the level of the umbilicus . pearson correlation coefficients and partial correlation coefficients were used to assess the relationship among wc , abdominal fat areas , and metabolic variables . the association of the different diagnostic criteria for central obesity with high vfa and with metabolic disease was analyzed by receiver operating characteristic ( roc ) curve analysis . kaplan - meier failure curves were used to estimate the cumulative incidence of hypertension , diabetes , and ms in individuals with and without central obesity defined by wc - ic or wc - mid cutoff values . wc - ic values were significantly higher than wc - mid in both sexes , and the differences between wc - ic and wc - mid were greater in women than in men ( p < 0.001 ) . the partial correlation coefficients adjusted for age in men and women were 0.91 and 0.834 , respectively . comparing participants with and without ct measurements , those who had ct evaluations showed slightly higher hdl - c ( 52 12 vs. 50 13 mg / dl ; p < 0.05 ) and ldl - c values ( 121 34 vs. 117 32 mg / dl ; p < 0.05 ) . women with ct measurement had higher wc - ic than women without ct measurement ( 85 9 vs. 83 9 cm ; p < 0.05 ) . as shown in table 2 , both wc - ic and wc - mid correlated significantly with bmi , total abdominal fat area , vfa , and sfa . wc - mid predicted high vfa ( vfa 50th percentile in the corresponding sex ) more often than wc - ic in women ( area under the roc [ auc ] 0.825 for wc - ic , 0.860 for wc - mid ; p = 0.0142 ) , but not in men ( auc 0.855 wc - ic , 0.865 wc - mid ; p = 0.454 ) . associations between wc - ic and wc - mid , bmi , and abdominal fat areas the data presented in supplementary table 1 show that both wc - ic and wc - mid correlated significantly with systolic and diastolic blood pressure , fpg , 2hpg , hba1c , tg , hdl - c , and hscrp in both sexes , and wc - mid was better correlated than wc - ic with these metabolic variables in both sexes . results in table 3 show that the identification of individuals with hypertension , diabetes , and ms by wc - ic and wc - mid was fair ( auc 0.680.7 for wc - ic and 0.70.75 for wc - mid ) . wc - mid had slightly better association with hypertension , diabetes , and ms than wc - ic ( p < 0.05 comparing auc ) . the optimal cutoffs for wc - ic and wc - mid varied , depending on which disease to identify . generally , wc - ic was more sensitive , whereas wc - mid was more specific . wc - mid had a higher age - adjusted auc than wc - ic for diabetes in men , hypertension in women , and ms in both sexes ( supplementary table 2 ) . the data in supplementary table 3 show that wc - mid at its optimal cutoffs had the highest auc for hypertension in females and diabetes and ms in both sexes . using the cutoffs of 90 and 80 cm ( males and females , respectively ) , wc - mid had significantly higher auc for hypertension and diabetes in women and for ms in both sexes ( all age - adjusted p < 0.05 ) ( supplementary table 3 ) . the differences in auc for hypertension , diabetes , and ms among four criteria were larger in women ( 0.040.07 ) than in men ( 0.020.05 ) . different definitions of central obesity to identify or predict hypertension , diabetes , or ms there were 1,503 subjects who stayed in the study for at least 12 months . among them , the data in table 3 indicate that the performance of wc - ic and wc - mid to predict incident hypertension , diabetes , and ms was fair ( auc 0.620.68 for wc - ic , 0.650.68 for wc - mid ) . the aucs for wc - ic and wc - mid for hypertension , diabetes , and ms were not statistically different ( age - adjusted p > 0.05 ) . the optimal cutoff values for wc - ic and wc - mid to predict different diseases varied , and wc - ic was more sensitive , whereas wc - mid was more specific . as demonstrated by the data in supplementary table 2 , there was no significant difference between wc - ic and wc - mid to predict hypertension , diabetes , or ms . however , wc - mid had slightly higher aucs than wc - ic for diabetes and for hypertension in women ( both age - adjusted p values 0.050.1 ) . data in supplementary table 3 show that the best criteria for highest auc depended on the disease to be predicted and the sex to be considered . using the cutoffs of 90/80 cm ( male / female ) , wc - ic and wc - mid showed similar auc for hypertension , diabetes , and ms in both sexes ( all age - adjusted p > 0.05 ; supplementary table 3 ) . there were 639 subjects , including 206 men and 433 women , who did not have hypertension at baseline . 1a and b , there was no difference in the incidence of hypertension in subjects with or without central obesity , neither by wc - ic or wc - mid criteria ( both p > 0.05 ) . 1c and d , the cumulative incidence of diabetes was significantly higher in the individuals who met wc - mid criteria for central obesity ( p = 0.003 ) and not for those with wc - ic criteria ( p = 0.112 ) . there were 587 subjects , including 179 men and 408 women , who had less than two components of ms at baseline . figure 1e and f shows that the cumulative incidence of ms was not significantly different in subjects who had central obesity by wc - ic criteria ( p = 0.988 ) or wc - mid criteria ( p = 0.223 ) . kaplan - meier curves for the cumulative incidence of developing hypertension ( a and b ) , diabetes ( c and d ) , or ms ( e and f ) by wc - ic ( a , c , and e ) or wc - mid ( b , d , and f ) to define central obesity . to the best of our knowledge , this is the first comprehensive study to compare different measurements of wc to define central obesity . we showed that wc - mid predicts high vfa better than wc - ic in women . correlation , as compared by auc , with hypertension , diabetes , and ms was better by wc - mid criteria than wc - ic criteria . however , the performance of wc - ic and wc - mid to predict hypertension , diabetes , and ms was similar , although only central obesity by wc - mid criteria , and not wc - ic criteria , predicted future diabetes incidence . overall , our findings suggest that wc - mid is a better measurement of central obesity than wc - ic . it is practical to keep the current cutoffs for central obesity ( i.e. using these cutoffs , in the current study , we found that the sensitivity of wc - ic measurement values for identifying or predicting hypertension , diabetes , and ms was greater than that of wc - mid measurements . since central obesity is a screening tool for metabolic diseases , higher sensitivity of wc - ic values may be a desirable attribute . furthermore , wc - ic can be more precisely located than wc - mid , which may make it more consistent during follow - up . therefore , in a recent scientific statement of the american heart association , wc - ic with cutoffs at 90/80 ( male / female ) cm for asians has been recommended to define central obesity ( 2 ) . in contrast , wc - mid correlated better to vfa and metabolic variables and worked better to identify and predict metabolic diseases in the current study . indeed , when optimal cutoffs were used , wc - mid showed better performance than wc - ic did , with more balanced sensitivity and specificity . furthermore , although measurement of wc - mid is a slightly more complex procedure , findings from the current study and from mason and katzmarzyk ( 27 ) have shown that the reproducibility of wc - mid measurement is also high . thus , if modification of the cutoffs for central obesity is to be considered , wc - mid is a better location of measurement than wc - ic . the impact of the location of wc measurement varies by sex . in the current study , the difference between wc - ic and wc - mid was larger in women ( 5.6 cm ) than in men ( 1.7 cm ) , which is in concordance with a report in caucasians ( 28 ) . this may explain why the differences between the correlation coefficients of vfa to wc - ic and wc - mid were larger in women than in men in present study ( table 2 ) . it could also explain why wc - mid in women , but not in men , showed significantly higher aucs than wc - ic for hypertension , diabetes , and ms when cutoffs of 90/80 ( male / female ) cm were used ( supplementary table 3 ) . also , the differences in auc among the four wc criteria used to identify these diseases were larger in women than in men ( supplementary table 3 ) , and similar findings were also reported in the previous study in caucasians ( 29 ) . in that study , the differences between wc - ic and wc - mid were 0.4 cm in men and 1.1 cm in women , and the differences in auc for components of ms among wc - ic , wc - mid , wc at umbilicus , and minimal wc were larger in women ( 0.0530.088 ) than in men ( 0.0030.029 ) . all of these findings suggest that the location of wc measurement has greater impact in women than in men . in the current study , the optimal cutoffs for both wc - ic and wc - mid were all different ( table 3 ) . in that study , optimal cutoffs were determined based on the performance for identifying at least one disease , including hypertension , diabetes , and dyslipidemia . moreover , there have been two chinese studies investigating the optimal cutoffs based on the relationship of wc to vfa ( 18,30 ) . ( 18 ) showed that subjects with vfa > 90 cm have a higher risk of future incidence of diabetes . in the current study , wc - mid was better correlated to vfa than wc - ic . fatty acids from vat drain to the liver , and the increased fat influx may increase hepatic tg content , resulting in increased hepatic glucose output and vldl tg production ( 34,35 ) . all of these mechanisms provide for a potentially pathogenic role of vat in the development of metabolic abnormalities , and indeed , increased vfa has been associated with increased risk of hypertension , diabetes , and dyslipidemia in humans ( 38,39 ) . however , wc has been shown to have a stronger correlation to sfa than vfa in caucasians , indicating that wc is a better index of sfa ( 28 ) . in present study , although wc also showed stronger correlation with sfa than vfa , the differences in the correlation coefficients were small , especially for wc - mid ( 0.010.04 in men and 00.03 in women ; table 2 ) . these findings suggest that wc , particularly wc - mid , can be viewed as an index of both sfa and abdominal vfa in asians . the strength of this study is in the completeness of its comparison of wc - ic and wc - mid . we compared the biologic roles of wc - mid and wc - ic through their relationships to vfa and metabolic variables . in contrast , this study was limited in that only wc - ic and wc - mid were compared and evaluated . however , since wc - ic or wc - mid have been the recommended locations by world health organization , ncep atp iii , and idf ( 68 ) , the findings of the current study are practical . moreover , the study subjects were not a random sample because the incidence of hypertension and diabetes were higher ( 5.1%/person - year and 2.8%/person - year , respectively ) . this may be due to a higher percentage of subjects who had prehypertension and prediabetes in the cohort ( 24 and 39% , respectively ) , since people at risk are more willing to be followed . in conclusion , wc - mid proved in this study to be a better measurement to define central obesity than wc - ic in asians , since wc - mid was more closely related to abdominal vfa and metabolic variables and had better results for identifying and predicting metabolic diseases . our data further indicate that there is a need to re - evaluate the location of wc measurement and cutoffs for central obesity in different ethnic groups .

avulsion injuries of the brachial plexus cause the most devastating palsies of the affected upper extremities . the treatment for brachial plexus avulsion lesions involves the use of neurotization procedures because neither direct repair nor interpositional nerve grafting can be performed for these irreparable preganglionic lesions . new treatment guidelines for total root avulsion enhance the combination of various types of neurotization with ipsilateral intraplexus and extraplexus nerve donors , contralateral cervical seventh ( cc7 ) root donor , and primary or secondary free functioning muscle transfer . a wide variety of potential donor nerves are available , including the intercostal nerves , the spinal accessory nerve , the phrenic nerve , partial ulnar nerve , partial median nerve , pectoral major , thoracodorsal nerve , and the partial ipsilateral , the hemi or total cc7 nerve roots . the search for novel therapeutic methods has led to the return of coaptation , the effectiveness of which has been confirmed by both experimental and clinical studies [ 1517 ] . collateral sprouting of the distal stump of a damaged nerve to an uninjured donor nerve is gaining popularity as a method for nerve regeneration . the process is induced by molecular changes in the microenvironment in which the nerve lesion occurred , sustained by wallerian degeneration , interruption of the normal neuronal turnover and local inflammatory response . . one of the most pressing unsolved questions is the origin of the regenerating axons after end - to - side neurorrhaphy . the window in the epineurium or the perineurium of the donor nerve improves the effectiveness of end - to - side nerve repair . however , creating an epineurial or perineural window is controversial due to the risk of donor nerve damage . the aim of the present study was to evaluate the histopathological and functional changes in the donor nerve from the c7 spinal nerve of the rabbit brachial plexus after a coaptation procedure . assessment was performed of avulsion of the ventral branches of the c5 and c6 spinal nerves treated by coaptation of these nerves to the c7 spinal nerve on the rabbit brachial plexus . due to the injury at the spinal nerve level , the short , wide trunk of the spinal nerves , and technical difficulties in performing neurorrhaphy in humans , end - to - side and side - to - side coaptations were evaluated . the experiments were approved by the second local animal ethics committee at the university of life sciences in wroclaw . the study was conducted on 22-week - old white new zealand rabbits ( oryctolagus cuniculus ) ( 22 females and 6 males ) that weighed an average of 33.5 kg . all surgical procedures were performed with a lateral approach to the left brachial plexus ( figure 1 ) . the ventral branches of spinal nerves c5 and c6 were exposed and incised at the spinal canal level . a 1-cm gap between the c5 and c6 spinal nerves and cranial trunk ( c5 , c6 ) was excised . an epineurial window on the cranial side of c7 was performed for end - to - side coaptation . for side - to - side coaptation , an additional epineurial window on the side of the cranial trunk was performed . the distal stump of the cranial trunk ( c5 and c6 ) was sutured to the window by two stitches ( ethilon 10 - 0 , ethicon ) in either the ets or sts group . twenty - eight right ( contralateral ) brachial plexuses were not treated ( controls ) . after 20 weeks , the left and right brachial plexuses were exposed to obtain the sampled nerves . premedication was performed with medetomidine ( cepetor ) at a dose of 150 g / kg body weight , butorphanol ( torbugesic ) at a dose of 0.2 mg / kg body weight and ketamine ( bioketan ) at a dose of 35 mg / kg body weight . general anesthesia was performed with propofol , which was continuously administered intravenously ( iv ) at a dose of 0.1 mg / kg / min . during the operation , the analgesic effect was supported by fentanyl at a dose of 23 mcg / kg . after the procedure , buprenorphine ( vetergesic ) was administered intramuscularly ( i m ) at a dose of 20 mcg / kg tid . the animals received meloxicam ( metacam ) at a dose of 0.2 mg / kg body weight for two days after surgery . adequate care was taken to minimize the pain and discomfort during and after the operation . all of the animals were anaesthetized with i m injections of medetomidine ( dose : 0.5 mg / kg ) , butorphanol ( 0.1 mg / kg ) and ketamine ( 25 mg / kg ) for the electromyographic evaluation . the electromyographic examination was performed at an ambient temperature of 22c using the nicolet viking quest portable system ( version 11.0 ) electrodiagnostic equipment . a standard electromyographic concentric needle electrode ( used as an active and recording electrode ) and a subdermal , the following muscles were analyzed to evaluate the nerve donor function : subscapularis , biceps brachii , teres major and triceps . the muscle innervation by nerves derived from the corresponding spinal nerves and segments of the spinal cord was assessed ( table 1 ) . higher values indicate a lower degree of muscle innervation ( table 2 , figure 2 ) . the proximal c7 trunk was compared with the region distal to the coapted area ( samples 1 and 2 ) , and the c7 trunk distal to the coapted area was compared with the contralateral healthy c7 nerve at the same level ( samples 2 and 3 ) and subjected to histomorphometric analysis ( figure 3 ) . the following parameters were analyzed : axon , myelin sheath and nerve fiber diameters ; g - ratio and nerve area ; number of axons ; and myelin fiber density ( mm ) . the obtained nerve specimens were immersion - fixed in 2.5% glutaraldehyde for 12 h at 4c and then washed with cacodylic buffer ( serva , heidelberg , germany ) . specimens were then post - fixed for 1 h in 1% osmium tetroxide ( dissolved in cacodylic buffer ) and subsequently washed with cacodylic buffer . finally , the specimens were dehydrated in alcohols and embedded in epon ( chempur , piekary slaskie , poland ) . power tome xl ( rmc products , tucson , az , usa ) was used to cut the fixed nerve specimens into 0.6-m thick sections , which were then stained with toluidine blue ( serva ) and mounted using euparal ( roth , mannheim , germany ) . the stained nerve cross - sections were examined under a bx41 light microscope that was equipped with the computer - assisted image analysis program cell ( olympus , tokyo , japan ) . for the analysis , non - overlapping photomicrographs at 630 magnification were taken manually . in each nerve cross - section , minimal diameters of the axons and nerve fiber ( axon and adjacent myelin ) were measured . only circular fibers were measured , allowing us to calculate the myelin sheath thickness of the nerve fibers using the following formula : nerve fiber diameter axon diameter . the degree of myelination was assessed using the g - ratio ( the ratio between the minimal diameters of axons and nerve fibers ) . the animal s skin was gently pinched by forceps until the first signs of discomfort to the animal , such as picking up the limbs , head turning or trembling skin , were observed . for this study , we prepared a 4-point grading scale : 0 no response ; 1 mild response , in which the animal exhibited a very weak reaction ; 2 moderate response , in which the animal exhibited a reaction in response to the stimuli ; and 3 a clear / significant response to the stimuli . the data were analyzed by prism 5.0 ( graphpad , la jolla , ca , usa ) statistical software . to compare the significance of the changes between the groups , student s t - test for independent samples was performed . the dissimilarities between the groups were scrutinized using the non - parametric kruskal - wallis test with dunn s post hoc analysis . the differences were considered significant at p<0.05 for student s t - test and at p<0.01 for the mann - whitney u test . the experiments were approved by the second local animal ethics committee at the university of life sciences in wroclaw . the study was conducted on 22-week - old white new zealand rabbits ( oryctolagus cuniculus ) ( 22 females and 6 males ) that weighed an average of 33.5 kg . all surgical procedures were performed with a lateral approach to the left brachial plexus ( figure 1 ) . the ventral branches of spinal nerves c5 and c6 were exposed and incised at the spinal canal level . a 1-cm gap between the c5 and c6 spinal nerves and cranial trunk ( c5 , c6 ) was excised . an epineurial window on the cranial side of c7 was performed for end - to - side coaptation . for side - to - side coaptation , an additional epineurial window on the side of the cranial trunk was performed . the distal stump of the cranial trunk ( c5 and c6 ) was sutured to the window by two stitches ( ethilon 10 - 0 , ethicon ) in either the ets or sts group . twenty - eight right ( contralateral ) brachial plexuses were not treated ( controls ) . after 20 weeks , the left and right brachial plexuses were exposed to obtain the sampled nerves . premedication was performed with medetomidine ( cepetor ) at a dose of 150 g / kg body weight , butorphanol ( torbugesic ) at a dose of 0.2 mg / kg body weight and ketamine ( bioketan ) at a dose of 35 mg / kg body weight . general anesthesia was performed with propofol , which was continuously administered intravenously ( iv ) at a dose of 0.1 mg / kg / min . during the operation , the analgesic effect was supported by fentanyl at a dose of 23 mcg / kg . after the procedure , buprenorphine ( vetergesic ) was administered intramuscularly ( i m ) at a dose of 20 mcg / kg tid . the animals received meloxicam ( metacam ) at a dose of 0.2 mg / kg body weight for two days after surgery . adequate care was taken to minimize the pain and discomfort during and after the operation . all of the animals were anaesthetized with i m injections of medetomidine ( dose : 0.5 mg / kg ) , butorphanol ( 0.1 mg / kg ) and ketamine ( 25 mg / kg ) for the electromyographic evaluation . the electromyographic examination was performed at an ambient temperature of 22c using the nicolet viking quest portable system ( version 11.0 ) electrodiagnostic equipment . a standard electromyographic concentric needle electrode ( used as an active and recording electrode ) and a subdermal , the following muscles were analyzed to evaluate the nerve donor function : subscapularis , biceps brachii , teres major and triceps . the muscle innervation by nerves derived from the corresponding spinal nerves and segments of the spinal cord was assessed ( table 1 ) . higher values indicate a lower degree of muscle innervation ( table 2 , figure 2 ) . the proximal c7 trunk was compared with the region distal to the coapted area ( samples 1 and 2 ) , and the c7 trunk distal to the coapted area was compared with the contralateral healthy c7 nerve at the same level ( samples 2 and 3 ) and subjected to histomorphometric analysis ( figure 3 ) . the following parameters were analyzed : axon , myelin sheath and nerve fiber diameters ; g - ratio and nerve area ; number of axons ; and myelin fiber density ( mm ) . the obtained nerve specimens were immersion - fixed in 2.5% glutaraldehyde for 12 h at 4c and then washed with cacodylic buffer ( serva , heidelberg , germany ) . specimens were then post - fixed for 1 h in 1% osmium tetroxide ( dissolved in cacodylic buffer ) and subsequently washed with cacodylic buffer . finally , the specimens were dehydrated in alcohols and embedded in epon ( chempur , piekary slaskie , poland ) . power tome xl ( rmc products , tucson , az , usa ) was used to cut the fixed nerve specimens into 0.6-m thick sections , which were then stained with toluidine blue ( serva ) and mounted using euparal ( roth , mannheim , germany ) . the stained nerve cross - sections were examined under a bx41 light microscope that was equipped with the computer - assisted image analysis program cell ( olympus , tokyo , japan ) . for the analysis , non - overlapping photomicrographs at 630 magnification were taken manually . in each nerve cross - section , minimal diameters of the axons and nerve fiber ( axon and adjacent myelin ) were measured . only circular fibers were measured , allowing us to calculate the myelin sheath thickness of the nerve fibers using the following formula : nerve fiber diameter axon diameter . the degree of myelination was assessed using the g - ratio ( the ratio between the minimal diameters of axons and nerve fibers ) . the animal s skin was gently pinched by forceps until the first signs of discomfort to the animal , such as picking up the limbs , head turning or trembling skin , were observed . for this study , we prepared a 4-point grading scale : 0 no response ; 1 mild response , in which the animal exhibited a very weak reaction ; 2 moderate response , in which the animal exhibited a reaction in response to the stimuli ; and 3 a clear / significant response to the stimuli . the data were analyzed by prism 5.0 ( graphpad , la jolla , ca , usa ) statistical software . to compare the significance of the changes between the groups , student s t - test for independent samples was performed . the dissimilarities between the groups were scrutinized using the non - parametric kruskal - wallis test with dunn s post hoc analysis . the differences were considered significant at p<0.05 for student s t - test and at p<0.01 for the mann - whitney u test . bar graphs present the results of the histomorphometric analyses of the sampled nerves ( axon , myelin sheath and nerve fiber diameters and the g - ratio ) ( figure 4 ) . in the control group ( sample 3 ) , the nerve area was 1.1304 mm , the number of axons was 3622.83 , and the myelin fiber density was 3078.2396.17 fiber / mm for c7 . in the ets group ( sample 2 ) , distal from the coaptation , the nerve area was 1.1267 mm , the number of axons was 3223.15 , and the myelin fiber density was 2977.2379.17 fiber / mm . in the sts group ( sample 2 ) , distal from the coaptation , the nerve area was 1.1255 mm , the number of axons was 3124.16 , and the myelin fiber density was 2864.8489.21 fiber / mm . a comparison of the tissue before coaptation ( proximal ) ( figure 5a ) and 2 cm from the coaptation ( distal ) ( figure 5b ) revealed statistically significant differences in the axon ( mw<0.0001 , p<0.01 ) , myelin sheath ( mw<0.0001 , p<0.01 ) and fiber ( mw<0.0001 , p<0.01 ) diameters in both the ets and sts groups . conversely , comparing the g - ratio for the ets proximal with the ets distal group did not indicate any prominent differences ( mw=0.78 , p<0.01 ) , whereas the sts proximal group significantly differed from the sts distal group ( mw=0.0001 , p<0.01 ) . a comparison of the sampled nerves 2 cm from the coaptation in the ets and sts groups with the control group ( figure 5c ) revealed significant differences in the following parameters : axon ( mw<0.0001 , p<0.01 ) , myelin sheath ( mw<0.0001 , p<0.05 ) and fiber diameters ( mw<0.0001 , p<0.01 ) . the g - ratio did not differ significantly between the ets group and the controls ( mw=0.1817 , p<0.01 ) , but statistically significant differences in the g - ratio were observed between the sts group and the controls ( mw=0.0064 , p<0.01 ) . the ets and sts groups significantly differed from the controls with respect to the following muscles : subscapularis ( t<0.001 , p<0.05 ) , biceps ( t<0.001 , p<0.05 ) and teres major ( t=0.005 , p<0.05 ) . however , there were no differences in the triceps ( in the ets group , we did not observe any changes ) . on the lateral side , the sensory function recovery was the fastest and was associated with innervation from c8 and t1 ( table 4 ) . bar graphs present the results of the histomorphometric analyses of the sampled nerves ( axon , myelin sheath and nerve fiber diameters and the g - ratio ) ( figure 4 ) . in the control group ( sample 3 ) , the nerve area was 1.1304 mm , the number of axons was 3622.83 , and the myelin fiber density was 3078.2396.17 fiber / mm for c7 . in the ets group ( sample 2 ) , distal from the coaptation , the nerve area was 1.1267 mm , the number of axons was 3223.15 , and the myelin fiber density was 2977.2379.17 fiber / mm . in the sts group ( sample 2 ) , distal from the coaptation , the nerve area was 1.1255 mm , the number of axons was 3124.16 , and the myelin fiber density was 2864.8489.21 fiber / mm . a comparison of the tissue before coaptation ( proximal ) ( figure 5a ) and 2 cm from the coaptation ( distal ) ( figure 5b ) revealed statistically significant differences in the axon ( mw<0.0001 , p<0.01 ) , myelin sheath ( mw<0.0001 , p<0.01 ) and fiber ( mw<0.0001 , p<0.01 ) diameters in both the ets and sts groups . conversely , comparing the g - ratio for the ets proximal with the ets distal group did not indicate any prominent differences ( mw=0.78 , p<0.01 ) , whereas the sts proximal group significantly differed from the sts distal group ( mw=0.0001 , p<0.01 ) . a comparison of the sampled nerves 2 cm from the coaptation in the ets and sts groups with the control group ( figure 5c ) revealed significant differences in the following parameters : axon ( mw<0.0001 , p<0.01 ) , myelin sheath ( mw<0.0001 , p<0.05 ) and fiber diameters ( mw<0.0001 , p<0.01 ) . the g - ratio did not differ significantly between the ets group and the controls ( mw=0.1817 , p<0.01 ) , but statistically significant differences in the g - ratio were observed between the sts group and the controls ( mw=0.0064 , p<0.01 ) . the ets and sts groups significantly differed from the controls with respect to the following muscles : subscapularis ( t<0.001 , p<0.05 ) , biceps ( t<0.001 , p<0.05 ) and teres major ( t=0.005 , p<0.05 ) . however , there were no differences in the triceps ( in the ets group , we did not observe any changes ) . on the lateral side , the sensory function recovery was the fastest and was associated with innervation from c8 and t1 ( table 4 ) . previous studies have evaluated donor function using coaptation on the level of individual peripheral nerves . in our study the risk of damage to the donor is related to collateral sprouting from c7 in response to avulsion of the ventral branches of the c5 and c6 spinal nerves treated by coaptation of these nerves to the c7 spinal nerve . collateral sprouting in end - to - side coaptation of the ventral branches of the c5 and c6 spinal nerves to the c7 spinal nerve in the rabbit brachial plexus was confirmed by electrophysiological , histomorphometric and behavioral results . to objectively analyze these results , all of the branches connecting the c5 , c6 , and c7 nerves below the site of coaptation were cut . electro - myography studies revealed denervation elements , such as fibrillations , positive sharp waves and repetitive complex discharges . the electrodiagnostic study revealed that the largest lesions were found in the biceps brachii muscle , which is innervated by the musculocutaneous nerve , whose fibers are derived from the c7 and c6 cervical nerves . no lesions were noted in the triceps muscle innervated by the radial nerve , which originates from the c7 , c8 , and t1 spinal nerves . no muscles are innervated solely by the c7 spinal nerve ; nevertheless , we made an assumption that the biceps brachii muscle and radial nerve are sufficient for c7 evaluation ( despite c5 and c6 damage ) . our research results indicate that sensory function returns 2 weeks after surgery and is fully restored within 3 weeks . no significant deficiency in sensory function was noted . in a study of damaged rat sural nerves , sensory function returned completely by 36 weeks after the end of the study . in our studies , the results indicate that the sensory function of the donor is impaired for a brief period . the skin pinch test is not an adequate tool to assess subtle changes , and damaged senses may have been overlooked in this study . with respect to the histomorphometric examination , the differences were statistically significant and did not translate into nerve conduction . we also noted differences between the g - ratio for side - to - side and end - to - side coaptation compared with the control group , although the differences were statistically significant only for the sts group ; overall , the differences in the g - ratio were less prominent than the other histomorphometric parameters . a g - ratio greater than 0.6 for the peripheral nerve indicates normal nerve conduction . the histological examination of other parameters changed according to the construction of the nerve and was dependent on the level of sample collection . in a study of reconstruction of the peroneal nerve , we examined the number of axons relative to the cross - section of the nerve donor . the results for the peroneal nerve were 0.2550.l11 mm for the cross - section , with an average of 33631997 axons . based on these results , the nerve area was 1.1304 mm and the number of axons was 3622.83 , which is sufficient to obtain good sensory results . donor nerve lesions are believed to be transient and to regress with time . functional recovery after peripheral nerve injury depends on the survival of the affected neurons and their capacity to regenerate the injured axons , as well as reinnervation of target tissues . the microenvironment in the nerve segment distal to the injury site , undergoing metabolic and structural changes , was a decisive factor in the regeneration of injured axons and functional reinnervation of the denervated target tissues . sensory exams have been used to classify injury severity . after an ets procedure in rats ( tibial nerve to peroneal nerve ) , signs of nerve damage were observed within two weeks . a five - fold increase in the percentage of denervated muscle fibers was noted relative to the control group , but the properties of the operated and healthy contralateral limb were comparable after six months . lundborg demonstrated wallerian degeneration in the donor nerve after a peroneal - to - posterior tibial coaptation procedure in rats 90 days after surgery . this process did not influence the limb function . in the medical literature , there are reports referring to lesions in the sensory and motor peripheral nerves , but there are few studies defining the effect of collateral sprouting on the donor nerve for mixed nerves . kovaci noted that motor neurons are more easily damaged than sensory neurons , which is likely due to their higher sensitivity . our study confirms this theory ; the returned sensory function was greater than the motor function . currently , the most widely used treatment for c5 or c6 avulsion is neurotization of the accessory nerve to the suprascapular nerve , the ulnar nerve fascicles to the motor branch biceps , and triceps branches to the axillary nerve . in this case , the long - term triceps strength is not diminished relative to the preoperative values after sectioning of the triceps motor branches ; however , strength equal to the normal contralateral side is not achieved during recovery . additionally , there are no apparent consequences of removing fascicles of the ulnar nerve with respect to grasp or pinch strength . in the case of cc7 donor site complications , transection of the posterior division of cc7 produces a weakness of muscles innervated by the radial nerve , especially the triceps and the wrist and finger extensors , although paralysis is not observed . ipsilateral c7 nerve root transfer may cause injury to the thoracodorsal nerve , which receives motor fascicles from the cervical nerves largely from c7 . the result of damage to the thoracodorsal nerve is weakness or paralysis of the latissimus dorsi muscle , which it innervates . the above examples describe the results of the contribution of the c7 fasciculus , which can represent a good donor but can cause some damage to the nerves . we believe the coaptation procedure may avoid nerve damage and that collateral sprouting of c7 can restore function . the sensory component of the c7 nerve root contributes less than 25% to the innervation of any given major upper extremity nerve : less than 1% , 6% , 16% and 19% to the musculocutaneous , ulnar , radial , and median nerves , respectively . when using the c7 nerve root as a donor , the central sensory deficit will be less than 25% . this is entirely within the ability of each major nerve to compensate and is likely a reason why c7 is a suitable donor for brachial plexus injury another important aspect of the procedure is using an appropriate portion of the c7 from the donor , as the requirements depend on the type of injury . for example , musculocutaneous nerve regeneration in neurotization with the posterior division or the anterior division of c7 is significantly superior to that with the anterolateral fascicles of the anterior division or the phrenic nerve . this study demonstrates that nerve topography is very important and may depend on the final outcome . therefore , coaptation with various individual segments requires further evaluation . donor assessment after coaptation treatment at the level of the spinal nerves is particularly difficult due to the absence of selective innervation of muscles and dermatomes for each spinal nerve . the only study directly evaluating nerve changes included histological examination , but in some cases , the structural changes do not necessarily translate into nerve conduction function . therefore , in our opinion , only the cumulative data from histology , electromyographic examination and sensory recovery allow the appropriate conclusions to be drawn . because the results of treating peripheral nerve injuries remain unsatisfactory , a more selective approach with respect to the spinal nerves , spinal trunks , or the peripheral nerves , as well as to the location of the injury and the method of repair and reconstruction , a possible solution is coaptation , but the consequences of using spinal nerves as donors are unknown . we believe that confirming the absence of disrupted nerve conduction at the level of the spinal nerves will facilitate further research of coaptation defects at these spinal nerve levels . we hope that this technique combined with neurotrophic procedures will provide an alternative to the previously used brachial plexus surgical techniques . however , we are aware that the primary issue is the translation of laboratory results into clinical practice . the coaptation procedure affects the nerve donor s histological structure , but this impact does not translate into detrimental effects at the level of nerve conduction or sensory function of the limb .

backgroundthe aim of the present study was to evaluate the donor nerve from the c7 spinal nerve of the rabbit brachial plexus after a coaptation procedure . assessment was performed of avulsion of the c5 and c6 spinal nerves treated by coaptation of these nerves to the c7 spinal nerve.material/methodsafter nerve injury , fourteen rabbits were treated by end - to - side coaptation ( ets ) , and fourteen animals were treated by side - to - side coaptation ( sts ) on the right brachial plexus . electrophysiological and histomorphometric analyses and the skin pinch test were used to evaluate the outcomes.resultsthere was no statistically significant difference in the g - ratio proximal and distal to the coaptation in the ets group , but the differences in the axon , myelin sheath and fiber diameters were statistically significant . the comparison of the ets and sts groups distal to the coaptation with the controls demonstrated statistically significant differences in the fiber , axon , and myelin sheath diameters . with respect to the g - ratio , the ets group exhibited no significant differences relative to the control , whereas the g - ratio in the sts group and the controls differed significantly . in the electrophysiological study , the ets and sts groups exhibited major changes in the biceps and subscapularis muscles.conclusionsthe coaptation procedure affects the histological structure of the nerve donor , but it does not translate into changes in nerve conduction or the sensory function of the limb . the donor nerve lesion in the ets group is transient and has minimal clinical relevance .

Background Material and Methods Experimental model Electrophysiological analysis Histomorphometric analysis of the sampled nerves The skin pinch test Statistical analysis Results Histomorphometric analyses of the sampled nerves Electrophysiological study Skin pinch test Discussion Conclusions

a wide variety of potential donor nerves are available , including the intercostal nerves , the spinal accessory nerve , the phrenic nerve , partial ulnar nerve , partial median nerve , pectoral major , thoracodorsal nerve , and the partial ipsilateral , the hemi or total cc7 nerve roots . the search for novel therapeutic methods has led to the return of coaptation , the effectiveness of which has been confirmed by both experimental and clinical studies [ 1517 ] . collateral sprouting of the distal stump of a damaged nerve to an uninjured donor nerve is gaining popularity as a method for nerve regeneration . the process is induced by molecular changes in the microenvironment in which the nerve lesion occurred , sustained by wallerian degeneration , interruption of the normal neuronal turnover and local inflammatory response . one of the most pressing unsolved questions is the origin of the regenerating axons after end - to - side neurorrhaphy . the window in the epineurium or the perineurium of the donor nerve improves the effectiveness of end - to - side nerve repair . however , creating an epineurial or perineural window is controversial due to the risk of donor nerve damage . the aim of the present study was to evaluate the histopathological and functional changes in the donor nerve from the c7 spinal nerve of the rabbit brachial plexus after a coaptation procedure . assessment was performed of avulsion of the ventral branches of the c5 and c6 spinal nerves treated by coaptation of these nerves to the c7 spinal nerve on the rabbit brachial plexus . due to the injury at the spinal nerve level , the short , wide trunk of the spinal nerves , and technical difficulties in performing neurorrhaphy in humans , end - to - side and side - to - side coaptations were evaluated . the ventral branches of spinal nerves c5 and c6 were exposed and incised at the spinal canal level . a 1-cm gap between the c5 and c6 spinal nerves and cranial trunk ( c5 , c6 ) was excised . an epineurial window on the cranial side of c7 was performed for end - to - side coaptation . for side - to - side coaptation , an additional epineurial window on the side of the cranial trunk was performed . the distal stump of the cranial trunk ( c5 and c6 ) was sutured to the window by two stitches ( ethilon 10 - 0 , ethicon ) in either the ets or sts group . after 20 weeks , the left and right brachial plexuses were exposed to obtain the sampled nerves . all of the animals were anaesthetized with i m injections of medetomidine ( dose : 0.5 mg / kg ) , butorphanol ( 0.1 mg / kg ) and ketamine ( 25 mg / kg ) for the electromyographic evaluation . a standard electromyographic concentric needle electrode ( used as an active and recording electrode ) and a subdermal , the following muscles were analyzed to evaluate the nerve donor function : subscapularis , biceps brachii , teres major and triceps . the muscle innervation by nerves derived from the corresponding spinal nerves and segments of the spinal cord was assessed ( table 1 ) . the proximal c7 trunk was compared with the region distal to the coapted area ( samples 1 and 2 ) , and the c7 trunk distal to the coapted area was compared with the contralateral healthy c7 nerve at the same level ( samples 2 and 3 ) and subjected to histomorphometric analysis ( figure 3 ) . the following parameters were analyzed : axon , myelin sheath and nerve fiber diameters ; g - ratio and nerve area ; number of axons ; and myelin fiber density ( mm ) . only circular fibers were measured , allowing us to calculate the myelin sheath thickness of the nerve fibers using the following formula : nerve fiber diameter axon diameter . the degree of myelination was assessed using the g - ratio ( the ratio between the minimal diameters of axons and nerve fibers ) . all surgical procedures were performed with a lateral approach to the left brachial plexus ( figure 1 ) . the ventral branches of spinal nerves c5 and c6 were exposed and incised at the spinal canal level . a 1-cm gap between the c5 and c6 spinal nerves and cranial trunk ( c5 , c6 ) was excised . an epineurial window on the cranial side of c7 was performed for end - to - side coaptation . for side - to - side coaptation , an additional epineurial window on the side of the cranial trunk was performed . the distal stump of the cranial trunk ( c5 and c6 ) was sutured to the window by two stitches ( ethilon 10 - 0 , ethicon ) in either the ets or sts group . after 20 weeks , the left and right brachial plexuses were exposed to obtain the sampled nerves . all of the animals were anaesthetized with i m injections of medetomidine ( dose : 0.5 mg / kg ) , butorphanol ( 0.1 mg / kg ) and ketamine ( 25 mg / kg ) for the electromyographic evaluation . a standard electromyographic concentric needle electrode ( used as an active and recording electrode ) and a subdermal , the following muscles were analyzed to evaluate the nerve donor function : subscapularis , biceps brachii , teres major and triceps . the muscle innervation by nerves derived from the corresponding spinal nerves and segments of the spinal cord was assessed ( table 1 ) . the proximal c7 trunk was compared with the region distal to the coapted area ( samples 1 and 2 ) , and the c7 trunk distal to the coapted area was compared with the contralateral healthy c7 nerve at the same level ( samples 2 and 3 ) and subjected to histomorphometric analysis ( figure 3 ) . the following parameters were analyzed : axon , myelin sheath and nerve fiber diameters ; g - ratio and nerve area ; number of axons ; and myelin fiber density ( mm ) . only circular fibers were measured , allowing us to calculate the myelin sheath thickness of the nerve fibers using the following formula : nerve fiber diameter axon diameter . the degree of myelination was assessed using the g - ratio ( the ratio between the minimal diameters of axons and nerve fibers ) . for this study , we prepared a 4-point grading scale : 0 no response ; 1 mild response , in which the animal exhibited a very weak reaction ; 2 moderate response , in which the animal exhibited a reaction in response to the stimuli ; and 3 a clear / significant response to the stimuli . bar graphs present the results of the histomorphometric analyses of the sampled nerves ( axon , myelin sheath and nerve fiber diameters and the g - ratio ) ( figure 4 ) . in the control group ( sample 3 ) , the nerve area was 1.1304 mm , the number of axons was 3622.83 , and the myelin fiber density was 3078.2396.17 fiber / mm for c7 . in the ets group ( sample 2 ) , distal from the coaptation , the nerve area was 1.1267 mm , the number of axons was 3223.15 , and the myelin fiber density was 2977.2379.17 fiber / mm . in the sts group ( sample 2 ) , distal from the coaptation , the nerve area was 1.1255 mm , the number of axons was 3124.16 , and the myelin fiber density was 2864.8489.21 fiber / mm . a comparison of the tissue before coaptation ( proximal ) ( figure 5a ) and 2 cm from the coaptation ( distal ) ( figure 5b ) revealed statistically significant differences in the axon ( mw<0.0001 , p<0.01 ) , myelin sheath ( mw<0.0001 , p<0.01 ) and fiber ( mw<0.0001 , p<0.01 ) diameters in both the ets and sts groups . conversely , comparing the g - ratio for the ets proximal with the ets distal group did not indicate any prominent differences ( mw=0.78 , p<0.01 ) , whereas the sts proximal group significantly differed from the sts distal group ( mw=0.0001 , p<0.01 ) . a comparison of the sampled nerves 2 cm from the coaptation in the ets and sts groups with the control group ( figure 5c ) revealed significant differences in the following parameters : axon ( mw<0.0001 , p<0.01 ) , myelin sheath ( mw<0.0001 , p<0.05 ) and fiber diameters ( mw<0.0001 , p<0.01 ) . the g - ratio did not differ significantly between the ets group and the controls ( mw=0.1817 , p<0.01 ) , but statistically significant differences in the g - ratio were observed between the sts group and the controls ( mw=0.0064 , p<0.01 ) . the ets and sts groups significantly differed from the controls with respect to the following muscles : subscapularis ( t<0.001 , p<0.05 ) , biceps ( t<0.001 , p<0.05 ) and teres major ( t=0.005 , p<0.05 ) . however , there were no differences in the triceps ( in the ets group , we did not observe any changes ) . on the lateral side , the sensory function recovery was the fastest and was associated with innervation from c8 and t1 ( table 4 ) . bar graphs present the results of the histomorphometric analyses of the sampled nerves ( axon , myelin sheath and nerve fiber diameters and the g - ratio ) ( figure 4 ) . in the control group ( sample 3 ) , the nerve area was 1.1304 mm , the number of axons was 3622.83 , and the myelin fiber density was 3078.2396.17 fiber / mm for c7 . in the ets group ( sample 2 ) , distal from the coaptation , the nerve area was 1.1267 mm , the number of axons was 3223.15 , and the myelin fiber density was 2977.2379.17 fiber / mm . in the sts group ( sample 2 ) , distal from the coaptation , the nerve area was 1.1255 mm , the number of axons was 3124.16 , and the myelin fiber density was 2864.8489.21 fiber / mm . a comparison of the tissue before coaptation ( proximal ) ( figure 5a ) and 2 cm from the coaptation ( distal ) ( figure 5b ) revealed statistically significant differences in the axon ( mw<0.0001 , p<0.01 ) , myelin sheath ( mw<0.0001 , p<0.01 ) and fiber ( mw<0.0001 , p<0.01 ) diameters in both the ets and sts groups . conversely , comparing the g - ratio for the ets proximal with the ets distal group did not indicate any prominent differences ( mw=0.78 , p<0.01 ) , whereas the sts proximal group significantly differed from the sts distal group ( mw=0.0001 , p<0.01 ) . a comparison of the sampled nerves 2 cm from the coaptation in the ets and sts groups with the control group ( figure 5c ) revealed significant differences in the following parameters : axon ( mw<0.0001 , p<0.01 ) , myelin sheath ( mw<0.0001 , p<0.05 ) and fiber diameters ( mw<0.0001 , p<0.01 ) . the g - ratio did not differ significantly between the ets group and the controls ( mw=0.1817 , p<0.01 ) , but statistically significant differences in the g - ratio were observed between the sts group and the controls ( mw=0.0064 , p<0.01 ) . the ets and sts groups significantly differed from the controls with respect to the following muscles : subscapularis ( t<0.001 , p<0.05 ) , biceps ( t<0.001 , p<0.05 ) and teres major ( t=0.005 , p<0.05 ) . however , there were no differences in the triceps ( in the ets group , we did not observe any changes ) . on the lateral side , the sensory function recovery was the fastest and was associated with innervation from c8 and t1 ( table 4 ) . in our study the risk of damage to the donor is related to collateral sprouting from c7 in response to avulsion of the ventral branches of the c5 and c6 spinal nerves treated by coaptation of these nerves to the c7 spinal nerve . collateral sprouting in end - to - side coaptation of the ventral branches of the c5 and c6 spinal nerves to the c7 spinal nerve in the rabbit brachial plexus was confirmed by electrophysiological , histomorphometric and behavioral results . to objectively analyze these results , all of the branches connecting the c5 , c6 , and c7 nerves below the site of coaptation were cut . the electrodiagnostic study revealed that the largest lesions were found in the biceps brachii muscle , which is innervated by the musculocutaneous nerve , whose fibers are derived from the c7 and c6 cervical nerves . no lesions were noted in the triceps muscle innervated by the radial nerve , which originates from the c7 , c8 , and t1 spinal nerves . no muscles are innervated solely by the c7 spinal nerve ; nevertheless , we made an assumption that the biceps brachii muscle and radial nerve are sufficient for c7 evaluation ( despite c5 and c6 damage ) . no significant deficiency in sensory function was noted . in our studies , the results indicate that the sensory function of the donor is impaired for a brief period . the skin pinch test is not an adequate tool to assess subtle changes , and damaged senses may have been overlooked in this study . with respect to the histomorphometric examination , the differences were statistically significant and did not translate into nerve conduction . we also noted differences between the g - ratio for side - to - side and end - to - side coaptation compared with the control group , although the differences were statistically significant only for the sts group ; overall , the differences in the g - ratio were less prominent than the other histomorphometric parameters . a g - ratio greater than 0.6 for the peripheral nerve indicates normal nerve conduction . the histological examination of other parameters changed according to the construction of the nerve and was dependent on the level of sample collection . in a study of reconstruction of the peroneal nerve , we examined the number of axons relative to the cross - section of the nerve donor . based on these results , the nerve area was 1.1304 mm and the number of axons was 3622.83 , which is sufficient to obtain good sensory results . donor nerve lesions are believed to be transient and to regress with time . functional recovery after peripheral nerve injury depends on the survival of the affected neurons and their capacity to regenerate the injured axons , as well as reinnervation of target tissues . the microenvironment in the nerve segment distal to the injury site , undergoing metabolic and structural changes , was a decisive factor in the regeneration of injured axons and functional reinnervation of the denervated target tissues . a five - fold increase in the percentage of denervated muscle fibers was noted relative to the control group , but the properties of the operated and healthy contralateral limb were comparable after six months . lundborg demonstrated wallerian degeneration in the donor nerve after a peroneal - to - posterior tibial coaptation procedure in rats 90 days after surgery . in the medical literature , there are reports referring to lesions in the sensory and motor peripheral nerves , but there are few studies defining the effect of collateral sprouting on the donor nerve for mixed nerves . currently , the most widely used treatment for c5 or c6 avulsion is neurotization of the accessory nerve to the suprascapular nerve , the ulnar nerve fascicles to the motor branch biceps , and triceps branches to the axillary nerve . in this case , the long - term triceps strength is not diminished relative to the preoperative values after sectioning of the triceps motor branches ; however , strength equal to the normal contralateral side is not achieved during recovery . additionally , there are no apparent consequences of removing fascicles of the ulnar nerve with respect to grasp or pinch strength . in the case of cc7 donor site complications , transection of the posterior division of cc7 produces a weakness of muscles innervated by the radial nerve , especially the triceps and the wrist and finger extensors , although paralysis is not observed . the above examples describe the results of the contribution of the c7 fasciculus , which can represent a good donor but can cause some damage to the nerves . the sensory component of the c7 nerve root contributes less than 25% to the innervation of any given major upper extremity nerve : less than 1% , 6% , 16% and 19% to the musculocutaneous , ulnar , radial , and median nerves , respectively . when using the c7 nerve root as a donor , the central sensory deficit will be less than 25% . this is entirely within the ability of each major nerve to compensate and is likely a reason why c7 is a suitable donor for brachial plexus injury another important aspect of the procedure is using an appropriate portion of the c7 from the donor , as the requirements depend on the type of injury . for example , musculocutaneous nerve regeneration in neurotization with the posterior division or the anterior division of c7 is significantly superior to that with the anterolateral fascicles of the anterior division or the phrenic nerve . donor assessment after coaptation treatment at the level of the spinal nerves is particularly difficult due to the absence of selective innervation of muscles and dermatomes for each spinal nerve . the only study directly evaluating nerve changes included histological examination , but in some cases , the structural changes do not necessarily translate into nerve conduction function . because the results of treating peripheral nerve injuries remain unsatisfactory , a more selective approach with respect to the spinal nerves , spinal trunks , or the peripheral nerves , as well as to the location of the injury and the method of repair and reconstruction , a possible solution is coaptation , but the consequences of using spinal nerves as donors are unknown . we believe that confirming the absence of disrupted nerve conduction at the level of the spinal nerves will facilitate further research of coaptation defects at these spinal nerve levels . the coaptation procedure affects the nerve donor s histological structure , but this impact does not translate into detrimental effects at the level of nerve conduction or sensory function of the limb .

slow waves and sleep spindles are the essential elements in the definition of sleep stages . they recently received additional attention because of their hypothesized involvement in processes related to neuronal plasticity , learning , and memory [ 24 ] . furthermore , slow oscillations ( < 1 hz ) are considered as a central feature of sleep . these intracortically generated fluctuations consist at the cellular level of rhythmic depolarizing components ( up states ) separated by prolonged hyperpolarizations ( down states ) . there is evidence that they are involved in the temporal organization of other sleep rhythms such as spindles and delta waves ( for a review , see ) . also , it is hypothesized that the slow oscillation is involved in processes of learning and memory during sleep . tononi and cirelli proposed that slow oscillations were an indication of synaptic downscaling in the cortex occurring during nonrapid eye movement sleep in order to counteract synaptic potentiation resulting from learning processes occurring during wakefulness [ 6 , 7 ] . evidence for this hypothesis comes from data of a motor learning task , arm immobilization , animal studies , and computational modeling . we shall address in this paper only one particular aspect in this context , which is , however , related to the interplay of sleep spindles and slow oscillations : i.e. , the temporal organization of spindles . it was postulated that these thalamically generated sleep spindles are grouped by cortically generated slow oscillations and more generally that the slow oscillations also organize the occurrence of other sleep oscillations . this hypothesis is based on experimental and theoretical evidence : sleep spindles lost their coherence and their temporal however , there are also inconsistencies in relation to this hypothesis : first , the frequency of the slow oscillations was originally considered as being only slightly < 1 hz . achermann and borbly , for instance , reported a peak in the power spectrum at 0.7 hz . this would correspond to a shorter typical interevent interval between sleep spindles than 4 s. thus , only approximately every third slow oscillation would trigger a spindle . second , the rhythmic occurrence of spindles is most pronounced in sleep stage 2 ( see fig . 4 ) , but slow oscillations are most prominent in deep sleep . thus , one should also take alternative explanations into account . in the present paper , we investigated whether the periodicity in the occurrence of sleep spindles and other sleep oscillations might be a pure stochastic effect that occurs without assuming an additional slow process . to test this hypothesis , we compared interevent interval distributions of the events detected in the human sleep eeg with events detected in artificial data generated with stationary stochastic processes , which resemble to some extent the short - term properties of the eeg signal . the analysis is based on a recently proposed method to detect and analyze oscillatory events in the human sleep eeg . eeg data were modeled on short time scales ( 1s ) by linear autoregressive ( ar ) models in a sliding window fashion . systems described by such linear models can be interpreted as a set of coupled stochastically driven harmonic oscillators with time - dependent frequencies and damping coefficients . in the case of the human sleep eeg , this frequency might represent resonances of the underlying thalamocortical networks . in , it was shown that the modulation of the frequency and the damping constant of the resonance of a simple excitatory inhibitory network can be detected from the mean activity of the network by applying this method . we analyzed 8-h sleep eeg data ( single channel , derivation c3a2 ) of eight healthy young male subjects ( mean age 24.1 0.6 years ) , each contributing four baseline nights [ 17 , 19 ] . the sampling rate was 128 hz and sleep stages were visually scored according to standard criteria . oscillatory events were detected by fitting ar models of order 8 to subsegments of 1 s in length in a sliding window fashion resulting in a time dependence of the parameters of the ar model . the signal generated by an ar(p)-model can be interpreted as the superposition of the output of n oscillators and p 2n relaxators . the p parameters of the ar(p)-model are transformed into n frequencies and damping constants of the oscillators , and p 2n damping constants of the purely decaying nonoscillating modes . thus , time - dependent frequencies and damping constants result from the sliding window approach . figure 1 shows a 20-s segment of eeg ( stage 2 ) and the time - dependent modules r and the corresponding frequencies \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$f\:$\end{document } of the poles . the damping constant is a function of r , = fs logr with fs denoting the sampling frequency . the case r = 1 corresponds to the limiting case of an undamped oscillation . the threshold for the event detection was set to r1 = 0.95 ( fig . = 0.9 was applied : in order to account for statistical fluctuations , the event was not necessarily terminated whenever r became smaller than r1 . it was considered as a single event if the corresponding r became larger than r1 again without falling below r2 . the value r1 was chosen such that clearly visible sleep spindles were reliably detected by the algorithm , while r2 had to be low enough so that a single spindle was detected as a single event , but high enough so that subsequent spindles were recognized as distinct events . the time between the first and the final crossing of the threshold r1 plus 1 s ( the length of the segment used to fit the ar model ) was considered as the duration of the oscillatory event . the time when r was maximal was defined as the time of occurrence of the event and the corresponding frequency the interevent intervals were estimated as the difference of the time of occurrence of successive oscillatory events . 1detection of oscillatory events in a 20-s eeg segment of stage 2 non - rem sleep . the upper panel shows the eeg signal , the middle panel shows r , and the lower panel the corresponding frequencies f , with the colors indicating corresponding oscillatory modes . the detection threshold r1 = 0.95 is indicated in the middle panel ( see section 2.2 ) detection of oscillatory events in a 20-s eeg segment of stage 2 non - rem sleep . the upper panel shows the eeg signal , the middle panel shows r , and the lower panel the corresponding frequencies f , with the colors indicating corresponding oscillatory modes . the detection threshold r1 = 0.95 is indicated in the middle panel ( see section 2.2 ) for the analysis of the interevent intervals and the event durations , we assigned the events into different frequency bands . we are aware that this is not an optimal approach because of interindividual variation in mean event frequencies and their dependence on sleep stage and cycle . however , at the moment we have no method available to analyze the events on the basis of individually determined time - dependent frequency bands . therefore , we used the frequency bands employed previously , i.e. , delta ( : 0 f < 4.5 hz ) , alpha ( : 8 f < 11.5 hz ) , and sigma ( : 11.5 f < 16 hz ; sleep spindles ) bands . figure 2 illustrates the detected events of a single night and the corresponding histogram of the event frequencies . 2detected oscillatory events in a single night ( black dots represent single events ) , hypnogram ( mt , movement time ; w , waking , rem , rapid eye movement sleep ; 1 to 4 , nonrapid eye movement sleep stages 1 to 4 ) , and corresponding histogram of the event frequencies . colors indicate the different frequency bands detected oscillatory events in a single night ( black dots represent single events ) , hypnogram ( mt , movement time ; w , waking , rem , rapid eye movement sleep ; 1 to 4 , nonrapid eye movement sleep stages 1 to 4 ) , and corresponding histogram of the event frequencies . colors indicate the different frequency bands artificial eeg data were generated using an ar(4 ) model with random inputs and parameters corresponding to a frequency in the spindle frequency band ( f = 14 hz ) and in the delta band ( f = 3 hz ) , assuming a sampling frequency of 128 hz . we restricted the analysis to data from an ar(4 ) model because the two higher frequencies that can be seen in fig . we analyzed data sets with different values of r for the spindle frequency because the spindle oscillations are our main focus . for each set of parameter values , a time series of 6 10 data points ( corresponding to approximately 13 h of eeg data ) was generated . 3twenty - second simulated ar(4 ) data with time - dependent modules and frequencies derived by fitting an ar(4 ) model similar to fig . 1 . the upper panel shows the signal , the middle panel shows r , and the lower panel the corresponding frequencies f , with the colors indicating corresponding oscillatory modes . the ar(4 ) model contained two oscillatory modes , one in the spindle frequency band , f = 14 hz , r = 0.94 , and one in the delta frequency band , f = 3hz , r = 0.91 twenty - second simulated ar(4 ) data with time - dependent modules and frequencies derived by fitting an ar(4 ) model similar to fig . 1 . the upper panel shows the signal , the middle panel shows r , and the lower panel the corresponding frequencies f , with the colors indicating corresponding oscillatory modes . the ar(4 ) model contained two oscillatory modes , one in the spindle frequency band , f = 14 hz , r = 0.94 , and one in the delta frequency band , f = 3hz , r = 0.91 we analyzed 8-h sleep eeg data ( single channel , derivation c3a2 ) of eight healthy young male subjects ( mean age 24.1 0.6 years ) , each contributing four baseline nights [ 17 , 19 ] . the sampling rate was 128 hz and sleep stages were visually scored according to standard criteria . oscillatory events were detected by fitting ar models of order 8 to subsegments of 1 s in length in a sliding window fashion resulting in a time dependence of the parameters of the ar model . the signal generated by an ar(p)-model can be interpreted as the superposition of the output of n oscillators and p 2n relaxators . the p parameters of the ar(p)-model are transformed into n frequencies and damping constants of the oscillators , and p 2n damping constants of the purely decaying nonoscillating modes . thus , time - dependent frequencies and damping constants result from the sliding window approach . figure 1 shows a 20-s segment of eeg ( stage 2 ) and the time - dependent modules r and the corresponding frequencies \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$f\:$\end{document } of the poles . the damping constant is a function of r , = fs logr with fs denoting the sampling frequency . the case r = 1 corresponds to the limiting case of an undamped oscillation . the threshold for the event detection was set to r1 = 0.95 ( fig . 1 ) . to determine the event duration , a second threshold r2 = 0.9 was applied : in order to account for statistical fluctuations , the event was not necessarily terminated whenever r became smaller than r1 . it was considered as a single event if the corresponding r became larger than r1 again without falling below r2 . the value r1 was chosen such that clearly visible sleep spindles were reliably detected by the algorithm , while r2 had to be low enough so that a single spindle was detected as a single event , but high enough so that subsequent spindles were recognized as distinct events . the time between the first and the final crossing of the threshold r1 plus 1 s ( the length of the segment used to fit the ar model ) was considered as the duration of the oscillatory event . the time when r was maximal was defined as the time of occurrence of the event and the corresponding frequency the interevent intervals were estimated as the difference of the time of occurrence of successive oscillatory events . 1detection of oscillatory events in a 20-s eeg segment of stage 2 non - rem sleep . the upper panel shows the eeg signal , the middle panel shows r , and the lower panel the corresponding frequencies f , with the colors indicating corresponding oscillatory modes . the detection threshold r1 = 0.95 is indicated in the middle panel ( see section 2.2 ) detection of oscillatory events in a 20-s eeg segment of stage 2 non - rem sleep . the upper panel shows the eeg signal , the middle panel shows r , and the lower panel the corresponding frequencies f , with the colors indicating corresponding oscillatory modes . the detection threshold r1 = 0.95 is indicated in the middle panel ( see section 2.2 ) for the analysis of the interevent intervals and the event durations , we assigned the events into different frequency bands . we are aware that this is not an optimal approach because of interindividual variation in mean event frequencies and their dependence on sleep stage and cycle . however , at the moment we have no method available to analyze the events on the basis of individually determined time - dependent frequency bands . therefore , we used the frequency bands employed previously , i.e. , delta ( : 0 f < 4.5 hz ) , alpha ( : 8 f < 11.5 hz ) , and sigma ( : 11.5 f < 16 hz ; sleep spindles ) bands . figure 2 illustrates the detected events of a single night and the corresponding histogram of the event frequencies . 2detected oscillatory events in a single night ( black dots represent single events ) , hypnogram ( mt , movement time ; w , waking , rem , rapid eye movement sleep ; 1 to 4 , nonrapid eye movement sleep stages 1 to 4 ) , and corresponding histogram of the event frequencies . colors indicate the different frequency bands detected oscillatory events in a single night ( black dots represent single events ) , hypnogram ( mt , movement time ; w , waking , rem , rapid eye movement sleep ; 1 to 4 , nonrapid eye movement sleep stages 1 to 4 ) , and corresponding histogram of the event frequencies . artificial eeg data were generated using an ar(4 ) model with random inputs and parameters corresponding to a frequency in the spindle frequency band ( f = 14 hz ) and in the delta band ( f = 3 hz ) , assuming a sampling frequency of 128 hz . we restricted the analysis to data from an ar(4 ) model because the two higher frequencies that can be seen in fig . we analyzed data sets with different values of r for the spindle frequency because the spindle oscillations are our main focus . for each set of parameter values , a time series of 6 10 data points ( corresponding to approximately 13 h of eeg data ) was generated . 3twenty - second simulated ar(4 ) data with time - dependent modules and frequencies derived by fitting an ar(4 ) model similar to fig . 1 . the upper panel shows the signal , the middle panel shows r , and the lower panel the corresponding frequencies f , with the colors indicating corresponding oscillatory modes . the ar(4 ) model contained two oscillatory modes , one in the spindle frequency band , f = 14 hz , r = 0.94 , and one in the delta frequency band , f = 3hz , r = 0.91 twenty - second simulated ar(4 ) data with time - dependent modules and frequencies derived by fitting an ar(4 ) model similar to fig . 1 . the upper panel shows the signal , the middle panel shows r , and the lower panel the corresponding frequencies f , with the colors indicating corresponding oscillatory modes . the ar(4 ) model contained two oscillatory modes , one in the spindle frequency band , f = 14 hz , r = 0.94 , and one in the delta frequency band , f = 3hz , r = 0.91 figure 4 shows the interevent interval distributions of sleep spindles ( 11.516 hz ) . the histograms of the empirical data were estimated based on the four recordings of all subjects ( pooled data ) . in sleep stage 2 , the maximum is at t 4 s and is much more pronounced than in deep sleep [ slow wave sleep ( sws ) ] . the distribution of the interevent intervals detected by our algorithm was similar to the distribution reported in for spindles identified by visual detection . figure 4 also contains the distributions of events detected from simulated data of stationary ar(4 ) models for two different values of the module r for the spindle frequency . events were detected with the same algorithm and parameters as for the eeg data . also , for the simulated data , the interevent interval distributions showed a peak around 4 s ( 2.5 s for r = 0.94 and between 2.5 and 4 for r = 0.89 ) . the results of the simulated data indicate that stationary stochastic processes also exhibit a periodicity in the occurrence of events with a maximum similar to empirical data without the assumption of an additional slow process grouping the events . thus , in order to understand the character of the maxima in the empirical distributions , it is necessary to investigate these distributions in more detail . 4normalized histograms ( pooled data ) of interevent intervals ( t ) in the spindle frequency range ( 11.516 hz ) in nonrapid eye movement sleep stage 2 ( st2 ; top left ) and slow wave sleep ( sws , stages 3 and 4 ; top right ) and of events in the spindle frequency range detected from data generated by stationary ar(4 ) models with r = 0.94 ( bottom left ) and r = 0.89 ( bottom right ) for the spindle frequency normalized histograms ( pooled data ) of interevent intervals ( t ) in the spindle frequency range ( 11.516 hz ) in nonrapid eye movement sleep stage 2 ( st2 ; top left ) and slow wave sleep ( sws , stages 3 and 4 ; top right ) and of events in the spindle frequency range detected from data generated by stationary ar(4 ) models with r = 0.94 ( bottom left ) and r = 0.89 ( bottom right ) for the spindle frequency in fig . 5 , an exponential decay for large interevent intervals was present in both the synthetic and the empirical data . for events occurring randomly with a certain constant rate ( poisson process ) , one would expect that the interevent intervals are exponentially distributed , with the exponent being equal to the event rate ( number of events per unit of time ) . in our situation , this can not be exactly the case because of the finite duration of the oscillatory events . however , for large interevent intervals compared to the event duration , this might be a reasonable assumption . the empirical distributions differ substantially from an exponential distribution not only for times below the maximum of the distribution but also for larger time intervals . thus , the region around the maximum can not be explained in the same way as the distributions for the ar processes . this is the first indication of an additional process contributing to the peak in the empirical interevent distributions . 5normalized histograms of interevent intervals as in fig . 4 but plotted on a semilogarithmic scale including additional values of r. the straight lines show fits to the exponential tails of these distributions normalized histograms of interevent intervals as in fig . 4 but plotted on a semilogarithmic scale including additional values of r. the straight lines show fits to the exponential tails of these distributions the systematic dependence of the exponential decay on r of the ar model and , therefore , on the damping of the spindle frequency pole , is illustrated in fig . 6a . for r 0.93 , the exponent becomes smaller with smaller r. a similar relationship holds for the event rate ( fig . the inverse relationship is observed only for large r , i.e. , lower damping leads to a decrease in the event rate , which is overcompensated by an increased event duration . the relationship between the exponent of the exponential decay and the event rate is shown for r 0.93 in fig . systematic deviations for larger event rates might be explained by more events of longer duration as illustrated in fig 7 6a exponents from the fit of the exponential tails of the interevent interval distributions ( fig . 5 ) of the simulated ar(4 ) data as a function of r of the spindle frequency pole . b event rate as a function of r. c exponent of the exponential decay ( mean sd ) as a function of the event rate shown only for 0.85 r 0.93 . values for the empirical distributions ( red ) from sleep spindles shown separately for nonrapid eye movement sleep stages 2 , 3 , and 4 . 7normalized histograms ( pooled data ) of the event duration ( td ) in the spindle frequency range ( 11.516 hz ) in nonrapid eye movement sleep stage 2 ( st2 ; top left ) and slow wave sleep ( sws , stages 3 and 4 ; top right ) and events in the spindle frequency range from a stationary ar(4 ) process with different values of r for the spindle frequency a exponents from the fit of the exponential tails of the interevent interval distributions ( fig . 5 ) of the simulated ar(4 ) data as a function of r of the spindle frequency pole . b event rate as a function of r. c exponent of the exponential decay ( mean sd ) as a function of the event rate shown only for 0.85 r 0.93 . values for the empirical distributions ( red ) from sleep spindles shown separately for nonrapid eye movement sleep stages 2 , 3 , and 4 . the straight line indicates the linear relationship as expected for a poisson process normalized histograms ( pooled data ) of the event duration ( td ) in the spindle frequency range ( 11.516 hz ) in nonrapid eye movement sleep stage 2 ( st2 ; top left ) and slow wave sleep ( sws , stages 3 and 4 ; top right ) and events in the spindle frequency range from a stationary ar(4 ) process with different values of r for the spindle frequency the exponent of the empirical data of sleep spindles ( fig . 6c ) suggests an increased damping ( corresponding to a smaller r ) with increasing sleep depth from stage 2 through stage 3 to stage 4 , which meets our expectations . the spindles exhibited , however , a larger exponent than expected from the resulting event rate . or , to put it the other way around , the event rate is smaller than one would expect from the exponent of the exponential decay . from the 4-s periodicity , one would have expected the contrary , i.e. , a larger event rate , because an interevent interval of 4 s corresponds to an event rate of 15 events per minute . a possible explanation for this discrepancy might be the estimation of the event density of spindles : the number of detected spindles during a particular sleep stage was divided by the total time spent in this stage . this event rate is not equal to the inverse of the mean of the interevent interval distribution because only interevent intervals with no sleep stage transition between the events were included . the normalized distribution of the event duration ( fig . 7 ) plotted on a logarithmic scale seems to be less specific for sleep spindles . first , the maximum around t = 4 s occurs in the simulated data for a wide range of values of r. second , there is no large difference between the distributions of the occurrence of spindles in sleep stage 2 and sws . there is a slightly larger probability to have longer spindles in sleep stage 2 compared to sws ( fig . 7 ) , which would correspond to a higher value of r if we assume that spindles were generated by a stationary ar process . note , however , that the probability of long spindles increased strongly in data generated by ar models of large r. this increase might be the reason for the slower exponential decrease in the interevent interval distribution for r = 0.95 compared to r = 0.93 . we have demonstrated that important properties of the temporal organization of the occurrence of sleep spindles are already present for oscillatory events detected in data generated with stationary stochastic processes . such properties are the position of the maximum of the interevent interval distribution , i.e. , the periodicity in the occurrence of events between 2 and 5 s and the maximum around 2 s in the distribution of event durations . a more detailed analysis , however , revealed that the exponential decay in the interevent distributions , which can be interpreted as reflecting the random occurrence of oscillatory events , starts in the case of the sleep spindles only at much longer interevent intervals compared to data from stationary ar processes . for interevent intervals around the maximum of 4 s , the empirical distributions of sleep spindles clearly differ from the distributions of the simulated events and show , in particular , no exponential decay . one could assume in a first approximation a gaussian distribution around 4 s , which would indicate that an additional process is responsible for the observed periodicity in the occurrence of spindles . including such a process in modeling the time series , i.e. , modeling explicitly also the dynamics of the parameters of the ar model , would result in a nonlinear model . the extent to which such a process is related to cortical slow oscillations has to be the subject of further research . in particular , the present analysis could be extended by ( 1 ) also varying the delta frequency and/or its damping in the simulated data and ( 2 ) taking into account that single sleep stages are not dynamically homogeneous , i.e. , not assuming that the spindles in a particular sleep stage were generated by a single stationary process but rather by a suitable ensemble of such processes . moreover , it is known that there are strong interindividual differences in the event rates of sleep spindles ( e.g. , ) . thus , one should also investigate to which extent interindividual differences in the interevent interval distributions are present . our analysis demonstrated , nonetheless , how phenomenological modeling of the short - term dynamics of the eeg using linear models can be used to detect and to analyze oscillatory events and to test hypotheses about the origin of some of their properties .

the sleep electroencephalogram ( eeg ) is characterized by typical oscillatory patterns such as sleep spindles and slow waves . recently , we proposed a method to detect and analyze these patterns using linear autoregressive models for short ( 1 s ) data segments . we analyzed the temporal organization of sleep spindles and discuss to what extent the observed interevent intervals correspond to properties of stationary stochastic processes and whether additional slow processes , such as slow oscillations , have to be assumed . we have found evidence for such an additional slow process , most pronounced in sleep stage 2 .

Introduction Data and Methods Data Oscillatory Events Simulated Data Results Discussion

slow waves and sleep spindles are the essential elements in the definition of sleep stages . furthermore , slow oscillations ( < 1 hz ) are considered as a central feature of sleep . these intracortically generated fluctuations consist at the cellular level of rhythmic depolarizing components ( up states ) separated by prolonged hyperpolarizations ( down states ) . there is evidence that they are involved in the temporal organization of other sleep rhythms such as spindles and delta waves ( for a review , see ) . also , it is hypothesized that the slow oscillation is involved in processes of learning and memory during sleep . tononi and cirelli proposed that slow oscillations were an indication of synaptic downscaling in the cortex occurring during nonrapid eye movement sleep in order to counteract synaptic potentiation resulting from learning processes occurring during wakefulness [ 6 , 7 ] . evidence for this hypothesis comes from data of a motor learning task , arm immobilization , animal studies , and computational modeling . we shall address in this paper only one particular aspect in this context , which is , however , related to the interplay of sleep spindles and slow oscillations : i.e. , the temporal organization of spindles . it was postulated that these thalamically generated sleep spindles are grouped by cortically generated slow oscillations and more generally that the slow oscillations also organize the occurrence of other sleep oscillations . this hypothesis is based on experimental and theoretical evidence : sleep spindles lost their coherence and their temporal however , there are also inconsistencies in relation to this hypothesis : first , the frequency of the slow oscillations was originally considered as being only slightly < 1 hz . achermann and borbly , for instance , reported a peak in the power spectrum at 0.7 hz . this would correspond to a shorter typical interevent interval between sleep spindles than 4 s. thus , only approximately every third slow oscillation would trigger a spindle . second , the rhythmic occurrence of spindles is most pronounced in sleep stage 2 ( see fig . 4 ) , but slow oscillations are most prominent in deep sleep . in the present paper , we investigated whether the periodicity in the occurrence of sleep spindles and other sleep oscillations might be a pure stochastic effect that occurs without assuming an additional slow process . to test this hypothesis , we compared interevent interval distributions of the events detected in the human sleep eeg with events detected in artificial data generated with stationary stochastic processes , which resemble to some extent the short - term properties of the eeg signal . the analysis is based on a recently proposed method to detect and analyze oscillatory events in the human sleep eeg . eeg data were modeled on short time scales ( 1s ) by linear autoregressive ( ar ) models in a sliding window fashion . we analyzed 8-h sleep eeg data ( single channel , derivation c3a2 ) of eight healthy young male subjects ( mean age 24.1 0.6 years ) , each contributing four baseline nights [ 17 , 19 ] . oscillatory events were detected by fitting ar models of order 8 to subsegments of 1 s in length in a sliding window fashion resulting in a time dependence of the parameters of the ar model . the signal generated by an ar(p)-model can be interpreted as the superposition of the output of n oscillators and p 2n relaxators . figure 1 shows a 20-s segment of eeg ( stage 2 ) and the time - dependent modules r and the corresponding frequencies \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$f\:$\end{document } of the poles . the case r = 1 corresponds to the limiting case of an undamped oscillation . = 0.9 was applied : in order to account for statistical fluctuations , the event was not necessarily terminated whenever r became smaller than r1 . the value r1 was chosen such that clearly visible sleep spindles were reliably detected by the algorithm , while r2 had to be low enough so that a single spindle was detected as a single event , but high enough so that subsequent spindles were recognized as distinct events . the time between the first and the final crossing of the threshold r1 plus 1 s ( the length of the segment used to fit the ar model ) was considered as the duration of the oscillatory event . the time when r was maximal was defined as the time of occurrence of the event and the corresponding frequency the interevent intervals were estimated as the difference of the time of occurrence of successive oscillatory events . 1detection of oscillatory events in a 20-s eeg segment of stage 2 non - rem sleep . the detection threshold r1 = 0.95 is indicated in the middle panel ( see section 2.2 ) detection of oscillatory events in a 20-s eeg segment of stage 2 non - rem sleep . the detection threshold r1 = 0.95 is indicated in the middle panel ( see section 2.2 ) for the analysis of the interevent intervals and the event durations , we assigned the events into different frequency bands . we are aware that this is not an optimal approach because of interindividual variation in mean event frequencies and their dependence on sleep stage and cycle . however , at the moment we have no method available to analyze the events on the basis of individually determined time - dependent frequency bands . therefore , we used the frequency bands employed previously , i.e. , delta ( : 0 f < 4.5 hz ) , alpha ( : 8 f < 11.5 hz ) , and sigma ( : 11.5 f < 16 hz ; sleep spindles ) bands . we analyzed data sets with different values of r for the spindle frequency because the spindle oscillations are our main focus . for each set of parameter values , a time series of 6 10 data points ( corresponding to approximately 13 h of eeg data ) was generated . 3twenty - second simulated ar(4 ) data with time - dependent modules and frequencies derived by fitting an ar(4 ) model similar to fig . 1 . the ar(4 ) model contained two oscillatory modes , one in the spindle frequency band , f = 14 hz , r = 0.94 , and one in the delta frequency band , f = 3hz , r = 0.91 twenty - second simulated ar(4 ) data with time - dependent modules and frequencies derived by fitting an ar(4 ) model similar to fig . 1 . the ar(4 ) model contained two oscillatory modes , one in the spindle frequency band , f = 14 hz , r = 0.94 , and one in the delta frequency band , f = 3hz , r = 0.91 we analyzed 8-h sleep eeg data ( single channel , derivation c3a2 ) of eight healthy young male subjects ( mean age 24.1 0.6 years ) , each contributing four baseline nights [ 17 , 19 ] . oscillatory events were detected by fitting ar models of order 8 to subsegments of 1 s in length in a sliding window fashion resulting in a time dependence of the parameters of the ar model . the p parameters of the ar(p)-model are transformed into n frequencies and damping constants of the oscillators , and p 2n damping constants of the purely decaying nonoscillating modes . figure 1 shows a 20-s segment of eeg ( stage 2 ) and the time - dependent modules r and the corresponding frequencies \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$f\:$\end{document } of the poles . the threshold for the event detection was set to r1 = 0.95 ( fig . to determine the event duration , a second threshold r2 = 0.9 was applied : in order to account for statistical fluctuations , the event was not necessarily terminated whenever r became smaller than r1 . the value r1 was chosen such that clearly visible sleep spindles were reliably detected by the algorithm , while r2 had to be low enough so that a single spindle was detected as a single event , but high enough so that subsequent spindles were recognized as distinct events . the time between the first and the final crossing of the threshold r1 plus 1 s ( the length of the segment used to fit the ar model ) was considered as the duration of the oscillatory event . the time when r was maximal was defined as the time of occurrence of the event and the corresponding frequency the interevent intervals were estimated as the difference of the time of occurrence of successive oscillatory events . 1detection of oscillatory events in a 20-s eeg segment of stage 2 non - rem sleep . the detection threshold r1 = 0.95 is indicated in the middle panel ( see section 2.2 ) detection of oscillatory events in a 20-s eeg segment of stage 2 non - rem sleep . the detection threshold r1 = 0.95 is indicated in the middle panel ( see section 2.2 ) for the analysis of the interevent intervals and the event durations , we assigned the events into different frequency bands . we are aware that this is not an optimal approach because of interindividual variation in mean event frequencies and their dependence on sleep stage and cycle . however , at the moment we have no method available to analyze the events on the basis of individually determined time - dependent frequency bands . therefore , we used the frequency bands employed previously , i.e. , delta ( : 0 f < 4.5 hz ) , alpha ( : 8 f < 11.5 hz ) , and sigma ( : 11.5 f < 16 hz ; sleep spindles ) bands . 2detected oscillatory events in a single night ( black dots represent single events ) , hypnogram ( mt , movement time ; w , waking , rem , rapid eye movement sleep ; 1 to 4 , nonrapid eye movement sleep stages 1 to 4 ) , and corresponding histogram of the event frequencies . artificial eeg data were generated using an ar(4 ) model with random inputs and parameters corresponding to a frequency in the spindle frequency band ( f = 14 hz ) and in the delta band ( f = 3 hz ) , assuming a sampling frequency of 128 hz . we analyzed data sets with different values of r for the spindle frequency because the spindle oscillations are our main focus . 3twenty - second simulated ar(4 ) data with time - dependent modules and frequencies derived by fitting an ar(4 ) model similar to fig . the ar(4 ) model contained two oscillatory modes , one in the spindle frequency band , f = 14 hz , r = 0.94 , and one in the delta frequency band , f = 3hz , r = 0.91 twenty - second simulated ar(4 ) data with time - dependent modules and frequencies derived by fitting an ar(4 ) model similar to fig . the upper panel shows the signal , the middle panel shows r , and the lower panel the corresponding frequencies f , with the colors indicating corresponding oscillatory modes . the ar(4 ) model contained two oscillatory modes , one in the spindle frequency band , f = 14 hz , r = 0.94 , and one in the delta frequency band , f = 3hz , r = 0.91 figure 4 shows the interevent interval distributions of sleep spindles ( 11.516 hz ) . in sleep stage 2 , the maximum is at t 4 s and is much more pronounced than in deep sleep [ slow wave sleep ( sws ) ] . the distribution of the interevent intervals detected by our algorithm was similar to the distribution reported in for spindles identified by visual detection . figure 4 also contains the distributions of events detected from simulated data of stationary ar(4 ) models for two different values of the module r for the spindle frequency . the results of the simulated data indicate that stationary stochastic processes also exhibit a periodicity in the occurrence of events with a maximum similar to empirical data without the assumption of an additional slow process grouping the events . 4normalized histograms ( pooled data ) of interevent intervals ( t ) in the spindle frequency range ( 11.516 hz ) in nonrapid eye movement sleep stage 2 ( st2 ; top left ) and slow wave sleep ( sws , stages 3 and 4 ; top right ) and of events in the spindle frequency range detected from data generated by stationary ar(4 ) models with r = 0.94 ( bottom left ) and r = 0.89 ( bottom right ) for the spindle frequency normalized histograms ( pooled data ) of interevent intervals ( t ) in the spindle frequency range ( 11.516 hz ) in nonrapid eye movement sleep stage 2 ( st2 ; top left ) and slow wave sleep ( sws , stages 3 and 4 ; top right ) and of events in the spindle frequency range detected from data generated by stationary ar(4 ) models with r = 0.94 ( bottom left ) and r = 0.89 ( bottom right ) for the spindle frequency in fig . 5 , an exponential decay for large interevent intervals was present in both the synthetic and the empirical data . for events occurring randomly with a certain constant rate ( poisson process ) , one would expect that the interevent intervals are exponentially distributed , with the exponent being equal to the event rate ( number of events per unit of time ) . however , for large interevent intervals compared to the event duration , this might be a reasonable assumption . this is the first indication of an additional process contributing to the peak in the empirical interevent distributions . 5normalized histograms of interevent intervals as in fig . 4 but plotted on a semilogarithmic scale including additional values of r. the straight lines show fits to the exponential tails of these distributions normalized histograms of interevent intervals as in fig . 5 ) of the simulated ar(4 ) data as a function of r of the spindle frequency pole . values for the empirical distributions ( red ) from sleep spindles shown separately for nonrapid eye movement sleep stages 2 , 3 , and 4 . 7normalized histograms ( pooled data ) of the event duration ( td ) in the spindle frequency range ( 11.516 hz ) in nonrapid eye movement sleep stage 2 ( st2 ; top left ) and slow wave sleep ( sws , stages 3 and 4 ; top right ) and events in the spindle frequency range from a stationary ar(4 ) process with different values of r for the spindle frequency a exponents from the fit of the exponential tails of the interevent interval distributions ( fig . 5 ) of the simulated ar(4 ) data as a function of r of the spindle frequency pole . values for the empirical distributions ( red ) from sleep spindles shown separately for nonrapid eye movement sleep stages 2 , 3 , and 4 . the straight line indicates the linear relationship as expected for a poisson process normalized histograms ( pooled data ) of the event duration ( td ) in the spindle frequency range ( 11.516 hz ) in nonrapid eye movement sleep stage 2 ( st2 ; top left ) and slow wave sleep ( sws , stages 3 and 4 ; top right ) and events in the spindle frequency range from a stationary ar(4 ) process with different values of r for the spindle frequency the exponent of the empirical data of sleep spindles ( fig . 6c ) suggests an increased damping ( corresponding to a smaller r ) with increasing sleep depth from stage 2 through stage 3 to stage 4 , which meets our expectations . from the 4-s periodicity , one would have expected the contrary , i.e. a possible explanation for this discrepancy might be the estimation of the event density of spindles : the number of detected spindles during a particular sleep stage was divided by the total time spent in this stage . this event rate is not equal to the inverse of the mean of the interevent interval distribution because only interevent intervals with no sleep stage transition between the events were included . 7 ) plotted on a logarithmic scale seems to be less specific for sleep spindles . first , the maximum around t = 4 s occurs in the simulated data for a wide range of values of r. second , there is no large difference between the distributions of the occurrence of spindles in sleep stage 2 and sws . there is a slightly larger probability to have longer spindles in sleep stage 2 compared to sws ( fig . 7 ) , which would correspond to a higher value of r if we assume that spindles were generated by a stationary ar process . we have demonstrated that important properties of the temporal organization of the occurrence of sleep spindles are already present for oscillatory events detected in data generated with stationary stochastic processes . a more detailed analysis , however , revealed that the exponential decay in the interevent distributions , which can be interpreted as reflecting the random occurrence of oscillatory events , starts in the case of the sleep spindles only at much longer interevent intervals compared to data from stationary ar processes . for interevent intervals around the maximum of 4 s , the empirical distributions of sleep spindles clearly differ from the distributions of the simulated events and show , in particular , no exponential decay . one could assume in a first approximation a gaussian distribution around 4 s , which would indicate that an additional process is responsible for the observed periodicity in the occurrence of spindles . the extent to which such a process is related to cortical slow oscillations has to be the subject of further research . in particular , the present analysis could be extended by ( 1 ) also varying the delta frequency and/or its damping in the simulated data and ( 2 ) taking into account that single sleep stages are not dynamically homogeneous , i.e. , not assuming that the spindles in a particular sleep stage were generated by a single stationary process but rather by a suitable ensemble of such processes . moreover , it is known that there are strong interindividual differences in the event rates of sleep spindles ( e.g. our analysis demonstrated , nonetheless , how phenomenological modeling of the short - term dynamics of the eeg using linear models can be used to detect and to analyze oscillatory events and to test hypotheses about the origin of some of their properties .

given local adaptation between a resident population and its environment , how can a nonadapted competitor invade ? invasion can easily be conceived when the invaders are more adapted than the residents . however , if the invader is at a disadvantage in the context of the resident 's environment , one solution could be to modify the environment into a configuration favouring the invaders . many organisms are indeed known to modify the environment , such action is also called niche construction ( odling - smee et al . . when the environmental modification is triggered by the organisms that benefit from it , natural selection can retain it as an extended phenotype ( dawkins 1982 ) . building nests , spider webs or beaver dams are examples of extended phenotypes improving the actors niche , whereas the secretion of toxins is an example of niche - deterioration ( although offering a relative inclusive fitness advantage to the actor ) , also referred to as interference competition , or allelopathy ( fitter 2003 ) . here we review three broad classes of microbial allelopathy , each associated with the release of a distinct class of targeted weapon : the release of chemical toxins that directly kill competitors . the release of viral propagules that directly kill competitors and subsequently amplify to kill more competitors . the release of chemical toxins which provoke an inflammatory response within a vertebrate host , which preferentially kills competitors . carrier lineages carry genes causing the explosive suicide of the host , with a low probability , the explosive suicide of the host , and the release of bactericidal toxins or bacteriocins ( small peptides or heat stable proteins , riley and wertz 2002 ) . furthermore , nonlysed carriers are immune to the bacteriocin , thanks to the specific antidote coding genes carried along with the bacteriocin genes . thus , the environmental change resulting from the release of bacteriocins can favour the carriers over any susceptible resident populations ( fig . this type of interference competition is found across a range of different bacterial species and analogous examples can even be found in eukaryotic yeasts ( wickner 1996 ; riley and wertz 2002 ) . schematic diagrams of ( a ) chemical , ( b ) viral and ( c ) immuno - manipulative weapons of mass microbial destruction . squares represent microbial densities of competing weapons - carriers ( c ) and susceptibles ( s ) . circles represent the density of weapon , the agent of environmental change ( b for bacteriocin , or other chemical toxin , v for temperate phage virus , and t for immuno - provocative toxin ) . the cylinder represents a multicellular host , the subject of immuno - manipulation by t. pointed arrows represent a positive relationship ( e.g. c increase the density of b ) , flat arrows represent a negative relationship ( e.g. b decrease the density of s ) . one well - documented example is that of escherichia coli strains producing colicins , bacteriocins active against other e. coli . previous experimental and theoretical studies of colicin - mediated bacterial competition have shown that colicin - producers can invade in a spatially structured environment and if unstructured , then only if sufficiently abundant ( chao and levin 1981 ; levin 1988 ; frank 1994 ; durrett and levin 1997 ) . in a spatially structured environment , the modification of the environment due to the secretion of colicins is a local phenomenon : with high local colicin concentrations killing neighbouring competitors , and thereby freeing nutrients for the invaders . invaders can then spread as a consequence of sequential waves of colicins followed by colicin - mediated resident death leaving a competitive vacuum ( chao and levin 1981 ; levin 1988 ; frank 1994 ; durrett and levin 1997 ) . resistance often comes at some cost to bacteria ( either metabolically through the production of an immunity compound or through the loss of membrane receptors ) , and so allows for the coexistence , in a spatially structured environment ( through negative frequency - dependent selection ) of producer , sensitive , and resistant bacteria ; producers out - compete sensitives , resistants beat producers , and sensitives beat resistants , creating a rock - paper - scissors dynamic ( czrn et al . 2002 ; kerr et al . 2002 the pleiotropic fitness cost associated with resistance is also likely to explain the diversity in bacteriocin types seen within a microbial species . being resistant to all bacteriocin types will often be too costly , so resistance to the most common bacteriocins will be frequent . this favours bacteria producing rare bacteriocins , which should then in turn become more widespread , thereby maintaining diversity ( west et al . more recently work on yeasts infected with double stranded rna viruses has helped to further elucidate the ecological conditions that favour the production of anti - competitor toxins . these viruses can be considered similar to bacteriocins as there is no known horizontal transmission of the virus between yeast cells : daughter cells are instead infected via the cytoplasm of their parents ( magliani et al . in saccharomyces cerevisiae , the k1 toxin system requires two viruses , one that encodes the toxin and immunity proteins and a second that is involved in encapsulating the virus . unlike most bacteriocins , lysis is not required to release the toxin , but there is a pronounced fitness cost when carrying the virus ( wloch - salamon et al . were able to show the importance of density in determining the success of toxin - producing strains . density dependence has previously been shown to be an important ecological determinant in allelopathic interactions ( brown et al . 2006 ) , but experimental work has focused on the frequency of the producing and sensitive strains . grieg and travisano ( 2008 ) demonstrated that toxin producers , even when at intermediate frequencies , were unable to invade if the overall microbial density was low . this effect was also observed in structured environments , which tend to favour toxin production . when both producers and sensitive strains are rare there will only be limited interaction between them and there will be little benefit in producing toxins . instead toxin producers will be at a disadvantage due to the metabolic and pleiotropic costs of producing toxins . nutrient availability , dispersal , and their effect on interference competition in yeast have also been considered experimentally . it has been suggested that toxin production might act as strategy of last resort among starving cells by killing competitors for nutrients ( ivanovska and hardwick 2005 ) . wloch - salamon et al . ( 2008 ) grew s. cerevisiae toxin - producing strains in competition with sensitives under low and high nutrient conditions . nutrient availability was manipulated by varying the rate of nutrient replacement , following initial growth to carrying capacity ( therefore controlling initial densities ) . wloch - salamon et al . ( 2008 ) found that toxin producers were able to out - compete sensitive cells only in high nutrient environments and were out - competed when grown under low nutrient conditions . they argued that the lack of toxin production seen in the low nutrient environment is unlikely to be solely due to a nutritional constraint , but rather due in addition to a physiological change in toxin production or resistance developing after the nutrients have been depleted ( wloch - salamon et al . they conclude that this acts as support for frank 's ( 1994 ) theoretical prediction that toxin production has evolved to occur as a competitive strategy under conditions where resources are abundant and growth is allowed . this makes it unlikely that toxin production is used to acquire resources from sensitive cells , but is more important in the invasion of high nutrient patches . they also looked at the effect on dispersal , finding that limited dispersal favoured toxin producers . a benefit was still observed in treatments with high dispersal , but it was insufficient to compensate for the resource cost of carrying the toxin - producing virus . these results are consistent with chao and levin 's ( 1981 ) results in spatially structured environments as dispersal will reduce the chances that related cells will profit from toxin production . there is still much about the ecology and evolution of interference competition through toxin production that remains to be properly understood . a huge diversity exists in the types of toxins used from bacteriocins to antibiotics : they can differ in the specificity of killing action , in their ability to diffuse , and even in their respective half - lives in the environment ( ito et al . 1970 ; michel - briand and baysse 2002 ; riley and wertz 2002 ; cascales et al . 2007 ) . all of these factors will greatly change how the environment is modified to allow toxin producers to invade . one constraint , however , that is faced by all toxin producers trying to invade a population is that for every halving of the invader population , the cost to individual invaders of successful environmental modification will double . given limited resources , these increasing costs will ultimately present a rarity threshold to invasion ( chao and levin 1981 ; levin 1988 ; frank 1994 ; durrett and levin 1997 ; gordon and riley 1999 ) . we have just seen that for chemical allelopathy in an unstructured environment , there exists a rarity threshold , below which allelopathic lineages can not invade . brown et al . ( 2006 ) demonstrated that this threshold can be reduced or eliminated for other forms of allelopathy . specifically , weapons that are capable of self - amplification can allow vanishingly rare carrier lineages to invade and replace residents . a microbial example is provided by bacteria and some of their phages ( viruses that can infect bacteria ) , which can function as bacteriocins ( bossi et al . 2003 ) but with the additional capacity of autocatalytic amplification on victims ( brown et al . more specifically , we focus on temperate phages , which are viruses that are able to be transmitted either vertically or horizontally . the most common is the lytic cycle ( rapid host lysis and production of numerous horizontally transmissible viral particles ) . very rarely , however , the phage can lysogenise the host , persisting in a dormant state while allowing the survival of the infected bacteria . this dormant phage is then replicated with the bacterial genome , and thus vertically transmitted upon bacterial division . furthermore , this vertically transmitted carried - phage provides immunity to its carrier - bacteria against further horizontal infection by this phage ( adams 1959 ; campbell 1996 ) . upon rare spontaneous induction of the carried - phage , viral progeny experimental competition of isogenic phage - carrying and susceptible bacterial lineages over a range of initial ratios illustrates that the ability of the released temperate phage 1b ) allows the phage - resistant carrier lineage to invade more rapidly when rare ( brown et al . autocatalytic environmental modification , such as the one triggered by the release of phage , can be seen as a public good that is expensive to produce initially but which can then be amplified at a negligible cost , therefore allowing an escape from the rarity threshold ( brown et al . indirect mutualist ( the enemy of your enemy is your friend ) interaction between bacteria and temperate phage can also be viewed in terms of parasite - mediated apparent competition , whereby the outcome of competition between two competitors is determined by their relative susceptibility to a shared parasite ( hudson and greenman 1998 ; tompkins et al . 2001 ; joo et al . for instance , one of the leading hypotheses for the replacement of resident red squirrels by invasive grey squirrels in the uk is the differential susceptibility to a parapoxvirus brought by the invasive greys ( rushton et al . however , unlike the one - off invasion of the uk by grey squirrels plus a parapoxvirus , food - borne pathogens such as e. coli o157::h7 and its suite of phages continuously invade new digestive tracts . we will propose below that this distinction between once - off lucky and recurrent both experimental and model results illustrate how the level of amplification is determined by the availability of susceptibles in the initial population ( brown et al . when susceptibles are sufficiently rare , the phage and colicin models converge as the phage ceases to gain any multiplicative advantage due to the rarity of susceptibles , acting purely through its direct killing effect . whereas phage can act essentially as a colicin - style nonmultiplicative killer when their carriers are numerically dominant , they do so in a less efficient manner due to their relatively wasteful capacity for replication ( e.g. effective colicin burst - size is at least 10-fold greater , gordon and riley 1999 ) . therefore for a high carrier frequency , colicin production is more efficient . given these differences between colicin and phage mediation , phage - carriers are more likely to be successful in invasive lineages , as they grow in the wake of the competitive vacuum left by their virulent indirect - mutualists . in contrast , a colicin - strategy specialising on killing without amplification by the susceptibles has an advantage over phage - carriers in a defensive context : their higher productivity ensures that they can not be as easily invaded when residents ( brown et al . we have focused solely on a single invasion event into a resident population consisting entirely of susceptibles . however , given the diversity of normal microbial communities within the gut , only a minority of the resident bacteria will be susceptible to any specific bacteriocin or phage . the carriers can only attack susceptibles , and resistants gain from the suppression of the susceptible population , along with the carriers . following the eradication of susceptibles , resistants will outcompete carriers due to the latter 's lytic behaviour , leaving an endpoint of pure resistants . this might not be the end of the game ; however , given that phage - resistant bacteria are likely to carry a cost relative to susceptibles ( resistance is often generated via the loss of useful membrane proteins that are recognized by both phages and bacteriocins ) . resistants could be outcompeted by susceptibles which are in turn outcompeted by carriers ( at least , in a structured environment for bacteriocin producers ) that are themselves outcompeted by these resistant bacteria , potentially promoting 2002 ; kerr et al . 2002 ; kirkup and riley 2004 ; brown et al . a possible mechanism allowing a broader niche emptying involves the production of toxins active against the animal host . if the toxin carrier had superior resistance to the resulting immunological response , manipulating the host immune state would then competitively benefit a suitably protected pathogen ( fig . consistently with this hypothesis , phages from e. coli o157::h7 carry genes coding for both immune - provoking toxins expressed just before lysis and for protection against the immune response ( plunkett et al . residents that are susceptible to the invaders phage can act as a phage - multiplying factory , and consequently multiply the phage - coded toxin as well ( gamage et al . 2003 ) . upon infection by the virus , susceptibles are turned into factories producing more viruses . if the virus also coded for niche - modifying proteins ( proteins impacting the extracellular environment ) , the susceptibles would then also become factories for such proteins , directly or indirectly triggering a change in the environment benefiting the phage carrier . the resultant immune response can then favour the protected invaders , at the expense of a broad spectrum of residents including those that resist the phage but are susceptible to the immune response . as genes allowing even further protection from the inflammatory response are also carried in the e. coli o157::h7 genome outside of the released phage ( hayashi et al . 2001 ) , even if rare residents are able to become lysogenised , they will not carry all the genes required for long - term persistence . we propose below that e. coli o157::h7 would thus preserve a competitive advantage during invasions . given the long co - existence of multiple phages within the e. coli o157::h7 lineage ( feng et al . 1998 ; ohnishi et al . 2001 ) these bacteria may have been proactive invaders for a long time . several more microbial examples of an immuno - manipulative anti - competitor strategy have been recently proposed ( lysenko et al . for example , lysenko et al . 2005 demonstrated in vivo that haemophilus influenze outcompetes streptococcus pneumoniae in the upper respiratory tract by recruiting and then by activating neutrophils and complement , to which streptococcus is more sensitive than haemophilus . ( 2006 ) demonstrated that more virulent strains of the rodent malaria parasite plasmodium chabaudi experience a stronger within - host competitive advantage in immuno - competent hosts , relative to competition in immunodeficient hosts . ( 2007 ) demonstrated that host inflammatory responses triggered by salmonella enterica serovar typhimurium aid its ability to invade resident gut microbiota . carrier lineages carry genes causing the explosive suicide of the host , with a low probability , the explosive suicide of the host , and the release of bactericidal toxins or bacteriocins ( small peptides or heat stable proteins , riley and wertz 2002 ) . furthermore , nonlysed carriers are immune to the bacteriocin , thanks to the specific antidote coding genes carried along with the bacteriocin genes . thus , the environmental change resulting from the release of bacteriocins can favour the carriers over any susceptible resident populations ( fig . this type of interference competition is found across a range of different bacterial species and analogous examples can even be found in eukaryotic yeasts ( wickner 1996 ; riley and wertz 2002 ) . schematic diagrams of ( a ) chemical , ( b ) viral and ( c ) immuno - manipulative weapons of mass microbial destruction . squares represent microbial densities of competing weapons - carriers ( c ) and susceptibles ( s ) . circles represent the density of weapon , the agent of environmental change ( b for bacteriocin , or other chemical toxin , v for temperate phage virus , and t for immuno - provocative toxin ) . the cylinder represents a multicellular host , the subject of immuno - manipulation by t. pointed arrows represent a positive relationship ( e.g. c increase the density of b ) , flat arrows represent a negative relationship ( e.g. b decrease the density of s ) . one well - documented example is that of escherichia coli strains producing colicins , bacteriocins active against other e. coli . previous experimental and theoretical studies of colicin - mediated bacterial competition have shown that colicin - producers can invade in a spatially structured environment and if unstructured , then only if sufficiently abundant ( chao and levin 1981 ; levin 1988 ; frank 1994 ; durrett and levin 1997 ) . in a spatially structured environment , the modification of the environment due to the secretion of colicins is a local phenomenon : with high local colicin concentrations killing neighbouring competitors , and thereby freeing nutrients for the invaders . invaders can then spread as a consequence of sequential waves of colicins followed by colicin - mediated resident death leaving a competitive vacuum ( chao and levin 1981 ; levin 1988 ; frank 1994 ; durrett and levin 1997 ) . the invasion of the bacteriocin producers favours the evolution of resistant bacteria . resistance often comes at some cost to bacteria ( either metabolically through the production of an immunity compound or through the loss of membrane receptors ) , and so allows for the coexistence , in a spatially structured environment ( through negative frequency - dependent selection ) of producer , sensitive , and resistant bacteria ; producers out - compete sensitives , resistants beat producers , and sensitives beat resistants , creating a rock - paper - scissors dynamic ( czrn et al . 2002 ; kerr et al . the pleiotropic fitness cost associated with resistance is also likely to explain the diversity in bacteriocin types seen within a microbial species . being resistant to all bacteriocin types will often be too costly , so resistance to the most common bacteriocins will be frequent . this favours bacteria producing rare bacteriocins , which should then in turn become more widespread , thereby maintaining diversity ( west et al . more recently work on yeasts infected with double stranded rna viruses has helped to further elucidate the ecological conditions that favour the production of anti - competitor toxins . these viruses can be considered similar to bacteriocins as there is no known horizontal transmission of the virus between yeast cells : daughter cells are instead infected via the cytoplasm of their parents ( magliani et al . in saccharomyces cerevisiae , the k1 toxin system requires two viruses , one that encodes the toxin and immunity proteins and a second that is involved in encapsulating the virus . unlike most bacteriocins , lysis is not required to release the toxin , but there is a pronounced fitness cost when carrying the virus ( wloch - salamon et al . were able to show the importance of density in determining the success of toxin - producing strains . density dependence has previously been shown to be an important ecological determinant in allelopathic interactions ( brown et al . 2006 ) , but experimental work has focused on the frequency of the producing and sensitive strains . grieg and travisano ( 2008 ) demonstrated that toxin producers , even when at intermediate frequencies , were unable to invade if the overall microbial density was low . this effect was also observed in structured environments , which tend to favour toxin production . when both producers and sensitive strains are rare there will only be limited interaction between them and there will be little benefit in producing toxins . instead toxin producers will be at a disadvantage due to the metabolic and pleiotropic costs of producing toxins . nutrient availability , dispersal , and their effect on interference competition in yeast have also been considered experimentally . it has been suggested that toxin production might act as strategy of last resort among starving cells by killing competitors for nutrients ( ivanovska and hardwick 2005 ) . wloch - salamon et al . ( 2008 ) grew s. cerevisiae toxin - producing strains in competition with sensitives under low and high nutrient conditions . nutrient availability was manipulated by varying the rate of nutrient replacement , following initial growth to carrying capacity ( therefore controlling initial densities ) . wloch - salamon et al . ( 2008 ) found that toxin producers were able to out - compete sensitive cells only in high nutrient environments and were out - competed when grown under low nutrient conditions . they argued that the lack of toxin production seen in the low nutrient environment is unlikely to be solely due to a nutritional constraint , but rather due in addition to a physiological change in toxin production or resistance developing after the nutrients have been depleted ( wloch - salamon et al . they conclude that this acts as support for frank 's ( 1994 ) theoretical prediction that toxin production has evolved to occur as a competitive strategy under conditions where resources are abundant and growth is allowed . this makes it unlikely that toxin production is used to acquire resources from sensitive cells , but is more important in the invasion of high nutrient patches . they also looked at the effect on dispersal , finding that limited dispersal favoured toxin producers . a benefit was still observed in treatments with high dispersal , but it was insufficient to compensate for the resource cost of carrying the toxin - producing virus . these results are consistent with chao and levin 's ( 1981 ) results in spatially structured environments as dispersal will reduce the chances that related cells will profit from toxin production . there is still much about the ecology and evolution of interference competition through toxin production that remains to be properly understood . a huge diversity exists in the types of toxins used from bacteriocins to antibiotics : they can differ in the specificity of killing action , in their ability to diffuse , and even in their respective half - lives in the environment ( ito et al . 1970 ; michel - briand and baysse 2002 ; riley and wertz 2002 ; cascales et al . all of these factors will greatly change how the environment is modified to allow toxin producers to invade . one constraint , however , that is faced by all toxin producers trying to invade a population is that for every halving of the invader population , the cost to individual invaders of successful environmental modification will double . given limited resources , these increasing costs will ultimately present a rarity threshold to invasion ( chao and levin 1981 ; levin 1988 ; frank 1994 ; durrett and levin 1997 ; gordon and riley 1999 ) . we have just seen that for chemical allelopathy in an unstructured environment , there exists a rarity threshold , below which allelopathic lineages can not invade . brown et al . ( 2006 ) demonstrated that this threshold can be reduced or eliminated for other forms of allelopathy . specifically , weapons that are capable of self - amplification can allow vanishingly rare carrier lineages to invade and replace residents . a microbial example is provided by bacteria and some of their phages ( viruses that can infect bacteria ) , which can function as bacteriocins ( bossi et al . 2003 ) but with the additional capacity of autocatalytic amplification on victims ( brown et al . more specifically , we focus on temperate phages , which are viruses that are able to be transmitted either vertically or horizontally . the most common is the lytic cycle ( rapid host lysis and production of numerous horizontally transmissible viral particles ) . very rarely , however , the phage can lysogenise the host , persisting in a dormant state while allowing the survival of the infected bacteria . this dormant phage is then replicated with the bacterial genome , and thus vertically transmitted upon bacterial division . furthermore , this vertically transmitted carried - phage provides immunity to its carrier - bacteria against further horizontal infection by this phage ( adams 1959 ; campbell 1996 ) . upon rare spontaneous induction of the carried - phage , viral progeny experimental competition of isogenic phage - carrying and susceptible bacterial lineages over a range of initial ratios illustrates that the ability of the released temperate phage 1b ) allows the phage - resistant carrier lineage to invade more rapidly when rare ( brown et al . autocatalytic environmental modification , such as the one triggered by the release of phage , can be seen as a public good that is expensive to produce initially but which can then be amplified at a negligible cost , therefore allowing an escape from the rarity threshold ( brown et al . indirect mutualist ( the enemy of your enemy is your friend ) interaction between bacteria and temperate phage can also be viewed in terms of parasite - mediated apparent competition , whereby the outcome of competition between two competitors is determined by their relative susceptibility to a shared parasite ( hudson and greenman 1998 ; tompkins et al . 2001 ; joo et al . for instance , one of the leading hypotheses for the replacement of resident red squirrels by invasive grey squirrels in the uk is the differential susceptibility to a parapoxvirus brought by the invasive greys ( rushton et al . however , unlike the one - off invasion of the uk by grey squirrels plus a parapoxvirus , food - borne pathogens such as e. coli o157::h7 and its suite of phages continuously invade new digestive tracts . we will propose below that this distinction between once - off lucky and recurrent proactive parasite - mediation can be explained in an evolutionary framework . both experimental and model results illustrate how the level of amplification is determined by the availability of susceptibles in the initial population ( brown et al . when susceptibles are sufficiently rare , the phage and colicin models converge as the phage ceases to gain any multiplicative advantage due to the rarity of susceptibles , acting purely through its direct killing effect . whereas phage can act essentially as a colicin - style nonmultiplicative killer when their carriers are numerically dominant , they do so in a less efficient manner due to their relatively wasteful capacity for replication ( e.g. effective colicin burst - size is at least 10-fold greater , gordon and riley 1999 ) . therefore for a high carrier frequency , colicin production is more efficient . given these differences between colicin and phage mediation , phage - carriers are more likely to be successful in invasive lineages , as they grow in the wake of the competitive vacuum left by their virulent indirect - mutualists . in contrast , a colicin - strategy specialising on killing without amplification by the susceptibles has an advantage over phage - carriers in a defensive context : their higher productivity ensures that they can not be as easily invaded when residents ( brown et al . to this point we have focused solely on a single invasion event into a resident population consisting entirely of susceptibles . however , given the diversity of normal microbial communities within the gut , only a minority of the resident bacteria will be susceptible to any specific bacteriocin or phage . consider a distinct resistant lineage , and the problem the carriers can only attack susceptibles , and resistants gain from the suppression of the susceptible population , along with the carriers . following the eradication of susceptibles , resistants will outcompete carriers due to the latter 's lytic behaviour , leaving an endpoint of pure resistants . this might not be the end of the game ; however , given that phage - resistant bacteria are likely to carry a cost relative to susceptibles ( resistance is often generated via the loss of useful membrane proteins that are recognized by both phages and bacteriocins ) . resistants could be outcompeted by susceptibles which are in turn outcompeted by carriers ( at least , in a structured environment for bacteriocin producers ) that are themselves outcompeted by these resistant bacteria , potentially promoting rock / paper / scissors dynamics ( czrn et al . 2002 ; kerr et al . 2002 ; kirkup and riley 2004 ; brown et al . a possible mechanism allowing a broader niche emptying involves the production of toxins active against the animal host . if the toxin carrier had superior resistance to the resulting immunological response , manipulating the host immune state would then competitively benefit a suitably protected pathogen ( fig . consistently with this hypothesis , phages from e. coli o157::h7 carry genes coding for both immune - provoking toxins expressed just before lysis and for protection against the immune response ( plunkett et al . residents that are susceptible to the invaders phage can act as a phage - multiplying factory , and consequently multiply the phage - coded toxin as well ( gamage et al . if the virus also coded for niche - modifying proteins ( proteins impacting the extracellular environment ) , the susceptibles would then also become factories for such proteins , directly or indirectly triggering a change in the environment benefiting the phage carrier . the resultant immune response can then favour the protected invaders , at the expense of a broad spectrum of residents including those that resist the phage but are susceptible to the immune response . as genes allowing even further protection from the inflammatory response are also carried in the e. coli o157::h7 genome outside of the released phage ( hayashi et al . 2001 ) , even if rare residents are able to become lysogenised , they will not carry all the genes required for long - term persistence . we propose below that e. coli o157::h7 would thus preserve a competitive advantage during invasions . given the long co - existence of multiple phages within the e. coli o157::h7 lineage ( feng et al . 1998 ; ohnishi et al . 2001 ) these bacteria may have been proactive invaders for a long time . several more microbial examples of an immuno - manipulative anti - competitor strategy have been recently proposed ( lysenko et al . 2005 , stecher et al . for example , lysenko et al . 2005 demonstrated in vivo that haemophilus influenze outcompetes streptococcus pneumoniae in the upper respiratory tract by recruiting and then by activating neutrophils and complement , to which streptococcus is more sensitive than haemophilus . similarly , raberg et al . ( 2006 ) demonstrated that more virulent strains of the rodent malaria parasite plasmodium chabaudi experience a stronger within - host competitive advantage in immuno - competent hosts , relative to competition in immunodeficient hosts . ( 2007 ) demonstrated that host inflammatory responses triggered by salmonella enterica serovar typhimurium aid its ability to invade resident gut microbiota . a broad strand of experimental and theoretical work on the ecology of bacteriocin - mediated competition highlights the importance of spatial structure in mediating the outcome of competition , with a broad consensus recognising that structured environments promote the invasion of rare killers by increasing the local density of chemical weapons to an effective dose ( chao and levin 1981 ; levin 1988 ; frank 1994 ; durrett and levin 1997 ; gordon and riley 1999 ) . the impact of spatial structure on the relative costs and benefits of investment in allelopathy also have strong implications on an evolutionary time scale . modelling work by gardner et al . ( 2004 ) showed that the evolution of bacteriocin production should be most favoured when producers and sensitives interact locally at intermediate frequencies . when toxin producers are locally scarce , they are unable to generate sufficient chemical weapons ( and therefore sufficient competitor deaths ) to compensate for the cost of production . in contrast when toxin producers are locally dominant , producing toxins has little benefit as there are few competitors to kill , and hence less available resources to gain from their death . only when producers are at intermediate local frequencies will bacteriocin production confer the greatest fitness advantage , by maximising gains from competitive escape for a given investment in killing . gardner et al . ( 2004 ) go on to illustrate that the production of anti - competitor chemical weapons can be understood as an example of microbial spite . a spiteful trait imposes costs on both actor and recipient ( hamilton 1964 , 116 ; hamilton 1970 ; gardner and west 2006 ; gardner et al . 2007 ) , and so allelopathy can be considered a spiteful trait as it has a negative fitness impact on the actor cell producing the toxin ( due to metabolic costs of toxin production , and for many bacteriocins , cell lysis so as to release the toxins ) and imposes a clear cost on recipient cells that are sensitive to the action of the toxin . for spite to evolve , hamilton 's rule rb > for a spiteful trait , c refers to the cost to the actor ( c > 0 ) and b refers to the cost ( or negative benefit , b < 0 ) to the recipients . given c > 0 > b , hamilton 's rule can be satisfied , only if relatedness r is negative . negative relatedness occurs between individuals that are less genetically similar ( at the social trait of interest ) than individuals chosen at random from the competitive arena ( hamilton 1970 ; grafen 1985 ; queller 1994 ) . thus , if a spiteful bacterium killed a random selection of neighbours ( who by definition have relatedness of zero , queller 1994 ) , then this trait would not be favoured . however , the specificity of bacteriocin action allows for the molecular discrimination between kin ( who carry appropriate immunity genes ) and non - kin ( who do not ) , and therefore the generation of negative relatedness between discriminately spiteful actor and unfortunate recipient . on the level of a clonal bacteriocin - producing lineage , bacteriocins are selfish ( i.e. returning direct benefits to the lineage through release from competition ) ; however bacteriocin production is certainly spiteful ( c > 0 > b ) at the level of a self - destructing bacterium producing the toxins ( gardner et al . the relative weighting of the indirect and direct effects of microbial spite change markedly when we consider the more complex dynamics created by temperate phage weapons amplifying on susceptibles ( dionisio 2007 ) , or immunological responses being ramped up by a sensitive multicellular host ( fig . 1 ) . in particular , the autocatalytic dynamic most evident in the amplification of phage on susceptibles will allow a greater destruction of competitors when the focal spiteful lineage is rare and sensitives are common , broadening the ecological conditions favouring microbial spite . of course , the temperate phage symbiont can not be considered a mere adaptation of its carrier . rather , one should consider the strategic interests of both players in such dangerous liaisons ( van baalen and jansen 2001 ) . the functional similarities from the carrier perspective between bacteriocins and temperate phage propagules ( bossi et al . 2003 ; brown et al . 2006 ; joo 2006 ) present an intrguing bridge between public goods ( weapons are a public good of the producer lineage , liberating them from competitors ) and symbionts ( brown and taddei 2007 ) . the impact of within - host competition on the evolution of virulence has been the subject of a diverse range of models , offering contrasting explanations for either an increase or a decrease in virulence as within - host diversity increases ( van baalen and sabelis 1995 ; frank 1996 ; brown et al . in contrast , in the case of chemical warfare overall virulence is predicted to be maximised at intermediate within - host diversities , at the point where spiteful killing is maximised and therefore overall microbial growth is most disrupted ( gardner et al . a partial test of this theory using photorhabdus spp . in caterpillars showed that single strain infections were more virulent than mixes of bacteriocin producer and a sensitive species of photorhabdus ( massey et al . further work looking at the effect of migration on virulence in a bacteria / nematode system found that virulence is reduced at high migration rates , leading to more local diversity and therefore more bacterial interference competition is likely to occur ( vigneux et al . the relationship between virulence and within - host diversity changes yet again when alternative mechanisms of microbial interference competition are considered . temperate phage mediated killing will create the greatest demographic disruption to overall microbial growth when phage carriers are scarce and susceptibles are common ( brown et al . when pro - inflammatory toxins are involved , virulence is likely to become decoupled from overall bacterial density , and be largely driven by the provocative actions of the toxin - producing lineage ( brown et al . in contrast , immuno - manipulation may reduce virulence in other systems , if the manipulation is carried out by resident chronic infections to skew or neutralise an inflammatory response ( riffkin et al . 2005 ; sansonetti and di santo 2007 ; alizon and van baalen 2008 ) or to increase resistance to superinfection ( brown and grenfell 2001 ) . more generally , the complex links between microbes and immunopathology are likely to limit the applicability of general rules for the evolution of virulence ( graham et al . 2005 ; day et al . 2007 ) . in this review we have briefly sketched three distinct mechanisms of microbial allelopathy , each with a distinct mediator of competition : chemicals , viruses or immunity . the study of chemically mediated microbial competition is by far the most advanced experimentally and theoretically , and can therefore serve as a template for future studies on virus- and immune - mediated allelopathy ( e.g. understanding and characterising density and frequency dependence , specificity and diversity ) . in return , the study of virus- and immune - mediated allelopathy bring to the fore issues of co - evolutionary dangerous liaisons between microbial provocator and their weapon ( virus or multicellular host ) that are also present , yet less evident , in chemically mediated competition . for instance , numerous bacteriocinogenic bacteria owe their allelopathy to mobile genetic elements , mostly plasmids but also transposons and even to extinct phages ( riley and wertz 2002 ) . a population that increases on arriving in a new habitat can do so in one of three ways . first , because of some chance preadaptation , mutation or environmental change , organisms find themselves favoured in their new environment ( sakai et al . parasites can play a determining role in these chance events , both through preadapted resistance ( rushton et al . 2000 ; tompkins et al . 2002 ) or through chance losses in parasitic environment ( competitive release hypothesis , torchin et al . second , organisms which exploit , through repeated generations , regularly appearing environmental disturbances ( for instance new forest patches following the death of a mature tree ) can evolve adaptations favouring their growth in these disturbed environments ( crawley 1997 ) . third , given certain genotypes become better adapted than their competitors to disturbed environments , they can then evolve adaptations to increase the environmental disturbance from which they benefit , i.e. become proactive invaders ( brown et al . cheatgrass , bromus tectorum , is one candidate example , a plant that not only grows well on niches emptied by fire , but also favours fire itself ( melgoza et al . similarly , bacteria inhabiting hosts where inflammation is recurrent will be selected to resist the associated immune response . given better adaptation to inflammation , selection could then favour the provocation of inflammation itself , through the acquisition of virulence factors . this principle of relative advantage suggests that invaders can gain by actively deteriorating their own environment . when the invader is a pathogen and the environment is a host , this strategy can have important implications for virulence . a broad strand of experimental and theoretical work on the ecology of bacteriocin - mediated competition highlights the importance of spatial structure in mediating the outcome of competition , with a broad consensus recognising that structured environments promote the invasion of rare killers by increasing the local density of chemical weapons to an effective dose ( chao and levin 1981 ; levin 1988 ; frank 1994 ; durrett and levin 1997 ; gordon and riley 1999 ) . the impact of spatial structure on the relative costs and benefits of investment in allelopathy also have strong implications on an evolutionary time scale . modelling work by gardner et al . ( 2004 ) showed that the evolution of bacteriocin production should be most favoured when producers and sensitives interact locally at intermediate frequencies . when toxin producers are locally scarce , they are unable to generate sufficient chemical weapons ( and therefore sufficient competitor deaths ) to compensate for the cost of production . in contrast when toxin producers are locally dominant , producing toxins has little benefit as there are few competitors to kill , and hence less available resources to gain from their death . only when producers are at intermediate local frequencies will bacteriocin production confer the greatest fitness advantage , by maximising gains from competitive escape for a given investment in killing . gardner et al . ( 2004 ) go on to illustrate that the production of anti - competitor chemical weapons can be understood as an example of microbial spite . a spiteful trait imposes costs on both actor and recipient ( hamilton 1964 , 116 ; hamilton 1970 ; gardner and west 2006 ; gardner et al . 2007 ) , and so allelopathy can be considered a spiteful trait as it has a negative fitness impact on the actor cell producing the toxin ( due to metabolic costs of toxin production , and for many bacteriocins , cell lysis so as to release the toxins ) and imposes a clear cost on recipient cells that are sensitive to the action of the toxin . for spite to evolve , hamilton 's rule rb > for a spiteful trait , c refers to the cost to the actor ( c > 0 ) and b refers to the cost ( or negative benefit , b < 0 ) to the recipients . given c > 0 > b , hamilton 's rule can be satisfied , only if relatedness r is negative . negative relatedness occurs between individuals that are less genetically similar ( at the social trait of interest ) than individuals chosen at random from the competitive arena ( hamilton 1970 ; grafen 1985 ; queller 1994 ) . thus , if a spiteful bacterium killed a random selection of neighbours ( who by definition have relatedness of zero , queller 1994 ) , then this trait would not be favoured . however , the specificity of bacteriocin action allows for the molecular discrimination between kin ( who carry appropriate immunity genes ) and non - kin ( who do not ) , and therefore the generation of negative relatedness between discriminately spiteful actor and unfortunate recipient . on the level of a clonal bacteriocin - producing lineage , bacteriocins are selfish ( i.e. returning direct benefits to the lineage through release from competition ) ; however bacteriocin production is certainly spiteful ( c > 0 > b ) at the level of a self - destructing bacterium producing the toxins ( gardner et al . the relative weighting of the indirect and direct effects of microbial spite change markedly when we consider the more complex dynamics created by temperate phage weapons amplifying on susceptibles ( dionisio 2007 ) , or immunological responses being ramped up by a sensitive multicellular host ( fig . 1 ) . in particular , the autocatalytic dynamic most evident in the amplification of phage on susceptibles will allow a greater destruction of competitors when the focal spiteful lineage is rare and sensitives are common , broadening the ecological conditions favouring microbial spite . of course , the temperate phage symbiont can not be considered a mere adaptation of its carrier . rather , one should consider the strategic interests of both players in such dangerous liaisons ( van baalen and jansen 2001 ) . the functional similarities from the carrier perspective between bacteriocins and temperate phage propagules ( bossi et al . 2003 ; brown et al . 2006 ; joo 2006 ) present an intrguing bridge between public goods ( weapons are a public good of the producer lineage , liberating them from competitors ) and symbionts ( brown and taddei 2007 ) . the impact of within - host competition on the evolution of virulence has been the subject of a diverse range of models , offering contrasting explanations for either an increase or a decrease in virulence as within - host diversity increases ( van baalen and sabelis 1995 ; frank 1996 ; brown et al . 2002 ; west and buckling 2003 ) . in contrast , in the case of chemical warfare overall virulence is predicted to be maximised at intermediate within - host diversities , at the point where spiteful killing is maximised and therefore overall microbial growth is most disrupted ( gardner et al . a partial test of this theory using photorhabdus spp . in caterpillars showed that single strain infections were more virulent than mixes of bacteriocin producer and a sensitive species of photorhabdus ( massey et al . further work looking at the effect of migration on virulence in a bacteria / nematode system found that virulence is reduced at high migration rates , leading to more local diversity and therefore more bacterial interference competition is likely to occur ( vigneux et al . the relationship between virulence and within - host diversity changes yet again when alternative mechanisms of microbial interference competition are considered . temperate phage mediated killing will create the greatest demographic disruption to overall microbial growth when phage carriers are scarce and susceptibles are common ( brown et al . when pro - inflammatory toxins are involved , virulence is likely to become decoupled from overall bacterial density , and be largely driven by the provocative actions of the toxin - producing lineage ( brown et al . in contrast , immuno - manipulation may reduce virulence in other systems , if the manipulation is carried out by resident chronic infections to skew or neutralise an inflammatory response ( riffkin et al . 1996 ; graham 2002 ; graham et al . 2005 ; sansonetti and di santo 2007 ; alizon and van baalen 2008 ) or to increase resistance to superinfection ( brown and grenfell 2001 ) . more generally , the complex links between microbes and immunopathology are likely to limit the applicability of general rules for the evolution of virulence ( graham et al . 2005 ; day et al . 2007 ) . in this review we have briefly sketched three distinct mechanisms of microbial allelopathy , each with a distinct mediator of competition : chemicals , viruses or immunity . the study of chemically mediated microbial competition is by far the most advanced experimentally and theoretically , and can therefore serve as a template for future studies on virus- and immune - mediated allelopathy ( e.g. understanding and characterising density and frequency dependence , specificity and diversity ) . in return , the study of virus- and immune - mediated allelopathy bring to the fore issues of co - evolutionary dangerous liaisons between microbial provocator and their weapon ( virus or multicellular host ) that are also present , yet less evident , in chemically mediated competition . for instance , numerous bacteriocinogenic bacteria owe their allelopathy to mobile genetic elements , mostly plasmids but also transposons and even to extinct phages ( riley and wertz 2002 ) . a population that increases on arriving in a new habitat can do so in one of three ways . first , because of some chance preadaptation , mutation or environmental change , organisms find themselves favoured in their new environment ( sakai et al . parasites can play a determining role in these chance events , both through preadapted resistance ( rushton et al . 2000 ; tompkins et al . 2002 ) or through chance losses in parasitic environment ( competitive release hypothesis , torchin et al . second , organisms which exploit , through repeated generations , regularly appearing environmental disturbances ( for instance new forest patches following the death of a mature tree ) can evolve adaptations favouring their growth in these disturbed environments ( crawley 1997 ) . third , given certain genotypes become better adapted than their competitors to disturbed environments , they can then evolve adaptations to increase the environmental disturbance from which they benefit , i.e. become proactive invaders ( brown et al . cheatgrass , bromus tectorum , is one candidate example , a plant that not only grows well on niches emptied by fire , but also favours fire itself ( melgoza et al . similarly , bacteria inhabiting hosts where inflammation is recurrent will be selected to resist the associated immune response . given better adaptation to inflammation , selection could then favour the provocation of inflammation itself , through the acquisition of virulence factors . this principle of relative advantage suggests that invaders can gain by actively deteriorating their own environment . when the invader is a pathogen and the environment is a host , this strategy can have important implications for virulence .

invading an occupied niche is a formidable ecological challenge , and one of particular human importance in the context of food - borne microbial pathogens . we discuss distinct categories of invader - triggered environmental change that facilitate invasion by emptying their niche of competitors . evidence is reviewed that gut bacteria use such strategies to manipulate their environment ( via bacteriocins , temperate phage viruses or immuno - manipulation ) at the expense of their competitors are reviewed . the possible virulence implications of microbial warfare among multiple co - infecting strains are diverse . killing competitors can reduce virulence by reducing overall microbial densities , or increase virulence if for example the allelopathic mechanism involves immuno - manipulation . finally , we place microbial anti - competitor strategies in a social evolution framework , highlighting how costly anti - competitor strategies can be understood as examples of microbial spite . we conclude by discussing other invasive species that have also developed such proactive strategies of invasion .

Introduction Chemical weapons Escaping the rarity threshold: biological weapons Provoking the superpower: immuno-manipulation Discussion Social evolution Virulence Proactive invaders

however , if the invader is at a disadvantage in the context of the resident 's environment , one solution could be to modify the environment into a configuration favouring the invaders . building nests , spider webs or beaver dams are examples of extended phenotypes improving the actors niche , whereas the secretion of toxins is an example of niche - deterioration ( although offering a relative inclusive fitness advantage to the actor ) , also referred to as interference competition , or allelopathy ( fitter 2003 ) . thus , the environmental change resulting from the release of bacteriocins can favour the carriers over any susceptible resident populations ( fig . schematic diagrams of ( a ) chemical , ( b ) viral and ( c ) immuno - manipulative weapons of mass microbial destruction . circles represent the density of weapon , the agent of environmental change ( b for bacteriocin , or other chemical toxin , v for temperate phage virus , and t for immuno - provocative toxin ) . the cylinder represents a multicellular host , the subject of immuno - manipulation by t. pointed arrows represent a positive relationship ( e.g. previous experimental and theoretical studies of colicin - mediated bacterial competition have shown that colicin - producers can invade in a spatially structured environment and if unstructured , then only if sufficiently abundant ( chao and levin 1981 ; levin 1988 ; frank 1994 ; durrett and levin 1997 ) . in a spatially structured environment , the modification of the environment due to the secretion of colicins is a local phenomenon : with high local colicin concentrations killing neighbouring competitors , and thereby freeing nutrients for the invaders . resistance often comes at some cost to bacteria ( either metabolically through the production of an immunity compound or through the loss of membrane receptors ) , and so allows for the coexistence , in a spatially structured environment ( through negative frequency - dependent selection ) of producer , sensitive , and resistant bacteria ; producers out - compete sensitives , resistants beat producers , and sensitives beat resistants , creating a rock - paper - scissors dynamic ( czrn et al . more recently work on yeasts infected with double stranded rna viruses has helped to further elucidate the ecological conditions that favour the production of anti - competitor toxins . these viruses can be considered similar to bacteriocins as there is no known horizontal transmission of the virus between yeast cells : daughter cells are instead infected via the cytoplasm of their parents ( magliani et al . unlike most bacteriocins , lysis is not required to release the toxin , but there is a pronounced fitness cost when carrying the virus ( wloch - salamon et al . when both producers and sensitive strains are rare there will only be limited interaction between them and there will be little benefit in producing toxins . nutrient availability , dispersal , and their effect on interference competition in yeast have also been considered experimentally . it has been suggested that toxin production might act as strategy of last resort among starving cells by killing competitors for nutrients ( ivanovska and hardwick 2005 ) . they also looked at the effect on dispersal , finding that limited dispersal favoured toxin producers . a huge diversity exists in the types of toxins used from bacteriocins to antibiotics : they can differ in the specificity of killing action , in their ability to diffuse , and even in their respective half - lives in the environment ( ito et al . a microbial example is provided by bacteria and some of their phages ( viruses that can infect bacteria ) , which can function as bacteriocins ( bossi et al . more specifically , we focus on temperate phages , which are viruses that are able to be transmitted either vertically or horizontally . very rarely , however , the phage can lysogenise the host , persisting in a dormant state while allowing the survival of the infected bacteria . for instance , one of the leading hypotheses for the replacement of resident red squirrels by invasive grey squirrels in the uk is the differential susceptibility to a parapoxvirus brought by the invasive greys ( rushton et al . however , unlike the one - off invasion of the uk by grey squirrels plus a parapoxvirus , food - borne pathogens such as e. coli o157::h7 and its suite of phages continuously invade new digestive tracts . we will propose below that this distinction between once - off lucky and recurrent both experimental and model results illustrate how the level of amplification is determined by the availability of susceptibles in the initial population ( brown et al . whereas phage can act essentially as a colicin - style nonmultiplicative killer when their carriers are numerically dominant , they do so in a less efficient manner due to their relatively wasteful capacity for replication ( e.g. residents that are susceptible to the invaders phage can act as a phage - multiplying factory , and consequently multiply the phage - coded toxin as well ( gamage et al . if the virus also coded for niche - modifying proteins ( proteins impacting the extracellular environment ) , the susceptibles would then also become factories for such proteins , directly or indirectly triggering a change in the environment benefiting the phage carrier . the resultant immune response can then favour the protected invaders , at the expense of a broad spectrum of residents including those that resist the phage but are susceptible to the immune response . several more microbial examples of an immuno - manipulative anti - competitor strategy have been recently proposed ( lysenko et al . 2005 demonstrated in vivo that haemophilus influenze outcompetes streptococcus pneumoniae in the upper respiratory tract by recruiting and then by activating neutrophils and complement , to which streptococcus is more sensitive than haemophilus . ( 2006 ) demonstrated that more virulent strains of the rodent malaria parasite plasmodium chabaudi experience a stronger within - host competitive advantage in immuno - competent hosts , relative to competition in immunodeficient hosts . carrier lineages carry genes causing the explosive suicide of the host , with a low probability , the explosive suicide of the host , and the release of bactericidal toxins or bacteriocins ( small peptides or heat stable proteins , riley and wertz 2002 ) . schematic diagrams of ( a ) chemical , ( b ) viral and ( c ) immuno - manipulative weapons of mass microbial destruction . squares represent microbial densities of competing weapons - carriers ( c ) and susceptibles ( s ) . circles represent the density of weapon , the agent of environmental change ( b for bacteriocin , or other chemical toxin , v for temperate phage virus , and t for immuno - provocative toxin ) . the cylinder represents a multicellular host , the subject of immuno - manipulation by t. pointed arrows represent a positive relationship ( e.g. previous experimental and theoretical studies of colicin - mediated bacterial competition have shown that colicin - producers can invade in a spatially structured environment and if unstructured , then only if sufficiently abundant ( chao and levin 1981 ; levin 1988 ; frank 1994 ; durrett and levin 1997 ) . in a spatially structured environment , the modification of the environment due to the secretion of colicins is a local phenomenon : with high local colicin concentrations killing neighbouring competitors , and thereby freeing nutrients for the invaders . resistance often comes at some cost to bacteria ( either metabolically through the production of an immunity compound or through the loss of membrane receptors ) , and so allows for the coexistence , in a spatially structured environment ( through negative frequency - dependent selection ) of producer , sensitive , and resistant bacteria ; producers out - compete sensitives , resistants beat producers , and sensitives beat resistants , creating a rock - paper - scissors dynamic ( czrn et al . more recently work on yeasts infected with double stranded rna viruses has helped to further elucidate the ecological conditions that favour the production of anti - competitor toxins . these viruses can be considered similar to bacteriocins as there is no known horizontal transmission of the virus between yeast cells : daughter cells are instead infected via the cytoplasm of their parents ( magliani et al . unlike most bacteriocins , lysis is not required to release the toxin , but there is a pronounced fitness cost when carrying the virus ( wloch - salamon et al . when both producers and sensitive strains are rare there will only be limited interaction between them and there will be little benefit in producing toxins . nutrient availability , dispersal , and their effect on interference competition in yeast have also been considered experimentally . it has been suggested that toxin production might act as strategy of last resort among starving cells by killing competitors for nutrients ( ivanovska and hardwick 2005 ) . they argued that the lack of toxin production seen in the low nutrient environment is unlikely to be solely due to a nutritional constraint , but rather due in addition to a physiological change in toxin production or resistance developing after the nutrients have been depleted ( wloch - salamon et al . this makes it unlikely that toxin production is used to acquire resources from sensitive cells , but is more important in the invasion of high nutrient patches . they also looked at the effect on dispersal , finding that limited dispersal favoured toxin producers . a huge diversity exists in the types of toxins used from bacteriocins to antibiotics : they can differ in the specificity of killing action , in their ability to diffuse , and even in their respective half - lives in the environment ( ito et al . ( 2006 ) demonstrated that this threshold can be reduced or eliminated for other forms of allelopathy . more specifically , we focus on temperate phages , which are viruses that are able to be transmitted either vertically or horizontally . very rarely , however , the phage can lysogenise the host , persisting in a dormant state while allowing the survival of the infected bacteria . upon rare spontaneous induction of the carried - phage , viral progeny experimental competition of isogenic phage - carrying and susceptible bacterial lineages over a range of initial ratios illustrates that the ability of the released temperate phage 1b ) allows the phage - resistant carrier lineage to invade more rapidly when rare ( brown et al . autocatalytic environmental modification , such as the one triggered by the release of phage , can be seen as a public good that is expensive to produce initially but which can then be amplified at a negligible cost , therefore allowing an escape from the rarity threshold ( brown et al . for instance , one of the leading hypotheses for the replacement of resident red squirrels by invasive grey squirrels in the uk is the differential susceptibility to a parapoxvirus brought by the invasive greys ( rushton et al . however , unlike the one - off invasion of the uk by grey squirrels plus a parapoxvirus , food - borne pathogens such as e. coli o157::h7 and its suite of phages continuously invade new digestive tracts . we will propose below that this distinction between once - off lucky and recurrent proactive parasite - mediation can be explained in an evolutionary framework . both experimental and model results illustrate how the level of amplification is determined by the availability of susceptibles in the initial population ( brown et al . whereas phage can act essentially as a colicin - style nonmultiplicative killer when their carriers are numerically dominant , they do so in a less efficient manner due to their relatively wasteful capacity for replication ( e.g. given these differences between colicin and phage mediation , phage - carriers are more likely to be successful in invasive lineages , as they grow in the wake of the competitive vacuum left by their virulent indirect - mutualists . in contrast , a colicin - strategy specialising on killing without amplification by the susceptibles has an advantage over phage - carriers in a defensive context : their higher productivity ensures that they can not be as easily invaded when residents ( brown et al . the resultant immune response can then favour the protected invaders , at the expense of a broad spectrum of residents including those that resist the phage but are susceptible to the immune response . as genes allowing even further protection from the inflammatory response are also carried in the e. coli o157::h7 genome outside of the released phage ( hayashi et al . several more microbial examples of an immuno - manipulative anti - competitor strategy have been recently proposed ( lysenko et al . 2005 demonstrated in vivo that haemophilus influenze outcompetes streptococcus pneumoniae in the upper respiratory tract by recruiting and then by activating neutrophils and complement , to which streptococcus is more sensitive than haemophilus . ( 2006 ) demonstrated that more virulent strains of the rodent malaria parasite plasmodium chabaudi experience a stronger within - host competitive advantage in immuno - competent hosts , relative to competition in immunodeficient hosts . ( 2004 ) go on to illustrate that the production of anti - competitor chemical weapons can be understood as an example of microbial spite . 2007 ) , and so allelopathy can be considered a spiteful trait as it has a negative fitness impact on the actor cell producing the toxin ( due to metabolic costs of toxin production , and for many bacteriocins , cell lysis so as to release the toxins ) and imposes a clear cost on recipient cells that are sensitive to the action of the toxin . given c > 0 > b , hamilton 's rule can be satisfied , only if relatedness r is negative . negative relatedness occurs between individuals that are less genetically similar ( at the social trait of interest ) than individuals chosen at random from the competitive arena ( hamilton 1970 ; grafen 1985 ; queller 1994 ) . returning direct benefits to the lineage through release from competition ) ; however bacteriocin production is certainly spiteful ( c > 0 > b ) at the level of a self - destructing bacterium producing the toxins ( gardner et al . the relative weighting of the indirect and direct effects of microbial spite change markedly when we consider the more complex dynamics created by temperate phage weapons amplifying on susceptibles ( dionisio 2007 ) , or immunological responses being ramped up by a sensitive multicellular host ( fig . in particular , the autocatalytic dynamic most evident in the amplification of phage on susceptibles will allow a greater destruction of competitors when the focal spiteful lineage is rare and sensitives are common , broadening the ecological conditions favouring microbial spite . of course , the temperate phage symbiont can not be considered a mere adaptation of its carrier . in contrast , in the case of chemical warfare overall virulence is predicted to be maximised at intermediate within - host diversities , at the point where spiteful killing is maximised and therefore overall microbial growth is most disrupted ( gardner et al . further work looking at the effect of migration on virulence in a bacteria / nematode system found that virulence is reduced at high migration rates , leading to more local diversity and therefore more bacterial interference competition is likely to occur ( vigneux et al . the relationship between virulence and within - host diversity changes yet again when alternative mechanisms of microbial interference competition are considered . temperate phage mediated killing will create the greatest demographic disruption to overall microbial growth when phage carriers are scarce and susceptibles are common ( brown et al . when pro - inflammatory toxins are involved , virulence is likely to become decoupled from overall bacterial density , and be largely driven by the provocative actions of the toxin - producing lineage ( brown et al . in contrast , immuno - manipulation may reduce virulence in other systems , if the manipulation is carried out by resident chronic infections to skew or neutralise an inflammatory response ( riffkin et al . in this review we have briefly sketched three distinct mechanisms of microbial allelopathy , each with a distinct mediator of competition : chemicals , viruses or immunity . a population that increases on arriving in a new habitat can do so in one of three ways . first , because of some chance preadaptation , mutation or environmental change , organisms find themselves favoured in their new environment ( sakai et al . 2002 ) or through chance losses in parasitic environment ( competitive release hypothesis , torchin et al . in contrast when toxin producers are locally dominant , producing toxins has little benefit as there are few competitors to kill , and hence less available resources to gain from their death . ( 2004 ) go on to illustrate that the production of anti - competitor chemical weapons can be understood as an example of microbial spite . 2007 ) , and so allelopathy can be considered a spiteful trait as it has a negative fitness impact on the actor cell producing the toxin ( due to metabolic costs of toxin production , and for many bacteriocins , cell lysis so as to release the toxins ) and imposes a clear cost on recipient cells that are sensitive to the action of the toxin . returning direct benefits to the lineage through release from competition ) ; however bacteriocin production is certainly spiteful ( c > 0 > b ) at the level of a self - destructing bacterium producing the toxins ( gardner et al . the relative weighting of the indirect and direct effects of microbial spite change markedly when we consider the more complex dynamics created by temperate phage weapons amplifying on susceptibles ( dionisio 2007 ) , or immunological responses being ramped up by a sensitive multicellular host ( fig . in particular , the autocatalytic dynamic most evident in the amplification of phage on susceptibles will allow a greater destruction of competitors when the focal spiteful lineage is rare and sensitives are common , broadening the ecological conditions favouring microbial spite . of course , the temperate phage symbiont can not be considered a mere adaptation of its carrier . the functional similarities from the carrier perspective between bacteriocins and temperate phage propagules ( bossi et al . in contrast , in the case of chemical warfare overall virulence is predicted to be maximised at intermediate within - host diversities , at the point where spiteful killing is maximised and therefore overall microbial growth is most disrupted ( gardner et al . further work looking at the effect of migration on virulence in a bacteria / nematode system found that virulence is reduced at high migration rates , leading to more local diversity and therefore more bacterial interference competition is likely to occur ( vigneux et al . the relationship between virulence and within - host diversity changes yet again when alternative mechanisms of microbial interference competition are considered . temperate phage mediated killing will create the greatest demographic disruption to overall microbial growth when phage carriers are scarce and susceptibles are common ( brown et al . when pro - inflammatory toxins are involved , virulence is likely to become decoupled from overall bacterial density , and be largely driven by the provocative actions of the toxin - producing lineage ( brown et al . in contrast , immuno - manipulation may reduce virulence in other systems , if the manipulation is carried out by resident chronic infections to skew or neutralise an inflammatory response ( riffkin et al . in this review we have briefly sketched three distinct mechanisms of microbial allelopathy , each with a distinct mediator of competition : chemicals , viruses or immunity . a population that increases on arriving in a new habitat can do so in one of three ways . first , because of some chance preadaptation , mutation or environmental change , organisms find themselves favoured in their new environment ( sakai et al . 2002 ) or through chance losses in parasitic environment ( competitive release hypothesis , torchin et al . third , given certain genotypes become better adapted than their competitors to disturbed environments , they can then evolve adaptations to increase the environmental disturbance from which they benefit , i.e. when the invader is a pathogen and the environment is a host , this strategy can have important implications for virulence .

quinazolinone derivatives represent one of the most active classes of compounds possessing a wide spectrum of biological activity . several reports have been published on the biological activities of quinazolinone derivatives , including their anti - inflammatory [ 17 ] , antimalarial [ 8 , 9 ] , antimicrobial , anti - fungal , antibacterial [ 1016 ] , anticonvulsant [ 1720 ] , and antitumor [ 21 , 22 ] activities . moreover , large number of quinazolinone derivatives having substitution at 2 and 3 position by different heterocyclic moieties increases anti - inflammatory potential of quinazolinone derivatives . similarly , various azetidinones [ 2325 ] and thiazolidinones [ 26 , 27 ] have been reported to possess potent anti - inflammatory activity . looking to the medicinal importance of 4(3h)-quinazolinone , 4-thiazolidinone , and azetidinones , we report here the synthesis of a new class of heterocyclic molecules in which all of these moieties are present and try to develop potential bioactive molecules . the structures of the compounds synthesized were assigned on the basis of elemental analysis , ir , h nmr , and mass spectral data . as shown in scheme 1 , compounds 2-methylsubstitued-4h-3,1-benzoxazin-4-ones ( 1,2 ) were synthesized by the known procedure of bogert . substituted anthranilic acid reacted with acetic anhydride to give 2-methylsubstituted-4h-3,1-benzoxazin-4-ones ( 1,2 ) , which further reacted with 2-amino phenyl amine in acetic acid to yield 3-(2-aminophenyl)-2-methyl-6-substituted quinazolin-4-ones ( 3,4 ) . compounds ( 3,4 ) when treated with different substituted aromatic aldehydes formed various substituted arylidene derivatives ( 510 ) . these arylidene derivatives on treatment with chloroacetylchloride in presence of triethylamine yielded 3-[2-(3-chloro-2-oxo-4-substituted phenylazetidin-1-yl)phenyl]-2-methyl-6-substitutedquinazolin-4-ones ( 1116 ) . on the other hand , these arylidene derivatives on reaction with thioglycolic acid and anhydrous zncl2 furnished 3-[2-(2-substitutedphenyl-4-oxo-1,3-thiazolidin-3-yl)phenyl]-2-methyl-6-substitutedquinazolin-4-ones . the structures of the compounds synthesized were assigned on the basis of elemental analysis , ir , h nmr , and mass spectral data . the experiments were performed with albino rats of the charles - foster stain of either sex , excluding pregnant females , of 7095 d. weighting 100150 g. food ( chaw pallet ) and water were given to the animals ad libitum . anti - inflammatory activity against carrageenan - induced rat 's paw oedema this study was done with the procedure of winter et al . . the rats were divided into three groups ( control , treated , and standard drug ) of six animals each . a freshly prepared suspension of carrageenan ( 1% in 0.9% saline ) 0.05 mn was injected under the planter aponeurosis of the right hind paw of each rat . test compounds and standard drug were administered orally to the animals of drug - treated groups and the standard drug group , respectively , 1 h before the carrageenan injection . the paw volume of each rat was measured before 1 and after 3 h of carrageenan treatment with the help of a plethysmometer . the percent anti - inflammatory activity was calculated according to the formula given below ( 1)percentage of inhibition of oedema=(1vtvc)100 , where vt and vc are the mean increase in paw volume of rats of the treated and the control group , respectively . this method is based on the property of the test compound to antagonize the phenyl quinone - induced pain syndrome in mice . groups of five mice were injected intraperitoneally with 0.25 ml of a 0.02% solution of phenylquinone in ethanol ( 5% ) 1 h after of oral administration of the test compound . the number of writhes induced in each mouse was counted for 5 min ( between 5 and 10 min ) after injection of an irritant . ( 2)% protection=(1mean number of writhes in mice of test groupsmean number of writhes in mice of control group ) 100 . melting points were determined in one - end - open capillary tube on thomas hoover melting apparatus and are uncorrected . the purities of compounds were checked by thin layer chromatography ( tlc ) using silica gel - g coated plats . the spots were developed in an iodine chamber and visualized under ultraviolet ( uv ) lamp . infrared ( ir ) absorption spectra were recorded on perkin - elmer rx-1 ftir spectrometer using potassium bromide ( kbr ) pellet , and wave numbers were given in cm . the h nmr spectra were recorded in cdcl3 on a bruker avance ii 400 nmr spectrometer ( 400 mhz ) . the c nmr spectra were recorded in cdcl3 on a bruker avance ii 400 nmr spectrometer operating at 100 mhz . the chemical shifts are reported in part per million ( ppm ) using trimethylsilane ( tms ) as an internal standard . elemental analysis ( c , h , n ) was performed on perkin - elmer 2400 analyzer , and values were either in 0.4% of the calculated . 3-(2-aminophenyl)-2-methyl - quinazolin-4-one ( 3 ) a mixture of 2-methyl-4h-3,1-benzoxazin-4-one ( 1 ) ( 0.01 mole ) with 2-aminophenylamine ( 0.01 mole ) in acetic acid ( 40 ml ) was refluxed for 7 hr . in the presence of sodium acetate . the solid thus obtained was filtered off and recrystallized from toluene to give compound 3 ( 78% ) . ir ( kbr ) max in cm : 3033 ( ch aromatic ) , 2920 ( ch n ) ; hnmr ( cdcl3 ) in ppm : 9.10 ( s , 2h , nh2 exchangeable ) , 6.897.61 ( m , 8h , ar h ) , 2.29 ( s , 3h , ch3 ) ; anal . calcd . for c15h13n3o : c , 71.71 ; h , 5.18 ; n , 16.73 : found : c , 71.85 ; h , 5.20 ; n , 16.68 : ms [ m ] at m / z 251 . 199c , ir ( kbr ) max in cm : 3030 ( ch aromatic ) , 2925 ( ch aliphatic ) , 1695 ( c = o ) , 1598 ( c = n ) ; h nmr ( cdcl3 ) in ppm : 9.06 ( s , 2h , nh2 exchangeable ) , 6.857.51 ( m , 7h , ar h ) , 2.24 ( s , 3h , ch3 ) ; anal . calcd . for c15h12n3obr : c , 54.55 ; h , 3.64 ; n , 12.73 : found : c , 54.38 ; h , 3.65 ; n , 12.76 : ms [ m ] at m / z 330 . 3- [ 2-(benzylideneamino)phenyl]-2-methyl - quinazolin-4-one ( 5 ) a solution of compound 3 ( 0.01 moles ) in ethanol ( 55 ml ) was refluxed with benzaldehyde ( 0.01 moles ) separately in the presence of glacial acetic acid ( 2.5 ml ) for 11 hr . the separated solid was filtered and recrystallized from methanol to give compound 29 ( 63% ) m.p . 118c : ir ( kbr ) max in cm : 3028 ( ch aromatic ) , 2930ch aliphatic ) , 1705 ( c = o ) , 1610 ( c = n ) ; hnmr ( cdcl3 ) in ppm : 6.797.63 ( m , 13h , ar h ) , 6.35 ( s , 1h , n = ch ) , 2.21 ( s , 3h , ch3 ) ; anal . caled . for c22h17n3o : c , 77.88 ; h , 5.01 ; n , 12.39 : found : c , 77.96 ; h , 5.00 ; n , 12.41 : ms [ m ] at m / z . 339 . 134c : ir ( kbr ) max in cm : 3028 ( ch aromatic ) , 2929 ( ch aliphatic ) , 1705 ( c = o ) , 1598 ( c = n ) 788 ( c cl ) ; h nmr ( cdcl3 ) in ppm : 6.827.65 ( m , 12h , ar h ) , 6.38 ( s , 1h , n = ch ) , 2.24 ( s , 3h , ch3 ) ; anal . : c,70.68 ; h , 4.28 ; n , 11.24 : found : c , 70.79 ; h , 4.27 ; n , 11.26 : ms [ m ] at m / z . 373.5 . 3-[2-(p - methoxybenzylideneamino)phenyl]-2-methyl - quinazolin-4-one ( 7 ) ( 60% ) . 148c : ir ( kbr ) max in cm : 3035(ch aromatic ) , 2932 ( ch aliphatic ) , 1700 ( c = o ) , 1600 ( c = n ) ; hnmr ( cdcl3 ) in ppm : 6.807.61 ( m , 12h , ar h ) , 6.40 ( s , 1h , n = ch ) , 3.48 ( s , 3h , och3 ) 2.26 ( s , 3h , ch3 ) ; anal . caled . for c23h19n3o2 : c , 74.80 ; h , 5.15 ; n , 11.38 : found : c , 74.96 ; h , 5.17 ; n , 11.29 : ms [ m ] at m / z . 369 . 215c : ir ( kbr ) max in cm : 3033 ( ch aromatic ) , 2926 ( ch aliphatic ) , 1698 ( c = o ) , 1606 ( c = n ) ; h nmr ( cdcl3 ) in ppm : 6.837.60 ( m , 12h , ar h ) , 6.43 ( s , 1h , n = ch ) , 2.22 ( s , 3h , ch3 ) ; anal . caled . for c22h16n3obr : c,63.16 ; h , 3.83 ; n , 10.05 : found : c , 63.29 ; h , 3.82 ; n , 10.07 : ms [ m ] at m / z . 418 . 234c : ir ( kbr ) max in cm : 3030 ( ch aromatic ) , 2930 ( ch aliphatic ) , 1695 ( c = o ) , 1601 ( c = n ) , 785 ( c cl ) ; hnmr ( cdcl3 ) in ppm : 6.817.59 ( m , 11h , ar h ) , 6.41 ( s , 1h , n = ch ) , 2.25 ( s , 3h , ch3 ) ; anal . : c , 58.34 ; h , 3.31 ; n , 9.28 : found : c , 58.55 ; h , 3.32 ; n , 9.30 : ms [ m ] at m / z . 452.5 . 223c : ir ( kbr ) max in cm : 3033 ( ch aromatic ) , 2926 ( ch aliphatic ) , 1698 ( c = o ) , 1595 ( c = n ) ; hnmr ( cdcl3 ) in ppm : 6.837.61 ( m , 11h , ar h ) , 6.44 ( s , 1h , n = ch ) , 3.44 ( s , 3h , och3 ) 2.25 ( s , 3h , ch3 ) ; anal . caled . for c23h18n3o2br : c , 61.61 ; h , 4.02 ; n , 9.38 : found : c , 61.52 ; h , 4.01 ; n , 9.36 : ms [ m ] at m / z . 448 3-[2-(3-chloro- 2-oxo- 4-phenylazetidin- 1-yl)-phenyl]-2-methyl - quinazol - in-4-one ( 11 ) to a solution of compound 5 ( 0.01 mole ) and triethylamine ( 0.5 ml ) in ethanol ( 55 ml ) was added in monochloroacetylchloride ( 0.014 mole ) at 50c . the reaction mixture was filtered to removed triethylamine hydrogen chloride and the resultant solution was poured onto crushed ice with constant stirring . the solid thus obtained was recrystallized from acetone to give desired compound 11 ( 60% ) m.p . ir ( kbr ) : max in cm : ir ( kbr ) max in cm : 3030 ( ch aromatic ) , 2925 ( ch aliphatic ) , 1700 ( c = o ) , 1595 ( c = n ) ; hnmr ( cdcl3 ) in ppm : 6.817.98 ( m , 13h , ar cl ) , 4.60 ( d , 1h , j = 9.3 hz , n ar ) , 2.21 ( s , 3h , ch3);. anal . calcd . for c24h18n3o2cl : c , 69.31 ; h , 4.33 ; n , 10.11 : found : c , 69.47 ; h , 4.32 ; n , 10.14 : ms [ m]at m / z 415.5 . the following compounds were prepared using a similar procedure described for compound 11 . 3-[2-(3-chloro- 2-oxo- 4-(p - chlorophenyl)azetidin- 1-yl)-phenyl]-2-methyl quinazolin-4-one ( 12 ) ( 58% ) ( methanol ) m.p . ir ( kbr ) : max in cm : ir ( kbr ) max in cm : 3033 ( ch aromatic ) , 2928 ( ch aliphatic ) , 1705 ( c = o ) , 1591 ( c = n ) , 788 ( c cl ) ; hnmr ( cdcl3 ) in ppm : 6.837.95 ( m , 12h , ar h ) , 6.56 ( d,1h , j = 8.8 hz , ch cl ) , 4.62 ( d , 1h , j = 9.0 hz , n ch ar ) , 2.25 ( s , 3h , ch3);. anal . calcd . for c24h17n3o2cl2 : c , 64.00 ; h , 3.78 ; n , 9.33 : found : c , 64.15 ; h , 3.80 ; n , 9.30 : ms [ m]at m / z 450 . 3-[2-(3-chloro- 2-oxo- 4-(p - methoxyphenyl)azetidin- 1-yl)-phenyl]-2-methyl quinazolin-4-one ( 13 ) ( 56% ) ( ethanol)m.p . ir ( kbr ) : max in cm : ir ( kbr ) max in cm : 3028 ( ch aromatic ) , 2930 ( ch n ) ; hnmr ( cdcl3 ) in ppm : 6.817.93 ( m , 12h , ar h ) , 6.54 ( d,1h , j = 9.0 hz , ch cl ) , 4.65 ( d , 1h , j = 9.3 hz , n ar ) , 3.47 ( s , 3h , och3 ) 2.23 ( s , 3h , ch3);. anal . calcd . for c25h20n3o3cl : c , 67.34 ; h , 4.49 ; n , 9.43 : found : c , 67.15 ; h , 4.50 ; n , 9.40 : ms [ m]at m / z 445.5 . 3- [ 2-(3-chloro-2-oxo-4-phenylazetidin-1-yl)-phenyl]-2-methyl-6-bromo - quinazolin-4-one ( 14 ) ( 58% ) ( methanol ) m.p . ir ( kbr ) : max in cm : ir ( kbr ) max in cm : 3030 ( ch aromatic ) , 2925 ( ch n ) ; hnmr ( cdcl3 ) in ppm : 6.807.92 ( m , 12h , ar h ) , 6.54 ( d , 1h , j = 9.2 hz , ch cl ) , 4.64 ( d , 1h , j = 9.1 hz , n ch ar ) , 2.22 ( s , 3h , ch3);. anal . calcd . for c24h17n3o2clbr : c , 58.24 ; h , 3.44 ; n , 8.49 : found : c , 58.36 ; h , 3.43 ; n , 8.51 : ms [ m]at m / z 494.5 . 3- [ 2-(3-chloro-2-oxo-4-(p - chlorophenyl)azetidin-1-yl)-phenyl]-2-methyl-6-bromo quinazolin-4-one ( 15 ) ( 54% ) ( ethanol ) m.p . ir ( kbr ) : max in cm : ir ( kbr ) max in cm : 3027 ( ch aromatic ) , 2930 ( ch aliphatic ) , 1710 ( c = o ) , 1598 ( c = n ) , 785 ( c cl ) ; hnmr ( cdcl3 ) in ppm : 6.797.90 ( m , 11h , ar h ) , 6.55 ( d , 1h , j = 9.3 hz , ch cl ) , 4.61 ( d , 1h , j = 8.8 hz , n ar ) , 2.20 ( s , 3h , ch3);. anal . calcd . for c24h16n3o2cl2br : c , 54.44 ; h , 3.02 ; n , 7.94 : found : c , 54.61 ; h , 3.01 ; n , 7.95 : ms [ m]at m / z 529 . 3- [ 2-(3-chloro-2-oxo-4-(p - methoxyphenyl)azetidin-1-yl)-phenyl]-2-methyl quinazolin-4-one ( 16 ) ( 60% ) ( acetone ) m.p . ir ( kbr ) : max in cm : ir ( kbr ) max in cm : 3030 ( ch aromatic ) , 2930 ( ch aliphatic ) , 1700 ( c = o ) , 1590 ( c = n ) ; hnmr ( cdcl3 ) in ppm : 6.807.92 ( m , 11h , ar h ) , 6.52 ( d , 1h , j = 9.2 hz , ch cl ) , 4.62 ( d , 1h , j = 9.0 hz , n ch ar ) , 3.49 ( s , 3h , och3 ) 2.25 ( s , 3h , ch3);. anal . calcd . for c25h19n3o3clbr : c , 57.20 ; h , 3.62 ; n , 8.01 : found : c , 57.31 ; h , 3.61 ; n , 8.06 : ms [ m]at m / z 524.5 . 3- [ 2-(2-phenyl-4-oxo-1,3-thiazolidin-3-yl)phenyl]-2-methyl-6-quinazolin-4-one ( 17 ) a mixture of compound 5(0.01 moles ) , thioglycolic acid ( 0.012 moles ) in absolute dioxane ( 75 ml ) and in the presence of anhydrous zncl2 was refluxed for 18 hr . the product finally obtained was dried and recrystallized from ethanol to yield compound 17 ( 55% ) m.p . ir ( kbr ) : max in cm : ir ( kbr ) max in cm3032 ( ch aromatic ) , 2931 ( ch aliphatic ) , 1705 ( c = o ) , 1595 ( c = n),685 ( c s c ) ; hnmr ( cdcl3 ) in ppm : 6.807.97 ( m , 13h , ar ar ) , 4.25 ( s , 2h , ch2 ) 2.25 ( s , 3h , ch3);. anal . calcd . for c24h19n3o2s : c , 69.73 ; h , 4.60 ; n , 10.17 : found : c , 69.82 ; h , 4.61 ; n , 10.22 : ms [ m]at m / z 413 . ir ( kbr ) : max in cm : ir ( kbr ) max in cm : 3030 ( ch aromatic ) , 2930 ( ch aliphatic ) , 1700 ( c = o ) , 1598 ( c = n ) , 784 ( c cl ) , 688 ( c s c ) ; hnmr ( cdcl3 ) in ppm : 6.837.96 ( m , 12h , ar ar ) , 4.21 ( s , 2h , ch2 ) 2.22 ( s , 3h , ch3);. anal . calcd . for c24h18n3o2scl : c , 64.36 ; h , 4.02 ; n , 9.39 : found : c,64.41 ; h , 4.03 ; n , 9.35 : ms [ m]at m / z 447.5 . 3-[2-(2-(p - methoxyphenyl)- 4-oxo- 1 , 3-thiazolidin- 3-yl)phenyl]-2-methyl quinazolin-4-one ( 19 ) ( 50% ) ( acetone ) m.p . ir ( kbr ) : max in cm : ir ( kbr ) max in cm : 3032 ( ch aromatic ) , 2927 ( ch aliphatic ) , 1700 ( c = o ) , 1600 ( c = n ) , 690 ( c s c ) , ; hnmr ( cdcl3 ) in ppm : 6.817.95 ( m , 12h , ar h ) , 4.52 ( s , 1h , n chvar ) , 4.23 ( s , 2h , ch2 ) 3.51 ( s , 3h , och3 ) , 2.20 ( s , 3h , ch3);. anal . calcd . for c25h21n3o3s : c , 67.72 ; h , 4.74 ; n , 9.48 : found : c,67.88 ; h , 4.73 ; n , 9.51 : ms [ m]at m / z 443 . 3- [ 2-(2-phenyl-4-oxo-1,3-thiazolidin-3-yl)phenyl]-2-methyl-6-bromo quinazolin-4-one ( 20 ) ( 55% ) ( ethanol ) m.p . ir ( kbr ) : max in cm : ir ( kbr ) max in cm : 3028 ( ch aromatic ) , 2933 ( ch aliphatic ) , 1705 ( c = o ) , 1600 ( c = n ) , 690 ( c s c ) ; h nmr ( cdcl3 ) in ppm : 6.817.94 ( m , 12h , ar ar ) , 4.22 ( s , 2h , ch2 ) 2.24 ( s , 3h , ch3 ) . calcd . for c24h18n3o2sbr : c , 58.54 ; h , 3.66 ; n , 8.54 : found : c,58.66 ; h , 3.67 ; n , 8.56 : ms [ m]at m / z 492.5 . 3-[2-(2-(p - chlorophenyl)- 4-oxo-1 , 3-thiazolidin- 3-yl)phenyl]-2-methyl-6-bromo quinazolin-4-one ( 21 ) ( 48% ) ( methanol ) m.p . ir ( kbr ) : max in cm : ir ( kbr ) max in cm : 3033 ( ch aromatic ) , 2925 ( ch aliphatic ) , 1705 ( c = o ) , 1595 ( c = n ) , 784 ( c cl ) , 685 ( c s c ) ; hnmr ( cdcl3 ) in ppm : 6.807.93 ( m , 11h , ar ar ) , 4.24 ( s , 2h , ch2 ) 2.26 ( s , 3h , ch3);. anal . calcd . for c24h17n3o2sclbr : c,54.70 ; h , 3.23 ; n , 7.98 : found : c , 54.86 ; h , 3.22 ; n , 7.97 : ms [ m]at m / z 526.5 . 3-[2-(2-(p - methoxyphenyl)-4-oxo-1,3-thiazolidin-3-yl)phenyl]-2-methyl-6-bromo quinazolin-4-one ( 22 ) ( 55% ) ( ethanol ) m.p . ir ( kbr ) : max in cm : ir ( kbr ) max in cm : 3031 ( ch aromatic ) , 2928 ( ch aliphatic ) , 1710 ( c = o ) , 1605 ( c = n ) , 685 ( c s c ) ; hnmr ( cdcl3 ) in ppm : 6.817.92 ( m , 11h , ar ar ) , 4.25 ( s , 2h , ch2 ) , 3.49 ( s , 3h , och3 ) , 2.26 ( s , 3h , ch3);. anal . calcd . for c25h20n3o3sbr : c , 57.47 ; h , 3.83 ; n , 8.05 : found : c , 57.62 ; h , 3.82 ; n , 8.03 : ms [ m]at m / z 522 . all the compounds ( 522 ) were screened for anti - inflammatory activity at 50 mg / kg p.o . using phenylbutazone as reference drug . all the tested compounds exhibited 15.1 to 32.5% edema inhibition at the dose of 50 mg / kg p.o . when the compounds were substituted with p - chlorophenyl group showed better anti - inflammatory activity than phenyl group . compound 9 , which was substituted by 2-(p - chlorobenzylideneamino)phenyl group at the iiird position of quinazolinone moiety showed better anti - inflammatory activity ( 20.4% ) than compounds 5 , 6 , 7 , 8 , and 10 . cyclizations of these compounds into azetidinones and thiazolidinones have shown better anti - inflammatory activity than corresponding arylidene derivatives . azetidinones ( 1116 ) and thiazolidinones ( 1722 ) have shown the anti - inflammatory activity from ( 24.627.3% ) and ( 22.932.5% ) respectively . in azetidinones , compound 15 showed better anti - inflammatory activity at the dose of 50 mg / kg p.o . thiazolidinones ( 1722 ) generally showed better anti - inflammatory then corresponding azetidinones ( 1116 ) . among all the tested compounds , 3-[2-(2-(p - chlorophenyl)-4-oxo-1,3-thiazolidin-3-yl)phenyl]-2-methyl-6-bromo quinazolin-4-one ( 21 ) showed better anti - inflammatory activity ( 32.5% ) . the compound 9 showed good analgesic activity ( 19.3% ) then compounds 5 , 6 , 7 , 8 , and 10 . the formation of azetidinones ( 1116 ) and thiazolidinones ( 1722 ) from compounds ( 510 ) showed better analgesic activity than corresponding arylidene derivatives . among these , it may be concluded that the compounds having 4-chlorophenyl group of quinazolinone moiety showed better anti - inflammatory activity than phenyl moiety . further , thiazolidinones showed better anti - inflammatory and analgesics activities in comparison to their corresponding azetidinones . among all the tested compounds , compound 21 showed better anti - inflammatory ( 32.5% ) and analgesics ( 29.6% ) activities than other compounds of series .

a series of 3-[2-(substitutedbenzylideneamino)phenyl]-2-methyl-6-substituted quinazolin-4-ones ( 510 ) , 3-[2-(3-chloro-2-oxo-4-substitutedphenylazetidin-1-yl)phenyl]-2-methyl-6-substitutedquinazolin-4-ones ( 1116 ) , and 3-[2-(2-substitutedphenyl-4-oxo-1,3-thiazolidin-3-yl)phentl]-2-methyl-6-substitutedquinazolin-4-ones ( 1722 ) have been synthesized in the present study . the structures of the synthesized compounds were assigned on the basis of elemental analysis , ir , 1h nmr , and mass spectral data . all the newly synthesized compounds were screened for anti - inflammatory and analgesic activities .

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

quinazolinone derivatives represent one of the most active classes of compounds possessing a wide spectrum of biological activity . several reports have been published on the biological activities of quinazolinone derivatives , including their anti - inflammatory [ 17 ] , antimalarial [ 8 , 9 ] , antimicrobial , anti - fungal , antibacterial [ 1016 ] , anticonvulsant [ 1720 ] , and antitumor [ 21 , 22 ] activities . moreover , large number of quinazolinone derivatives having substitution at 2 and 3 position by different heterocyclic moieties increases anti - inflammatory potential of quinazolinone derivatives . similarly , various azetidinones [ 2325 ] and thiazolidinones [ 26 , 27 ] have been reported to possess potent anti - inflammatory activity . looking to the medicinal importance of 4(3h)-quinazolinone , 4-thiazolidinone , and azetidinones , we report here the synthesis of a new class of heterocyclic molecules in which all of these moieties are present and try to develop potential bioactive molecules . the structures of the compounds synthesized were assigned on the basis of elemental analysis , ir , h nmr , and mass spectral data . substituted anthranilic acid reacted with acetic anhydride to give 2-methylsubstituted-4h-3,1-benzoxazin-4-ones ( 1,2 ) , which further reacted with 2-amino phenyl amine in acetic acid to yield 3-(2-aminophenyl)-2-methyl-6-substituted quinazolin-4-ones ( 3,4 ) . compounds ( 3,4 ) when treated with different substituted aromatic aldehydes formed various substituted arylidene derivatives ( 510 ) . these arylidene derivatives on treatment with chloroacetylchloride in presence of triethylamine yielded 3-[2-(3-chloro-2-oxo-4-substituted phenylazetidin-1-yl)phenyl]-2-methyl-6-substitutedquinazolin-4-ones ( 1116 ) . on the other hand , these arylidene derivatives on reaction with thioglycolic acid and anhydrous zncl2 furnished 3-[2-(2-substitutedphenyl-4-oxo-1,3-thiazolidin-3-yl)phenyl]-2-methyl-6-substitutedquinazolin-4-ones . the structures of the compounds synthesized were assigned on the basis of elemental analysis , ir , h nmr , and mass spectral data . the experiments were performed with albino rats of the charles - foster stain of either sex , excluding pregnant females , of 7095 d. weighting 100150 g. food ( chaw pallet ) and water were given to the animals ad libitum . anti - inflammatory activity against carrageenan - induced rat 's paw oedema this study was done with the procedure of winter et al . the rats were divided into three groups ( control , treated , and standard drug ) of six animals each . a freshly prepared suspension of carrageenan ( 1% in 0.9% saline ) 0.05 mn was injected under the planter aponeurosis of the right hind paw of each rat . the percent anti - inflammatory activity was calculated according to the formula given below ( 1)percentage of inhibition of oedema=(1vtvc)100 , where vt and vc are the mean increase in paw volume of rats of the treated and the control group , respectively . this method is based on the property of the test compound to antagonize the phenyl quinone - induced pain syndrome in mice . groups of five mice were injected intraperitoneally with 0.25 ml of a 0.02% solution of phenylquinone in ethanol ( 5% ) 1 h after of oral administration of the test compound . the purities of compounds were checked by thin layer chromatography ( tlc ) using silica gel - g coated plats . infrared ( ir ) absorption spectra were recorded on perkin - elmer rx-1 ftir spectrometer using potassium bromide ( kbr ) pellet , and wave numbers were given in cm . elemental analysis ( c , h , n ) was performed on perkin - elmer 2400 analyzer , and values were either in 0.4% of the calculated . in the presence of sodium acetate . ir ( kbr ) max in cm : 3033 ( ch aromatic ) , 2920 ( ch n ) ; hnmr ( cdcl3 ) in ppm : 9.10 ( s , 2h , nh2 exchangeable ) , 6.897.61 ( m , 8h , ar h ) , 2.29 ( s , 3h , ch3 ) ; anal . 199c , ir ( kbr ) max in cm : 3030 ( ch aromatic ) , 2925 ( ch aliphatic ) , 1695 ( c = o ) , 1598 ( c = n ) ; h nmr ( cdcl3 ) in ppm : 9.06 ( s , 2h , nh2 exchangeable ) , 6.857.51 ( m , 7h , ar h ) , 2.24 ( s , 3h , ch3 ) ; anal . 3- [ 2-(benzylideneamino)phenyl]-2-methyl - quinazolin-4-one ( 5 ) a solution of compound 3 ( 0.01 moles ) in ethanol ( 55 ml ) was refluxed with benzaldehyde ( 0.01 moles ) separately in the presence of glacial acetic acid ( 2.5 ml ) for 11 hr . 118c : ir ( kbr ) max in cm : 3028 ( ch aromatic ) , 2930ch aliphatic ) , 1705 ( c = o ) , 1610 ( c = n ) ; hnmr ( cdcl3 ) in ppm : 6.797.63 ( m , 13h , ar h ) , 6.35 ( s , 1h , n = ch ) , 2.21 ( s , 3h , ch3 ) ; anal . 134c : ir ( kbr ) max in cm : 3028 ( ch aromatic ) , 2929 ( ch aliphatic ) , 1705 ( c = o ) , 1598 ( c = n ) 788 ( c cl ) ; h nmr ( cdcl3 ) in ppm : 6.827.65 ( m , 12h , ar h ) , 6.38 ( s , 1h , n = ch ) , 2.24 ( s , 3h , ch3 ) ; anal . 148c : ir ( kbr ) max in cm : 3035(ch aromatic ) , 2932 ( ch aliphatic ) , 1700 ( c = o ) , 1600 ( c = n ) ; hnmr ( cdcl3 ) in ppm : 6.807.61 ( m , 12h , ar h ) , 6.40 ( s , 1h , n = ch ) , 3.48 ( s , 3h , och3 ) 2.26 ( s , 3h , ch3 ) ; anal . 215c : ir ( kbr ) max in cm : 3033 ( ch aromatic ) , 2926 ( ch aliphatic ) , 1698 ( c = o ) , 1606 ( c = n ) ; h nmr ( cdcl3 ) in ppm : 6.837.60 ( m , 12h , ar h ) , 6.43 ( s , 1h , n = ch ) , 2.22 ( s , 3h , ch3 ) ; anal . 234c : ir ( kbr ) max in cm : 3030 ( ch aromatic ) , 2930 ( ch aliphatic ) , 1695 ( c = o ) , 1601 ( c = n ) , 785 ( c cl ) ; hnmr ( cdcl3 ) in ppm : 6.817.59 ( m , 11h , ar h ) , 6.41 ( s , 1h , n = ch ) , 2.25 ( s , 3h , ch3 ) ; anal . 223c : ir ( kbr ) max in cm : 3033 ( ch aromatic ) , 2926 ( ch aliphatic ) , 1698 ( c = o ) , 1595 ( c = n ) ; hnmr ( cdcl3 ) in ppm : 6.837.61 ( m , 11h , ar h ) , 6.44 ( s , 1h , n = ch ) , 3.44 ( s , 3h , och3 ) 2.25 ( s , 3h , ch3 ) ; anal . ir ( kbr ) : max in cm : ir ( kbr ) max in cm : 3030 ( ch aromatic ) , 2925 ( ch aliphatic ) , 1700 ( c = o ) , 1595 ( c = n ) ; hnmr ( cdcl3 ) in ppm : 6.817.98 ( m , 13h , ar cl ) , 4.60 ( d , 1h , j = 9.3 hz , n ar ) , 2.21 ( s , 3h , ch3);. the following compounds were prepared using a similar procedure described for compound 11 . ir ( kbr ) : max in cm : ir ( kbr ) max in cm : 3033 ( ch aromatic ) , 2928 ( ch aliphatic ) , 1705 ( c = o ) , 1591 ( c = n ) , 788 ( c cl ) ; hnmr ( cdcl3 ) in ppm : 6.837.95 ( m , 12h , ar h ) , 6.56 ( d,1h , j = 8.8 hz , ch cl ) , 4.62 ( d , 1h , j = 9.0 hz , n ch ar ) , 2.25 ( s , 3h , ch3);. ir ( kbr ) : max in cm : ir ( kbr ) max in cm : 3028 ( ch aromatic ) , 2930 ( ch n ) ; hnmr ( cdcl3 ) in ppm : 6.817.93 ( m , 12h , ar h ) , 6.54 ( d,1h , j = 9.0 hz , ch cl ) , 4.65 ( d , 1h , j = 9.3 hz , n ar ) , 3.47 ( s , 3h , och3 ) 2.23 ( s , 3h , ch3);. ir ( kbr ) : max in cm : ir ( kbr ) max in cm : 3030 ( ch aromatic ) , 2925 ( ch n ) ; hnmr ( cdcl3 ) in ppm : 6.807.92 ( m , 12h , ar h ) , 6.54 ( d , 1h , j = 9.2 hz , ch cl ) , 4.64 ( d , 1h , j = 9.1 hz , n ch ar ) , 2.22 ( s , 3h , ch3);. ir ( kbr ) : max in cm : ir ( kbr ) max in cm : 3027 ( ch aromatic ) , 2930 ( ch aliphatic ) , 1710 ( c = o ) , 1598 ( c = n ) , 785 ( c cl ) ; hnmr ( cdcl3 ) in ppm : 6.797.90 ( m , 11h , ar h ) , 6.55 ( d , 1h , j = 9.3 hz , ch cl ) , 4.61 ( d , 1h , j = 8.8 hz , n ar ) , 2.20 ( s , 3h , ch3);. ir ( kbr ) : max in cm : ir ( kbr ) max in cm : 3030 ( ch aromatic ) , 2930 ( ch aliphatic ) , 1700 ( c = o ) , 1590 ( c = n ) ; hnmr ( cdcl3 ) in ppm : 6.807.92 ( m , 11h , ar h ) , 6.52 ( d , 1h , j = 9.2 hz , ch cl ) , 4.62 ( d , 1h , j = 9.0 hz , n ch ar ) , 3.49 ( s , 3h , och3 ) 2.25 ( s , 3h , ch3);. 3- [ 2-(2-phenyl-4-oxo-1,3-thiazolidin-3-yl)phenyl]-2-methyl-6-quinazolin-4-one ( 17 ) a mixture of compound 5(0.01 moles ) , thioglycolic acid ( 0.012 moles ) in absolute dioxane ( 75 ml ) and in the presence of anhydrous zncl2 was refluxed for 18 hr . ir ( kbr ) : max in cm : ir ( kbr ) max in cm3032 ( ch aromatic ) , 2931 ( ch aliphatic ) , 1705 ( c = o ) , 1595 ( c = n),685 ( c s c ) ; hnmr ( cdcl3 ) in ppm : 6.807.97 ( m , 13h , ar ar ) , 4.25 ( s , 2h , ch2 ) 2.25 ( s , 3h , ch3);. ir ( kbr ) : max in cm : ir ( kbr ) max in cm : 3030 ( ch aromatic ) , 2930 ( ch aliphatic ) , 1700 ( c = o ) , 1598 ( c = n ) , 784 ( c cl ) , 688 ( c s c ) ; hnmr ( cdcl3 ) in ppm : 6.837.96 ( m , 12h , ar ar ) , 4.21 ( s , 2h , ch2 ) 2.22 ( s , 3h , ch3);. ir ( kbr ) : max in cm : ir ( kbr ) max in cm : 3032 ( ch aromatic ) , 2927 ( ch aliphatic ) , 1700 ( c = o ) , 1600 ( c = n ) , 690 ( c s c ) , ; hnmr ( cdcl3 ) in ppm : 6.817.95 ( m , 12h , ar h ) , 4.52 ( s , 1h , n chvar ) , 4.23 ( s , 2h , ch2 ) 3.51 ( s , 3h , och3 ) , 2.20 ( s , 3h , ch3);. ir ( kbr ) : max in cm : ir ( kbr ) max in cm : 3028 ( ch aromatic ) , 2933 ( ch aliphatic ) , 1705 ( c = o ) , 1600 ( c = n ) , 690 ( c s c ) ; h nmr ( cdcl3 ) in ppm : 6.817.94 ( m , 12h , ar ar ) , 4.22 ( s , 2h , ch2 ) 2.24 ( s , 3h , ch3 ) . ir ( kbr ) : max in cm : ir ( kbr ) max in cm : 3033 ( ch aromatic ) , 2925 ( ch aliphatic ) , 1705 ( c = o ) , 1595 ( c = n ) , 784 ( c cl ) , 685 ( c s c ) ; hnmr ( cdcl3 ) in ppm : 6.807.93 ( m , 11h , ar ar ) , 4.24 ( s , 2h , ch2 ) 2.26 ( s , 3h , ch3);. ir ( kbr ) : max in cm : ir ( kbr ) max in cm : 3031 ( ch aromatic ) , 2928 ( ch aliphatic ) , 1710 ( c = o ) , 1605 ( c = n ) , 685 ( c s c ) ; hnmr ( cdcl3 ) in ppm : 6.817.92 ( m , 11h , ar ar ) , 4.25 ( s , 2h , ch2 ) , 3.49 ( s , 3h , och3 ) , 2.26 ( s , 3h , ch3);. all the compounds ( 522 ) were screened for anti - inflammatory activity at 50 mg / kg p.o . all the tested compounds exhibited 15.1 to 32.5% edema inhibition at the dose of 50 mg / kg p.o . when the compounds were substituted with p - chlorophenyl group showed better anti - inflammatory activity than phenyl group . compound 9 , which was substituted by 2-(p - chlorobenzylideneamino)phenyl group at the iiird position of quinazolinone moiety showed better anti - inflammatory activity ( 20.4% ) than compounds 5 , 6 , 7 , 8 , and 10 . cyclizations of these compounds into azetidinones and thiazolidinones have shown better anti - inflammatory activity than corresponding arylidene derivatives . azetidinones ( 1116 ) and thiazolidinones ( 1722 ) have shown the anti - inflammatory activity from ( 24.627.3% ) and ( 22.932.5% ) respectively . in azetidinones , compound 15 showed better anti - inflammatory activity at the dose of 50 mg / kg p.o . thiazolidinones ( 1722 ) generally showed better anti - inflammatory then corresponding azetidinones ( 1116 ) . among all the tested compounds , 3-[2-(2-(p - chlorophenyl)-4-oxo-1,3-thiazolidin-3-yl)phenyl]-2-methyl-6-bromo quinazolin-4-one ( 21 ) showed better anti - inflammatory activity ( 32.5% ) . the formation of azetidinones ( 1116 ) and thiazolidinones ( 1722 ) from compounds ( 510 ) showed better analgesic activity than corresponding arylidene derivatives . among these , it may be concluded that the compounds having 4-chlorophenyl group of quinazolinone moiety showed better anti - inflammatory activity than phenyl moiety . further , thiazolidinones showed better anti - inflammatory and analgesics activities in comparison to their corresponding azetidinones . among all the tested compounds , compound 21 showed better anti - inflammatory ( 32.5% ) and analgesics ( 29.6% ) activities than other compounds of series .

it is estimated that chronic hepatitis c ( chc ) affects more than 180 million people worldwide , which is about 3% of the world population ( 1 ) . an increase in hepatitis c virus ( hcv)-related morbidities and mortalities has been observed in recent years . in addition , increasing rates of advanced liver disease as a result of hcv infection are expected to be observed in the next decade , and developing countries , which have a higher prevalence of hcv infection , will experience the major burden of end - stage liver diseases ( esld ) throughout the world ( 2 - 4 ) . therefore , it is obvious that this infection is a major concern of health policy makers . fortunately , the risk of developing hepatocellular carcinoma ( hcc ) as one of the main complications of hcv infection can be reduced by 75% with successful treatment ( 5 ) , and today , newly introduced treatment strategies have provided opportunities to manage and control this public health concern ( 6 , 7 ) . in the present study , we conducted an electronic search of available literature published to find pertinent contents reporting novel hcv treatments toward clearance in all hcv infected patient groups and cost benefits of new treatments in the era of daas . to identify articles , the search was begun among all peer - reviewed journal indexed in pubmed , scopus and google scholar . the literature search was done by using the following key words : " hepatitis c virus , hcv , hcv genotypes , special groups , hiv / hcv co - infections , thalassemia , hemophilia , hemodialysis , liver transplantation , mass screening , diagnosis and treatment outcome " . the search results were investigated carefully , and then most relevant results were strongly considered for including in this study by all authors and consulting the supervisor of the study ( sma ) . it began with interferon ( ifn ) mono - therapy , with less than 20% sustained virological response ( svr ) . milestones include the addition of ribavirin ( rbv ) to the treatment protocol and providing pegylated - ifn ( pegifn ) as an alternative treatment ( 8 - 10 ) . treatment with pegifn / rbv was the standard of care for about 10 years , and it allowed about 50% of subjects with hcv genotype 1 infection to attain svr ( 10 - 12 ) . the success rate of treatment with this regimen is very dependent on patient characteristics , including age , body mass index , ethnicity , and genetic factors such as polymorphisms near the interferon lambda 3 ( ifnl3 ) gene ( 13 , 14 ) . viral factors , especially hcv genotype , also affect the response to hcv treatment ( 15 ) , and there are always additional factors that should be taken into account in each treatment approach , including treatment success rate , duration , cost , and side effects . in light of these concerns , knowledge of the hcv replication cycle and the role of viral proteins in the virulence of hcv have resulted in targeting of the viral proteins involved in the hcv life cycle to develop new hcv treatments . in 2011 , the first generation of direct acting antivirals ( daas ) boceprevir ( boc ) and telaprevir ( tvr ) were introduced and added to the previous pegifn / rbv regimen ( 17 , 18 ) . these new triple therapy strategies led to higher svr , but they were still ifn - based and had severe adverse effects . triple therapy with boc or tvr quickly fell out of favor due to the introduction of a new wave of more efficient daas beginning in 2013 , which changed the standards for hcv treatment . in december 2013 , the fda approved sofosbuvir ( sof ) in combination with pegifn / rbv for treatment of hcv genotype 1 infection . this approach achieved a response rate of over 85% ; however , the presence of unfavorable treatment predictors such as cirrhosis , previous history of treatment , and unfavorable host genetics can influence the success rate of treatment with sof / pegifn / rbv ( 19 ) . sof in combination with rbv and/or pegifn was also approved for treatment of hcv genotypes 2 , 3 , and 4 , with limited efficacy for hcv genotype 3 ( 20 ) . in october 2014 , the fda approved ledipasvir ( ldv ) in combination with sof for treatment of hcv genotype 1 , which achieved more than 95% efficacy ( 21 , 22 ) . fortunately , in addition to being more effective than the previous sof / pegifn / rbv regimen , sof / ldv is influenced little by patient characteristics . in december 2014 , the fda approved another ifn - free daa regimen a combination of ombitasvir / paritaprevir - r / dasabuvir ( a three direct acting antiviral , or 3d ) for treatment of hcv genotype 1 infection , with an efficacy rate of over 95% ( 23 ) . finally , in january 2016 , the fda approved combination therapy with grazoprevir / elbasvir ( gzr / ebr ) , with about a 95% svr rate ( 24 ) . other regimens containing daas , such as simeprevir ( smv ) and daclatasvir ( dcv ) , have also been approved for treatment of hcv infection since 2013 ( 25 , 26 ) . the use of new treatment strategies has provided opportunities to eradicate hcv infection . however , effective treatment is not enough ; there are still major issues that must be taken into account , some of which we address in this paper . although an effective treatment regimen is a necessary tool in infection eradication , identifying infected individuals is also of vital importance . subjects with hcv infection can remain asymptomatic for a long time , so they may be unaware of their infection . during this period , infection can progress to advanced liver disease , and the patient may spread the virus to others . surprisingly , about 75% of patients with hcv in the united states are unaware of being infected ( 27 ) . another challenge to developing screening strategies for hcv is the occult nature of this infection , which can not be identified with routine diagnostic tests ( 28 , 29 ) . we believe that health policy makers should design powerful screening programs to identify hcv - infected subjects . risk factors for hcv infection include intravenous drug use ( ivdu ) , history of unsafe injection practices , use of blood and/or blood products before the introduction of blood donor screening for hcv between 1992 and 1996 ( depending on the national policies of different countries ) , being homeless , and history of imprisonment ( 30 ) . the prevalence of these risk factors varies among different populations and countries ; population - based studies have determined that different geographical regions have unique risk factors for hcv infection . for example , baby boomers , persons born between 1945 and 1965 in the united states , account for about 70% of hcv infections in the us ( 31 ) . in addition , previous parenteral therapy for schistosomiasis is a significant predictor of hcv infection in egypt ( 32 ) . however , in some countries , further population - based studies are required to determine the special risk factors related to each area . once this is done , subjects with these risk factors can be recognized as special groups and singled out for screening . these population - based studies should be conducted alongside the implementation of new prevention and treatment strategies in order to track changes in geographically - based risk factors and changes in the prevalence of hcv among subjects with these risk factors . these studies will also help in the evaluation of screening strategies . as a result of geographical differences in risk factors and prevalence , a different screening strategy is needed for each country , and this should be supported by governmental and non - governmental organizations . governments should pay attention to this important issue and fund screening , and health policy makers should design a risk - based , powerful screening method . furthermore , the quality of these activities and screening methods should be evaluated by research projects and epidemiological population - based studies , which can guide policy makers . ultimately , medical practitioners and healthcare personnel have an important role in this regard , and they must support screening projects . risk - based screening strategies can identify about 86% of patients with hcv ( 33 ) . some patients do not have traditional risk factors and therefore can not be identified by risk - based screening . therefore , worldwide clinical screening , together with the approach of risk - based screening ( 34 ) , is recommended . however , the feasibility of such an approach is highly dependent on the economic situation of a given country . one of the main inconveniences of new treatment strategies is their current high cost . for example , the cost of a 12-week treatment with sof is 85,000 - 110,000 usd . new treatment approaches have shorter durations , minimal adverse effects , and higher efficacy ; however , they are very expensive . strategies should be applied to reduce treatment costs and provide wide access to new treatments , especially in low- and middle - income countries , in which about 80% of patients with chc live ( 5 , 35 , 36 ) . people with inherited bleeding disorders ( such as hemophilia ) , people with inherited hemoglobin disorders ( such as thalassemia ) , patients under hemodialysis , patients with organ transplantation ( especially liver- and kidney - transplant patients ) and hcv patients co - infected with hiv are some of the groups that need special attention and priority in treatment ( 37 ) . special patient groups with chc are at increased risk of death or complications ( 38 ) . a basic question arises here : what considerations need to be made for special patient groups in the era of daas ? these populations are the most challenging to treat , and they require massive attention . because of faster progression to both esld and hcc in special patients , hcv is a significant cause of morbidity and mortality in these populations ( 39 ) . hiv / hcv co - infection : hiv and hcv co - infection poses a challenge because it is widespread , particularly among ivdus , and it exhibits lower rates of spontaneous hcv clearance , poor response to treatment of chronic hcv in the pre - daa era , and more rapid progression of hcv - related liver diseases such as cirrhosis and hcc ( 40 ) . global statistics indicate that four to five million people are co - infected with hiv / hcv ( 41 ) . adoption of interferon - based hcv treatments ( following the pegifn / rbv and first - generation hcv protease inhibitors ) resulted in lower svr in those with hiv / hcv co - infection than in those with hcv mono - infection ( 40 ) . furthermore , hcv treatment regimens involve serious adverse effects and pharmacokinetic drug interactions with hiv - antiretroviral drugs ( 42 - 44 ) . the development and approval of new oral regimens of daas has created an opportunity to improve hcv treatment efficacy and safety for hiv / hcv co - infected patients . however , drug interactions between hcv daas and hiv - antiretroviral agents are still a major problem . for example , smv and 3d regimens are contra - indicated in hiv patients receiving many protease inhibitors . today , sof plays a key role in treating hiv / hcv co - infection ; it has a lower degree of interaction with hiv - antiretroviral drugs . the combination of sof / ldv or sof / dcv provides high rates of svr in hiv / hcv co - infected patients . recently , the use of sof / ldv and sof / dcv has been shown to be effective and safe in patients with hiv / hcv co - infection , achieving svr rates of 98% and 96% - 98% , respectively ( 45 , 46 ) . although there has been much progress in the field of hiv / hcv co - infection treatment , this patient population still needs more attention . thalassemia and hemophilia : hcv infection is one of the most common infections following the use of blood and blood products among thalassemia and hemophilia patients ( 47 ) . the treatment of thalassemia patients infected with hcv is a very controversial issue . dual therapy with pegifn / rbv , use of protease inhibitors ( boc and tvr ) , and pegifn / rbv - based triple therapy leads to rbv - associated life - threatening anemia in many thalassemia patients ( 48 ) . on the other hand , the elimination of rbv and the use of low - dose rbv in the treatment of thalassemia patients seems to result in low svr rates ( 49 ) . nevertheless , because of the severe adverse effects of rbv - based treatments in these patients unfortunately , there are currently no clinical trials evaluating the effect of daa use to treat hcv infection in thalassemia patients . although the combination of pegifn and rbv is still used in treatment of hcv - infected thalassemia patients ( 15 ) , a few thalassemia cases have been treated with sof - based treatment in our clinic ( middle east liver disease center ) and experienced a favorable treatment response . however , clinical trials with new daas are required to evaluate this approach . liver failure due to hcv infection is one of the common causes of death in patients with hemophilia . anti - hcv therapy plays a vital role in the interruption of the hcv infection pathway in order to prevent cirrhosis and hcc . the current standard of care for treatment of hcv in hemophilia is pegifn / rbv , which achieves svr in 61% of patients ( 51 ) . however , the side effects of the pegifn / rbv regimen , including thrombocytopenia and excessive bleeding , should be considered . a recent open - label study showed that out of 14 hemophilia patients infected with hcv and treated with sof / ldv , all achieved svr ( 100% ) ( 52 ) . despite the increasing use of highly effective anti - hcv agents with minimal side effects to treat hemophilia patients , hemophiliacs still constitute a unique patient population that requires special consideration . hemodialysis : hcv infection is one of the most common infections transmitted by the parenteral route in patients receiving maintenance hemodialysis ( 53 ) . the use of rbv is problematic in this group . receiving an ifn - free and , if possible , rbv - free regimen is a fundamental , urgent need in patients under hemodialysis ( 51 ) . in persons with renal impairment receiving chronic hemodialysis , options for hcv treatment however , asunaprevir , dcv , smv , gzr / ebr , and 3d regimens are cleared by hepatic metabolism and can be used in patients with renal disease ( 54 ) . liver transplantation : hepatitis c recurrence is common after liver transplantation when patients are transplanted with detectable viral loads . recurrence of hcv following liver transplantation may accelerate graft injury , which is difficult to treat with ifn / rbv therapy ( 55 ) . antiviral treatment before transplantation can prevent hcv recurrence . ifn - based regimens are poorly tolerated and are either ineffectual or contra - indicated in most liver - transplant patients ( 56 ) . in contrast , sof - based regimens have satisfactory virological response in more than 80% of post - transplant patients ( 57 ) . favorable response to the currently available therapies and new highly effective treatments for hcv revealed that hcv clearance could be significantly improved in special patient groups . daa - based therapies stand to achieve a very high rate of treatment success with minimal side effects . the introduction of new therapeutic agents does not detract from the importance of preventive strategies , including the development of an hcv vaccine ( 58 ) . it seems that an effective vaccine is achievable in the near future , and as bill gates has said , treatment without prevention is simply unsustainable . on the other hand , reduction of harmful behaviors should be the main strategy to reduce the prevalence of hcv infection in certain high - risk groups , such as ivdus and inmates ( 27 , 30 ) . furthermore , public knowledge and awareness are vital to the eradication of every disease , and ignorance will prevent future eradication of hcv . it began with interferon ( ifn ) mono - therapy , with less than 20% sustained virological response ( svr ) . milestones include the addition of ribavirin ( rbv ) to the treatment protocol and providing pegylated - ifn ( pegifn ) as an alternative treatment ( 8 - 10 ) . treatment with pegifn / rbv was the standard of care for about 10 years , and it allowed about 50% of subjects with hcv genotype 1 infection to attain svr ( 10 - 12 ) . the success rate of treatment with this regimen is very dependent on patient characteristics , including age , body mass index , ethnicity , and genetic factors such as polymorphisms near the interferon lambda 3 ( ifnl3 ) gene ( 13 , 14 ) . viral factors , especially hcv genotype , also affect the response to hcv treatment ( 15 ) , and there are always additional factors that should be taken into account in each treatment approach , including treatment success rate , duration , cost , and side effects . in light of these concerns , knowledge of the hcv replication cycle and the role of viral proteins in the virulence of hcv have resulted in targeting of the viral proteins involved in the hcv life cycle to develop new hcv treatments . in 2011 , the first generation of direct acting antivirals ( daas ) boceprevir ( boc ) and telaprevir ( tvr ) were introduced and added to the previous pegifn / rbv regimen ( 17 , 18 ) . these new triple therapy strategies led to higher svr , but they were still ifn - based and had severe adverse effects . triple therapy with boc or tvr quickly fell out of favor due to the introduction of a new wave of more efficient daas beginning in 2013 , which changed the standards for hcv treatment . in december 2013 , the fda approved sofosbuvir ( sof ) in combination with pegifn / rbv for treatment of hcv genotype 1 infection . this approach achieved a response rate of over 85% ; however , the presence of unfavorable treatment predictors such as cirrhosis , previous history of treatment , and unfavorable host genetics can influence the success rate of treatment with sof / pegifn / rbv ( 19 ) . sof in combination with rbv and/or pegifn was also approved for treatment of hcv genotypes 2 , 3 , and 4 , with limited efficacy for hcv genotype 3 ( 20 ) . in october 2014 , the fda approved ledipasvir ( ldv ) in combination with sof for treatment of hcv genotype 1 , which achieved more than 95% efficacy ( 21 , 22 ) . fortunately , in addition to being more effective than the previous sof / pegifn / rbv regimen , sof / ldv is influenced little by patient characteristics . in december 2014 , the fda approved another ifn - free daa regimen a combination of ombitasvir / paritaprevir - r / dasabuvir ( a three direct acting antiviral , or 3d ) for treatment of hcv genotype 1 infection , with an efficacy rate of over 95% ( 23 ) . finally , in january 2016 , the fda approved combination therapy with grazoprevir / elbasvir ( gzr / ebr ) , with about a 95% svr rate ( 24 ) . other regimens containing daas , such as simeprevir ( smv ) and daclatasvir ( dcv ) , have also been approved for treatment of hcv infection since 2013 ( 25 , 26 ) . the use of new treatment strategies has provided opportunities to eradicate hcv infection . however , effective treatment is not enough ; there are still major issues that must be taken into account , some of which we address in this paper . although an effective treatment regimen is a necessary tool in infection eradication , identifying infected individuals is also of vital importance . subjects with hcv infection can remain asymptomatic for a long time , so they may be unaware of their infection . during this period , infection can progress to advanced liver disease , and the patient may spread the virus to others . surprisingly , about 75% of patients with hcv in the united states are unaware of being infected ( 27 ) . another challenge to developing screening strategies for hcv is the occult nature of this infection , which can not be identified with routine diagnostic tests ( 28 , 29 ) . we believe that health policy makers should design powerful screening programs to identify hcv - infected subjects . risk factors for hcv infection include intravenous drug use ( ivdu ) , history of unsafe injection practices , use of blood and/or blood products before the introduction of blood donor screening for hcv between 1992 and 1996 ( depending on the national policies of different countries ) , being homeless , and history of imprisonment ( 30 ) . the prevalence of these risk factors varies among different populations and countries ; population - based studies have determined that different geographical regions have unique risk factors for hcv infection . for example , baby boomers , persons born between 1945 and 1965 in the united states , account for about 70% of hcv infections in the us ( 31 ) . in addition , previous parenteral therapy for schistosomiasis is a significant predictor of hcv infection in egypt ( 32 ) . however , in some countries , further population - based studies are required to determine the special risk factors related to each area . once this is done , subjects with these risk factors can be recognized as special groups and singled out for screening . these population - based studies should be conducted alongside the implementation of new prevention and treatment strategies in order to track changes in geographically - based risk factors and changes in the prevalence of hcv among subjects with these risk factors . these studies will also help in the evaluation of screening strategies . as a result of geographical differences in risk factors and prevalence , a different screening strategy is needed for each country , and this should be supported by governmental and non - governmental organizations . governments should pay attention to this important issue and fund screening , and health policy makers should design a risk - based , powerful screening method . furthermore , the quality of these activities and screening methods should be evaluated by research projects and epidemiological population - based studies , which can guide policy makers . ultimately , medical practitioners and healthcare personnel have an important role in this regard , and they must support screening projects . risk - based screening strategies can identify about 86% of patients with hcv ( 33 ) . some patients do not have traditional risk factors and therefore can not be identified by risk - based screening . therefore , worldwide clinical screening , together with the approach of risk - based screening ( 34 ) , is recommended . however , the feasibility of such an approach is highly dependent on the economic situation of a given country . one of the main inconveniences of new treatment strategies is their current high cost . for example , the cost of a 12-week treatment with sof is 85,000 - 110,000 usd . new treatment approaches have shorter durations , minimal adverse effects , and higher efficacy ; however , they are very expensive . strategies should be applied to reduce treatment costs and provide wide access to new treatments , especially in low- and middle - income countries , in which about 80% of patients with chc live ( 5 , 35 , 36 ) . people with inherited bleeding disorders ( such as hemophilia ) , people with inherited hemoglobin disorders ( such as thalassemia ) , patients under hemodialysis , patients with organ transplantation ( especially liver- and kidney - transplant patients ) and hcv patients co - infected with hiv are some of the groups that need special attention and priority in treatment ( 37 ) . special patient groups with chc are at increased risk of death or complications ( 38 ) . a basic question arises here : what considerations need to be made for special patient groups in the era of daas ? these populations are the most challenging to treat , and they require massive attention . because of faster progression to both esld and hcc in special patients , hcv is a significant cause of morbidity and mortality in these populations ( 39 ) . hiv / hcv co - infection : hiv and hcv co - infection poses a challenge because it is widespread , particularly among ivdus , and it exhibits lower rates of spontaneous hcv clearance , poor response to treatment of chronic hcv in the pre - daa era , and more rapid progression of hcv - related liver diseases such as cirrhosis and hcc ( 40 ) . global statistics indicate that four to five million people are co - infected with hiv / hcv ( 41 ) . adoption of interferon - based hcv treatments ( following the pegifn / rbv and first - generation hcv protease inhibitors ) resulted in lower svr in those with hiv / hcv co - infection than in those with hcv mono - infection ( 40 ) . furthermore , hcv treatment regimens involve serious adverse effects and pharmacokinetic drug interactions with hiv - antiretroviral drugs ( 42 - 44 ) . the development and approval of new oral regimens of daas has created an opportunity to improve hcv treatment efficacy and safety for hiv / hcv co - infected patients . however , drug interactions between hcv daas and hiv - antiretroviral agents are still a major problem . for example , smv and 3d regimens are contra - indicated in hiv patients receiving many protease inhibitors . today , sof plays a key role in treating hiv / hcv co - infection ; it has a lower degree of interaction with hiv - antiretroviral drugs . the combination of sof / ldv or sof / dcv provides high rates of svr in hiv / hcv co - infected patients . recently , the use of sof / ldv and sof / dcv has been shown to be effective and safe in patients with hiv / hcv co - infection , achieving svr rates of 98% and 96% - 98% , respectively ( 45 , 46 ) . although there has been much progress in the field of hiv / hcv co - infection treatment , this patient population still needs more attention . thalassemia and hemophilia : hcv infection is one of the most common infections following the use of blood and blood products among thalassemia and hemophilia patients ( 47 ) . the treatment of thalassemia patients infected with hcv is a very controversial issue . dual therapy with pegifn / rbv , use of protease inhibitors ( boc and tvr ) , and pegifn / rbv - based triple therapy leads to rbv - associated life - threatening anemia in many thalassemia patients ( 48 ) . on the other hand , the elimination of rbv and the use of low - dose rbv in the treatment of thalassemia patients seems to result in low svr rates ( 49 ) . nevertheless , because of the severe adverse effects of rbv - based treatments in these patients , it is very important to utilize rbv - free regimens . unfortunately , there are currently no clinical trials evaluating the effect of daa use to treat hcv infection in thalassemia patients . although the combination of pegifn and rbv is still used in treatment of hcv - infected thalassemia patients ( 15 ) , a few thalassemia cases have been treated with sof - based treatment in our clinic ( middle east liver disease center ) and experienced a favorable treatment response . however , clinical trials with new daas are required to evaluate this approach . liver failure due to hcv infection is one of the common causes of death in patients with hemophilia . anti - hcv therapy plays a vital role in the interruption of the hcv infection pathway in order to prevent cirrhosis and hcc . the current standard of care for treatment of hcv in hemophilia is pegifn / rbv , which achieves svr in 61% of patients ( 51 ) . however , the side effects of the pegifn / rbv regimen , including thrombocytopenia and excessive bleeding , should be considered . a recent open - label study showed that out of 14 hemophilia patients infected with hcv and treated with sof / ldv , all achieved svr ( 100% ) ( 52 ) . despite the increasing use of highly effective anti - hcv agents with minimal side effects to treat hemophilia patients , hemophiliacs still constitute a unique patient population that requires special consideration . hemodialysis : hcv infection is one of the most common infections transmitted by the parenteral route in patients receiving maintenance hemodialysis ( 53 ) . receiving an ifn - free and , if possible , rbv - free regimen is a fundamental , urgent need in patients under hemodialysis ( 51 ) . in persons with renal impairment receiving chronic hemodialysis , options for hcv treatment however , asunaprevir , dcv , smv , gzr / ebr , and 3d regimens are cleared by hepatic metabolism and can be used in patients with renal disease ( 54 ) . liver transplantation : hepatitis c recurrence is common after liver transplantation when patients are transplanted with detectable viral loads . recurrence of hcv following liver transplantation may accelerate graft injury , which is difficult to treat with ifn / rbv therapy ( 55 ) . antiviral treatment before transplantation can prevent hcv recurrence . ifn - based regimens are poorly tolerated and are either ineffectual or contra - indicated in most liver - transplant patients ( 56 ) . in contrast , sof - based regimens have satisfactory virological response in more than 80% of post - transplant patients ( 57 ) . favorable response to the currently available therapies and new highly effective treatments for hcv revealed that hcv clearance could be significantly improved in special patient groups . daa - based therapies stand to achieve a very high rate of treatment success with minimal side effects . the introduction of new therapeutic agents does not detract from the importance of preventive strategies , including the development of an hcv vaccine ( 58 ) . it seems that an effective vaccine is achievable in the near future , and as bill gates has said , treatment without prevention is simply unsustainable . studies on vaccine development should be prioritized . on the other hand , reduction of harmful behaviors should be the main strategy to reduce the prevalence of hcv infection in certain high - risk groups , such as ivdus and inmates ( 27 , 30 ) . furthermore , public knowledge and awareness are vital to the eradication of every disease , and ignorance will prevent future eradication of hcv . new treatments have a higher rate of success , less severe side effects , and a shorter duration of therapy . the main goal for the hepatology and infectious disease communities is the eradication of hcv in the next 20 years ; however , hcv eradication will be an uphill battle . the next steps are ( 1 ) finding and treating patients with hcv infection in the general population ; ( 2 ) improving the availability and affordability of effective treatments in developing countries , which will bear the majority of the burden of liver diseases in the next decade without proper management ; ( 3 ) combatting hcv infection in special groups , such as patients with thalassemia , hiv / hcv co - infection , kidney disease , and liver - transplant patients ; and ( 4 ) concentration on prevention alongside treatment , always remembering that prevention is better than a cure .

contextafter the introduction of safe and highly effective hepatitis c virus ( hcv ) treatments , eradication of hcv in the next 20 years is the ultimate goal . since 2011 , the advent of first generation direct acting antivirals ( daas ) were started and followed by the introduction of a new wave of daas in 2013 which exhibit outstanding efficacy . it is obvious that the eradication of hepatitis c is not restricted to development of daas.evidence acquisitionan electronic search of available literature published was conducted in all peer - reviewed journal indexed in pubmed , scopus and google scholar . the literature search was done among articles related treatment of hepatitis c with daas in different patient groups with mass screening of the patients and cost benefit of new treatments as main key words.resultsthere are major steps that should be taken to eradicate hcv , including ( 1 ) the development of screening strategies , particularly for groups such as intravenous drug users and recipients of blood or blood products before the introduction of hcv screening in donors ; ( 2 ) the development of strategies to overcome issues with the high cost of recently introduced treatments ; ( 3 ) special attention to special patient groups , such as hiv / hcv co - infection , hemophilia , thalassemia , hemodialysis , and liver - transplant patients ; and ( 4 ) development of preventive strategies , such as the development of an efficient hcv vaccine , special attention to harm reduction in high - risk groups , and promotion of mass awareness of hcv.conclusionsthe eradication of hcv will require significant governmental financial investment for screening , prevention , and treatment of infected patients . although , we have a long way to eradication of hcv , the next steps could be including proper planning to patient finding , availability of new treatments to all patients and development of hcv prevention strategies such as vaccines .

1. Context 2. Evidence Acquisition 3. Results 3.1. The Evolution of Hepatitis C Treatment 3.2. Next Steps for Eradication of HCV Infection 3.3. The Necessity of Mass Screening 3.4. Cost/Benefits of New Treatment Strategies 3.5. Special Patient Groups Need Special Attention 3.6. Prevention Strategies 4. Conclusions

it is estimated that chronic hepatitis c ( chc ) affects more than 180 million people worldwide , which is about 3% of the world population ( 1 ) . an increase in hepatitis c virus ( hcv)-related morbidities and mortalities has been observed in recent years . in addition , increasing rates of advanced liver disease as a result of hcv infection are expected to be observed in the next decade , and developing countries , which have a higher prevalence of hcv infection , will experience the major burden of end - stage liver diseases ( esld ) throughout the world ( 2 - 4 ) . therefore , it is obvious that this infection is a major concern of health policy makers . fortunately , the risk of developing hepatocellular carcinoma ( hcc ) as one of the main complications of hcv infection can be reduced by 75% with successful treatment ( 5 ) , and today , newly introduced treatment strategies have provided opportunities to manage and control this public health concern ( 6 , 7 ) . in the present study , we conducted an electronic search of available literature published to find pertinent contents reporting novel hcv treatments toward clearance in all hcv infected patient groups and cost benefits of new treatments in the era of daas . to identify articles , the search was begun among all peer - reviewed journal indexed in pubmed , scopus and google scholar . the literature search was done by using the following key words : " hepatitis c virus , hcv , hcv genotypes , special groups , hiv / hcv co - infections , thalassemia , hemophilia , hemodialysis , liver transplantation , mass screening , diagnosis and treatment outcome " . the success rate of treatment with this regimen is very dependent on patient characteristics , including age , body mass index , ethnicity , and genetic factors such as polymorphisms near the interferon lambda 3 ( ifnl3 ) gene ( 13 , 14 ) . viral factors , especially hcv genotype , also affect the response to hcv treatment ( 15 ) , and there are always additional factors that should be taken into account in each treatment approach , including treatment success rate , duration , cost , and side effects . in light of these concerns , knowledge of the hcv replication cycle and the role of viral proteins in the virulence of hcv have resulted in targeting of the viral proteins involved in the hcv life cycle to develop new hcv treatments . in 2011 , the first generation of direct acting antivirals ( daas ) boceprevir ( boc ) and telaprevir ( tvr ) were introduced and added to the previous pegifn / rbv regimen ( 17 , 18 ) . triple therapy with boc or tvr quickly fell out of favor due to the introduction of a new wave of more efficient daas beginning in 2013 , which changed the standards for hcv treatment . in december 2013 , the fda approved sofosbuvir ( sof ) in combination with pegifn / rbv for treatment of hcv genotype 1 infection . this approach achieved a response rate of over 85% ; however , the presence of unfavorable treatment predictors such as cirrhosis , previous history of treatment , and unfavorable host genetics can influence the success rate of treatment with sof / pegifn / rbv ( 19 ) . sof in combination with rbv and/or pegifn was also approved for treatment of hcv genotypes 2 , 3 , and 4 , with limited efficacy for hcv genotype 3 ( 20 ) . in october 2014 , the fda approved ledipasvir ( ldv ) in combination with sof for treatment of hcv genotype 1 , which achieved more than 95% efficacy ( 21 , 22 ) . in december 2014 , the fda approved another ifn - free daa regimen a combination of ombitasvir / paritaprevir - r / dasabuvir ( a three direct acting antiviral , or 3d ) for treatment of hcv genotype 1 infection , with an efficacy rate of over 95% ( 23 ) . other regimens containing daas , such as simeprevir ( smv ) and daclatasvir ( dcv ) , have also been approved for treatment of hcv infection since 2013 ( 25 , 26 ) . the use of new treatment strategies has provided opportunities to eradicate hcv infection . however , effective treatment is not enough ; there are still major issues that must be taken into account , some of which we address in this paper . another challenge to developing screening strategies for hcv is the occult nature of this infection , which can not be identified with routine diagnostic tests ( 28 , 29 ) . risk factors for hcv infection include intravenous drug use ( ivdu ) , history of unsafe injection practices , use of blood and/or blood products before the introduction of blood donor screening for hcv between 1992 and 1996 ( depending on the national policies of different countries ) , being homeless , and history of imprisonment ( 30 ) . for example , baby boomers , persons born between 1945 and 1965 in the united states , account for about 70% of hcv infections in the us ( 31 ) . these population - based studies should be conducted alongside the implementation of new prevention and treatment strategies in order to track changes in geographically - based risk factors and changes in the prevalence of hcv among subjects with these risk factors . these studies will also help in the evaluation of screening strategies . governments should pay attention to this important issue and fund screening , and health policy makers should design a risk - based , powerful screening method . however , the feasibility of such an approach is highly dependent on the economic situation of a given country . one of the main inconveniences of new treatment strategies is their current high cost . for example , the cost of a 12-week treatment with sof is 85,000 - 110,000 usd . strategies should be applied to reduce treatment costs and provide wide access to new treatments , especially in low- and middle - income countries , in which about 80% of patients with chc live ( 5 , 35 , 36 ) . people with inherited bleeding disorders ( such as hemophilia ) , people with inherited hemoglobin disorders ( such as thalassemia ) , patients under hemodialysis , patients with organ transplantation ( especially liver- and kidney - transplant patients ) and hcv patients co - infected with hiv are some of the groups that need special attention and priority in treatment ( 37 ) . special patient groups with chc are at increased risk of death or complications ( 38 ) . a basic question arises here : what considerations need to be made for special patient groups in the era of daas ? hiv / hcv co - infection : hiv and hcv co - infection poses a challenge because it is widespread , particularly among ivdus , and it exhibits lower rates of spontaneous hcv clearance , poor response to treatment of chronic hcv in the pre - daa era , and more rapid progression of hcv - related liver diseases such as cirrhosis and hcc ( 40 ) . global statistics indicate that four to five million people are co - infected with hiv / hcv ( 41 ) . adoption of interferon - based hcv treatments ( following the pegifn / rbv and first - generation hcv protease inhibitors ) resulted in lower svr in those with hiv / hcv co - infection than in those with hcv mono - infection ( 40 ) . the development and approval of new oral regimens of daas has created an opportunity to improve hcv treatment efficacy and safety for hiv / hcv co - infected patients . today , sof plays a key role in treating hiv / hcv co - infection ; it has a lower degree of interaction with hiv - antiretroviral drugs . the combination of sof / ldv or sof / dcv provides high rates of svr in hiv / hcv co - infected patients . recently , the use of sof / ldv and sof / dcv has been shown to be effective and safe in patients with hiv / hcv co - infection , achieving svr rates of 98% and 96% - 98% , respectively ( 45 , 46 ) . although there has been much progress in the field of hiv / hcv co - infection treatment , this patient population still needs more attention . thalassemia and hemophilia : hcv infection is one of the most common infections following the use of blood and blood products among thalassemia and hemophilia patients ( 47 ) . on the other hand , the elimination of rbv and the use of low - dose rbv in the treatment of thalassemia patients seems to result in low svr rates ( 49 ) . anti - hcv therapy plays a vital role in the interruption of the hcv infection pathway in order to prevent cirrhosis and hcc . the current standard of care for treatment of hcv in hemophilia is pegifn / rbv , which achieves svr in 61% of patients ( 51 ) . however , the side effects of the pegifn / rbv regimen , including thrombocytopenia and excessive bleeding , should be considered . in persons with renal impairment receiving chronic hemodialysis , options for hcv treatment however , asunaprevir , dcv , smv , gzr / ebr , and 3d regimens are cleared by hepatic metabolism and can be used in patients with renal disease ( 54 ) . ifn - based regimens are poorly tolerated and are either ineffectual or contra - indicated in most liver - transplant patients ( 56 ) . favorable response to the currently available therapies and new highly effective treatments for hcv revealed that hcv clearance could be significantly improved in special patient groups . the introduction of new therapeutic agents does not detract from the importance of preventive strategies , including the development of an hcv vaccine ( 58 ) . on the other hand , reduction of harmful behaviors should be the main strategy to reduce the prevalence of hcv infection in certain high - risk groups , such as ivdus and inmates ( 27 , 30 ) . furthermore , public knowledge and awareness are vital to the eradication of every disease , and ignorance will prevent future eradication of hcv . the success rate of treatment with this regimen is very dependent on patient characteristics , including age , body mass index , ethnicity , and genetic factors such as polymorphisms near the interferon lambda 3 ( ifnl3 ) gene ( 13 , 14 ) . viral factors , especially hcv genotype , also affect the response to hcv treatment ( 15 ) , and there are always additional factors that should be taken into account in each treatment approach , including treatment success rate , duration , cost , and side effects . in light of these concerns , knowledge of the hcv replication cycle and the role of viral proteins in the virulence of hcv have resulted in targeting of the viral proteins involved in the hcv life cycle to develop new hcv treatments . in 2011 , the first generation of direct acting antivirals ( daas ) boceprevir ( boc ) and telaprevir ( tvr ) were introduced and added to the previous pegifn / rbv regimen ( 17 , 18 ) . triple therapy with boc or tvr quickly fell out of favor due to the introduction of a new wave of more efficient daas beginning in 2013 , which changed the standards for hcv treatment . in december 2013 , the fda approved sofosbuvir ( sof ) in combination with pegifn / rbv for treatment of hcv genotype 1 infection . this approach achieved a response rate of over 85% ; however , the presence of unfavorable treatment predictors such as cirrhosis , previous history of treatment , and unfavorable host genetics can influence the success rate of treatment with sof / pegifn / rbv ( 19 ) . sof in combination with rbv and/or pegifn was also approved for treatment of hcv genotypes 2 , 3 , and 4 , with limited efficacy for hcv genotype 3 ( 20 ) . in october 2014 , the fda approved ledipasvir ( ldv ) in combination with sof for treatment of hcv genotype 1 , which achieved more than 95% efficacy ( 21 , 22 ) . in december 2014 , the fda approved another ifn - free daa regimen a combination of ombitasvir / paritaprevir - r / dasabuvir ( a three direct acting antiviral , or 3d ) for treatment of hcv genotype 1 infection , with an efficacy rate of over 95% ( 23 ) . other regimens containing daas , such as simeprevir ( smv ) and daclatasvir ( dcv ) , have also been approved for treatment of hcv infection since 2013 ( 25 , 26 ) . the use of new treatment strategies has provided opportunities to eradicate hcv infection . however , effective treatment is not enough ; there are still major issues that must be taken into account , some of which we address in this paper . another challenge to developing screening strategies for hcv is the occult nature of this infection , which can not be identified with routine diagnostic tests ( 28 , 29 ) . risk factors for hcv infection include intravenous drug use ( ivdu ) , history of unsafe injection practices , use of blood and/or blood products before the introduction of blood donor screening for hcv between 1992 and 1996 ( depending on the national policies of different countries ) , being homeless , and history of imprisonment ( 30 ) . these population - based studies should be conducted alongside the implementation of new prevention and treatment strategies in order to track changes in geographically - based risk factors and changes in the prevalence of hcv among subjects with these risk factors . these studies will also help in the evaluation of screening strategies . as a result of geographical differences in risk factors and prevalence , a different screening strategy is needed for each country , and this should be supported by governmental and non - governmental organizations . governments should pay attention to this important issue and fund screening , and health policy makers should design a risk - based , powerful screening method . one of the main inconveniences of new treatment strategies is their current high cost . for example , the cost of a 12-week treatment with sof is 85,000 - 110,000 usd . strategies should be applied to reduce treatment costs and provide wide access to new treatments , especially in low- and middle - income countries , in which about 80% of patients with chc live ( 5 , 35 , 36 ) . people with inherited bleeding disorders ( such as hemophilia ) , people with inherited hemoglobin disorders ( such as thalassemia ) , patients under hemodialysis , patients with organ transplantation ( especially liver- and kidney - transplant patients ) and hcv patients co - infected with hiv are some of the groups that need special attention and priority in treatment ( 37 ) . special patient groups with chc are at increased risk of death or complications ( 38 ) . a basic question arises here : what considerations need to be made for special patient groups in the era of daas ? hiv / hcv co - infection : hiv and hcv co - infection poses a challenge because it is widespread , particularly among ivdus , and it exhibits lower rates of spontaneous hcv clearance , poor response to treatment of chronic hcv in the pre - daa era , and more rapid progression of hcv - related liver diseases such as cirrhosis and hcc ( 40 ) . global statistics indicate that four to five million people are co - infected with hiv / hcv ( 41 ) . adoption of interferon - based hcv treatments ( following the pegifn / rbv and first - generation hcv protease inhibitors ) resulted in lower svr in those with hiv / hcv co - infection than in those with hcv mono - infection ( 40 ) . the development and approval of new oral regimens of daas has created an opportunity to improve hcv treatment efficacy and safety for hiv / hcv co - infected patients . today , sof plays a key role in treating hiv / hcv co - infection ; it has a lower degree of interaction with hiv - antiretroviral drugs . the combination of sof / ldv or sof / dcv provides high rates of svr in hiv / hcv co - infected patients . recently , the use of sof / ldv and sof / dcv has been shown to be effective and safe in patients with hiv / hcv co - infection , achieving svr rates of 98% and 96% - 98% , respectively ( 45 , 46 ) . although there has been much progress in the field of hiv / hcv co - infection treatment , this patient population still needs more attention . thalassemia and hemophilia : hcv infection is one of the most common infections following the use of blood and blood products among thalassemia and hemophilia patients ( 47 ) . on the other hand , the elimination of rbv and the use of low - dose rbv in the treatment of thalassemia patients seems to result in low svr rates ( 49 ) . the current standard of care for treatment of hcv in hemophilia is pegifn / rbv , which achieves svr in 61% of patients ( 51 ) . however , the side effects of the pegifn / rbv regimen , including thrombocytopenia and excessive bleeding , should be considered . hemodialysis : hcv infection is one of the most common infections transmitted by the parenteral route in patients receiving maintenance hemodialysis ( 53 ) . in persons with renal impairment receiving chronic hemodialysis , options for hcv treatment however , asunaprevir , dcv , smv , gzr / ebr , and 3d regimens are cleared by hepatic metabolism and can be used in patients with renal disease ( 54 ) . ifn - based regimens are poorly tolerated and are either ineffectual or contra - indicated in most liver - transplant patients ( 56 ) . favorable response to the currently available therapies and new highly effective treatments for hcv revealed that hcv clearance could be significantly improved in special patient groups . the introduction of new therapeutic agents does not detract from the importance of preventive strategies , including the development of an hcv vaccine ( 58 ) . it seems that an effective vaccine is achievable in the near future , and as bill gates has said , treatment without prevention is simply unsustainable . on the other hand , reduction of harmful behaviors should be the main strategy to reduce the prevalence of hcv infection in certain high - risk groups , such as ivdus and inmates ( 27 , 30 ) . furthermore , public knowledge and awareness are vital to the eradication of every disease , and ignorance will prevent future eradication of hcv . new treatments have a higher rate of success , less severe side effects , and a shorter duration of therapy . the main goal for the hepatology and infectious disease communities is the eradication of hcv in the next 20 years ; however , hcv eradication will be an uphill battle . the next steps are ( 1 ) finding and treating patients with hcv infection in the general population ; ( 2 ) improving the availability and affordability of effective treatments in developing countries , which will bear the majority of the burden of liver diseases in the next decade without proper management ; ( 3 ) combatting hcv infection in special groups , such as patients with thalassemia , hiv / hcv co - infection , kidney disease , and liver - transplant patients ; and ( 4 ) concentration on prevention alongside treatment , always remembering that prevention is better than a cure .

human and mouse peripheral blood was obtained from five independent healthy blood donors and four c57 black mice , respectively . human and mouse mononuclear cells ( pbmcs ) were isolated by density gradient ( ficoll / histopaque-1077 ; sigma , st . louis , mo ; or lympholyte - m ; cederlane laboratories , hornby , on , canada , respectively ) and cultured in rpmi-1640 containing 10% fbs ( gibco brl , grand island , ny ) . human islets were obtained from the european consortium for islet transplantation ( ecit ) for the research distribution program ( human islet isolation facility , san raffaele institute , milano , italy ) . islet preparations , with purity < 70% , not suitable for transplantation , were obtained and cultured in cmrl medium-1066 ( invitrogen , carlsbad , ca ) with 10% fbs , as previously described ( 11 ) . cell cultures were treated with recombinant trail ( 1 g / ml ) , prepared as described ( 12 ) , for 72 h. cell viability was monitored over time by trypan blue dye exclusion , and apoptosis was monitored by double staining with annexin v / propidium iodide , as previously described ( 13 ) . validation of array results and investigation of specific gene expression were carried out in rna samples with the real - time thermal analyzer rotor - gene 6000 ( corbett , cambridge u.k . ) , by using sybr green based technology and the rt - pcr primer set for human socs1 cdna ( sabioscience , frederick , md ) or mouse socs1 and vascular cell adhesion molecule ( vcam)-1 cdnas ( qiagen , hilden , germany ) . gene expression of the target sequences was normalized in relation to the expression of endogenous controls . ( see also the online appendix , available at http://diabetes.diabetesjournals.org/cgi/content/full/db09-1771/dc1 . ) for western blot analyses , pbmc samples were lysed as previously described ( 14 ) . equal amounts of protein for each sample were migrated in acrylamide gels and blotted onto nitrocellulose filters . the following antibodies were used : monoclonal antibody anti - socs1 and anti - tubulin ( purchased from santa cruz biotechnology , santa cruz , ca , and sigma , respectively ) . after incubation with peroxidase - conjugated anti - mouse igg , specific reactions were revealed with the ecl detection kit ( amersham pharmacia biotech , piscataway , nj ) . animal care and treatments were conducted in conformity with institutional guidelines in compliance with national and international laws and policies ( eec council directive 86/609 , 1987 ) . a total of 24 6-week - old c57 black mice were rendered diabetic by five daily intraperitoneal injections of stz ( sigma ) at a dose of 50 mg / kg . half of the mice treated with stz ( n = 12 ) were also co - injected daily with recombinant trail ( 20 g / day ) ( 12 ) . control mice received the vehicle citrate buffer alone ( mock treated ; n = 10 ) . an additional group of nondiabetic mice ( n = 10 ) was treated with recombinant trail ( 20 g / day ) for 5 consecutive days . the animals had unrestricted access to water and were maintained on a 12-h light - dark cycle in a non pathogen - free environment on standard mouse food ( harlan nossan correzzana , milan , italy ) . serum glucose concentrations were determined weekly by an autoanalyzer technique ( hitachi 717 ; tokyo , japan ) . after 6 weeks , the animals were anesthetized by an intraperitoneal injection of pentobarbital sodium ( 60 mg / kg ) and killed for blood harvesting and histological examination of pancreas . blood was immediately centrifuged to remove cells , and serum was collected for enzyme - linked immunosorbent assay ( elisa ) assays . insulin , tnf- , and osteoprotegerin ( opg ) were measured in serum samples using the insulin mouse ultrasensitive elisa kit ( drg instruments , marburg , germany ) and the mouse tnf- and opg elisa kits ( both purchased from r&d system , minneapolis , mn ) , according to the manufacturer 's instructions . pancreas samples were frozen for rna extraction and were embedded in paraffin for histological examination . the distribution and morphology of pancreatic islets were assessed by analyzing 4-m - thick cross - sectional serial sections after staining with hematoxylin and eosin . pancreatic islets were manually traced on the computer , and area measurements were performed , using a video - based image analysis program ( mcid ; imaging research , st . data were calculated and shown as mean sd or as median and interquartile range ( iqr ) , according to the distribution . comparisons between stz - treated animals and normal controls were performed with student t test and with test . differences in parameters mean values across study phases were analyzed using anova for repeated measures . human and mouse peripheral blood was obtained from five independent healthy blood donors and four c57 black mice , respectively . human and mouse mononuclear cells ( pbmcs ) were isolated by density gradient ( ficoll / histopaque-1077 ; sigma , st . louis , mo ; or lympholyte - m ; cederlane laboratories , hornby , on , canada , respectively ) and cultured in rpmi-1640 containing 10% fbs ( gibco brl , grand island , ny ) . human islets were obtained from the european consortium for islet transplantation ( ecit ) for the research distribution program ( human islet isolation facility , san raffaele institute , milano , italy ) . islet preparations , with purity < 70% , not suitable for transplantation , were obtained and cultured in cmrl medium-1066 ( invitrogen , carlsbad , ca ) with 10% fbs , as previously described ( 11 ) . cell cultures were treated with recombinant trail ( 1 g / ml ) , prepared as described ( 12 ) , for 72 h. cell viability was monitored over time by trypan blue dye exclusion , and apoptosis was monitored by double staining with annexin v / propidium iodide , as previously described ( 13 ) . validation of array results and investigation of specific gene expression were carried out in rna samples with the real - time thermal analyzer rotor - gene 6000 ( corbett , cambridge u.k . ) , by using sybr green based technology and the rt - pcr primer set for human socs1 cdna ( sabioscience , frederick , md ) or mouse socs1 and vascular cell adhesion molecule ( vcam)-1 cdnas ( qiagen , hilden , germany ) . gene expression of the target sequences was normalized in relation to the expression of endogenous controls . ( see also the online appendix , available at http://diabetes.diabetesjournals.org/cgi/content/full/db09-1771/dc1 . ) for western blot analyses , pbmc samples were lysed as previously described ( 14 ) . equal amounts of protein for each sample were migrated in acrylamide gels and blotted onto nitrocellulose filters . the following antibodies were used : monoclonal antibody anti - socs1 and anti - tubulin ( purchased from santa cruz biotechnology , santa cruz , ca , and sigma , respectively ) . after incubation with peroxidase - conjugated anti - mouse igg , specific reactions were revealed with the ecl detection kit ( amersham pharmacia biotech , piscataway , nj ) . animal care and treatments were conducted in conformity with institutional guidelines in compliance with national and international laws and policies ( eec council directive 86/609 , 1987 ) . a total of 24 6-week - old c57 black mice were rendered diabetic by five daily intraperitoneal injections of stz ( sigma ) at a dose of 50 mg / kg . half of the mice treated with stz ( n = 12 ) were also co - injected daily with recombinant trail ( 20 g / day ) ( 12 ) . control mice received the vehicle citrate buffer alone ( mock treated ; n = 10 ) . an additional group of nondiabetic mice ( n = 10 ) was treated with recombinant trail ( 20 g / day ) for 5 consecutive days . the animals had unrestricted access to water and were maintained on a 12-h light - dark cycle in a non pathogen - free environment on standard mouse food ( harlan nossan correzzana , milan , italy ) . serum glucose concentrations were determined weekly by an autoanalyzer technique ( hitachi 717 ; tokyo , japan ) . after 6 weeks , the animals were anesthetized by an intraperitoneal injection of pentobarbital sodium ( 60 mg / kg ) and killed for blood harvesting and histological examination of pancreas . blood was immediately centrifuged to remove cells , and serum was collected for enzyme - linked immunosorbent assay ( elisa ) assays . insulin , tnf- , and osteoprotegerin ( opg ) were measured in serum samples using the insulin mouse ultrasensitive elisa kit ( drg instruments , marburg , germany ) and the mouse tnf- and opg elisa kits ( both purchased from r&d system , minneapolis , mn ) , according to the manufacturer 's instructions . pancreas samples were frozen for rna extraction and were embedded in paraffin for histological examination . the distribution and morphology of pancreatic islets were assessed by analyzing 4-m - thick cross - sectional serial sections after staining with hematoxylin and eosin . pancreatic islets were manually traced on the computer , and area measurements were performed , using a video - based image analysis program ( mcid ; imaging research , st . data were calculated and shown as mean sd or as median and interquartile range ( iqr ) , according to the distribution . comparisons between stz - treated animals and normal controls were performed with student t test and with test . differences in parameters mean values across study phases were analyzed using anova for repeated measures . starting from preliminary cdna microarray analyses of human pbmc cultures exposed to recombinant trail , which identified socs1 as one of the most upregulated genes in response to trail , we validated this observation by quantitative rt - pcr for socs1 in both human pbmcs and mouse pbmcs ( fig . the significant upregulation of the steady - state mrna levels of socs1 was confirmed at the protein level by analyzing socs1 protein by western blot ( fig . because socs1 is known to downregulate the sensitivity of pancreatic -cells to interferon- ( ifn- ) and tnf- ( 79 ) , the effect of trail was next analyzed in human pancreatic islets . in vitro treatment with recombinant trail upregulated socs1 mrna expression also in human islets ( fig . levels of socs1 mrna and protein were analyzed by quantitative rt - pcr ( a and c ) and western blot ( b ) analyses , respectively . in a and c , after normalization to the level of the housekeeping genes , results were expressed as fold of socs1 mrna induction in trail - treated cultures with respect to the control untreated cultures . data are reported as means sd of experiments , each performed in triplicate . in b , the levels of socs1 protein were assessed by western blot analysis in cell lysates . representative examples of western blot results obtained from two ( pb # 1 and pb # 2 ) out of five independent human pbmc cultures are shown . after densitometric analyses , results were expressed as fold of socs1 protein induction in trail - treated cultures with respect to the control untreated cultures . * p < 0.05 with respect to untreated cells . having demonstrated that recombinant trail promotes the expression of socs1 in cultured human and mouse pbmcs , as well as in purified human islets , next experiments were carried out to evaluate the in vivo effect of trail in a relevant animal model of type 1 diabetes . five consecutive intraperitoneal daily injections of low - dose ( 50 mg / kg ) stz were administered to 6-week - old mice ( n = 24 ) that were monitored for 6 weeks , in comparison with a group of vehicle - treated control mice ( n = 10 ) ( fig . half of stz - injected mice ( n = 12 ) also received five intraperitoneal daily injections of recombinant trail ( 20 g / injection ) ( fig . it should be noticed that injection of recombinant trail performed in a group of nondiabetic mice ( n = 10 ) , following the same schedule of diabetic mice ( 20 g / injection per 5 days , fig . 2a ) , did not affect any of the parameters examined in this study ( glycemia , body weight , insulinemia ) . these animals were indistinguishable from vehicle - injected control mice and therefore have been omitted from the figures . effect of in vivo injection of recombinant trail on body weight and glycemia in diabetic mice . a : schematic representation of the treatment protocol for each mice group . b : kinetics of blood glucose levels , reported as fold of increase with respect to time 0 , for each group of mice . c : body weight variation at 6 weeks is reported as percentage of increase with respect with time 0 . horizontal bars are median , upper and lower edges of box are 75th and 25th percentiles , and lines extending from box are 10th and 90th percentiles . mice treated with stz alone displayed a significant increase of blood glucose levels from week 2 onward ( fig . 2b ; from 98 + 19 mg / dl at week 0 , up to 600 + 15 mg / dl at week 6 ) . mice co - injected with stz+trail showed significantly ( p < 0.05 ) lower blood glucose levels with respect to the animals injected with stz alone , although the blood glucose levels of this group remained higher than in controls ( fig . in addition , mice co - injected with stz+trail gained significantly ( p < 0.05 ) more weight than animals treated with stz alone , although the body weight of this group of animals remained lower with respect to mock - treated animals ( fig . 2c ) . at the end of the experimental period ( 6 weeks ) , animals were killed for blood harvesting , to be used for the determination of the circulating levels of insulin , tnf- , and opg and for histopathological examination of the pancreas ( fig . 3 ) . while insulinemia was significantly ( p < 0.05 ) higher in mice co - injected with stz+trail with respect to mice injected with stz alone ( fig . 3a ) , the levels of the inflammatory markers tnf- and opg ( 8,9,1517 ) were lower ( p < 0.05 ) in szt+trail - injected animals ( fig . 3b ) . because repeated injections of recombinant trail attenuated hyperglycemia and increased insulinemia in the majority of diabetic mice , we next examined the total area and the morphology of pancreatic islets . while stz induced a dramatic decrease in the number and size of pancreatic islets ( fig . 3c ) , the total area of pancreatic islets was significant higher ( p < 0.05 ) in szt+trail - injected animals than in stz - injected mice ( fig . effect of in vivo injection of recombinant trail on circulating levels of insulin and inflammatory cytokines in diabetic mice . at the end of the experimental period ( 6 weeks ) , blood was harvested for determination of the levels of insulin ( a ) and tnf- and opg ( b ) by elisa . in c , representative hematoxylin - eosin stained histological sections from pancreas of c57 black mice were treated as indicated . original magnification : 10. arrows indicate islets . in d , islet area was measured in each mice group , as described in research design and methods , by analyzing at least three independent histological pancreatic sections for each mouse ( arbitrary units [ a.u . ] ) . in a , b , and d , horizontal bars are median , upper and lower edges of box are 75th and 25th percentiles , and lines extending from box are 10th and 90th percentiles . ( a high - quality digital representation of this figure is available in the online issue . ) because the data illustrated above suggested that trail treatment decreases systemic markers of inflammation ( tnf- and opg ) , in the last group of experiments , we have analyzed the expression of vcam-1 , a well - defined marker of insulitis ( 9 ) , and socs1 at the pancreatic level . in vivo treatment with recombinant trail 1 ) , socs1 mrna levels were higher in the pancreas of animals treated with stz+trail than in animals treated with stz alone ( fig . 4 ) . effect of in vivo trail treatment on socs1 and vcam-1 rna levels in the pancreas . levels of socs1 and vcam-1 were analyzed by quantitative rt - pcr on rna extracted from pancreas of mice killed at week 6 . data are reported as means sd of quantitative rt - pcr results performed on at least five pancreata for each experimental group . starting from preliminary cdna microarray analyses of human pbmc cultures exposed to recombinant trail , which identified socs1 as one of the most upregulated genes in response to trail , we validated this observation by quantitative rt - pcr for socs1 in both human pbmcs and mouse pbmcs ( fig . the significant upregulation of the steady - state mrna levels of socs1 was confirmed at the protein level by analyzing socs1 protein by western blot ( fig . because socs1 is known to downregulate the sensitivity of pancreatic -cells to interferon- ( ifn- ) and tnf- ( 79 ) , the effect of trail was next analyzed in human pancreatic islets . in vitro treatment with recombinant trail upregulated socs1 mrna expression also in human islets ( fig . levels of socs1 mrna and protein were analyzed by quantitative rt - pcr ( a and c ) and western blot ( b ) analyses , respectively . in a and c , after normalization to the level of the housekeeping genes , results were expressed as fold of socs1 mrna induction in trail - treated cultures with respect to the control untreated cultures . data are reported as means sd of experiments , each performed in triplicate . in b , the levels of socs1 protein were assessed by western blot analysis in cell lysates . representative examples of western blot results obtained from two ( pb # 1 and pb # 2 ) out of five independent human pbmc cultures are shown . after densitometric analyses , results were expressed as fold of socs1 protein induction in trail - treated cultures with respect to the control untreated cultures . having demonstrated that recombinant trail promotes the expression of socs1 in cultured human and mouse pbmcs , as well as in purified human islets , next experiments were carried out to evaluate the in vivo effect of trail in a relevant animal model of type 1 diabetes . five consecutive intraperitoneal daily injections of low - dose ( 50 mg / kg ) stz were administered to 6-week - old mice ( n = 24 ) that were monitored for 6 weeks , in comparison with a group of vehicle - treated control mice ( n = 10 ) ( fig . half of stz - injected mice ( n = 12 ) also received five intraperitoneal daily injections of recombinant trail ( 20 g / injection ) ( fig . it should be noticed that injection of recombinant trail performed in a group of nondiabetic mice ( n = 10 ) , following the same schedule of diabetic mice ( 20 g / injection per 5 days , fig . 2a ) , did not affect any of the parameters examined in this study ( glycemia , body weight , insulinemia ) . these animals were indistinguishable from vehicle - injected control mice and therefore have been omitted from the figures . effect of in vivo injection of recombinant trail on body weight and glycemia in diabetic mice . b : kinetics of blood glucose levels , reported as fold of increase with respect to time 0 , for each group of mice . c : body weight variation at 6 weeks is reported as percentage of increase with respect with time 0 . horizontal bars are median , upper and lower edges of box are 75th and 25th percentiles , and lines extending from box are 10th and 90th percentiles . mice treated with stz alone displayed a significant increase of blood glucose levels from week 2 onward ( fig . 2b ; from 98 + 19 mg / dl at week 0 , up to 600 + 15 mg / dl at week 6 ) . mice co - injected with stz+trail showed significantly ( p < 0.05 ) lower blood glucose levels with respect to the animals injected with stz alone , although the blood glucose levels of this group remained higher than in controls ( fig . in addition , mice co - injected with stz+trail gained significantly ( p < 0.05 ) more weight than animals treated with stz alone , although the body weight of this group of animals remained lower with respect to mock - treated animals ( fig . at the end of the experimental period ( 6 weeks ) , animals were killed for blood harvesting , to be used for the determination of the circulating levels of insulin , tnf- , and opg and for histopathological examination of the pancreas ( fig . 3 ) . while insulinemia was significantly ( p < 0.05 ) higher in mice co - injected with stz+trail with respect to mice injected with stz alone ( fig . 3a ) , the levels of the inflammatory markers tnf- and opg ( 8,9,1517 ) were lower ( p < 0.05 ) in szt+trail - injected animals ( fig . 3b ) . because repeated injections of recombinant trail attenuated hyperglycemia and increased insulinemia in the majority of diabetic mice , we next examined the total area and the morphology of pancreatic islets . while stz induced a dramatic decrease in the number and size of pancreatic islets ( fig . 3c ) , the total area of pancreatic islets was significant higher ( p < 0.05 ) in szt+trail - injected animals than in stz - injected mice ( fig . 3d ) . effect of in vivo injection of recombinant trail on circulating levels of insulin and inflammatory cytokines in diabetic mice . at the end of the experimental period ( 6 weeks ) , blood was harvested for determination of the levels of insulin ( a ) and tnf- and opg ( b ) by elisa . in c , representative hematoxylin - eosin stained histological sections from pancreas of c57 black mice were treated as indicated . original magnification : 10. arrows indicate islets . in d , islet area was measured in each mice group , as described in research design and methods , by analyzing at least three independent histological pancreatic sections for each mouse ( arbitrary units [ a.u . ] ) . in a , b , and d , horizontal bars are median , upper and lower edges of box are 75th and 25th percentiles , and lines extending from box are 10th and 90th percentiles . ( a high - quality digital representation of this figure is available in the online issue . ) because the data illustrated above suggested that trail treatment decreases systemic markers of inflammation ( tnf- and opg ) , in the last group of experiments , we have analyzed the expression of vcam-1 , a well - defined marker of insulitis ( 9 ) , and socs1 at the pancreatic level . in vivo treatment with recombinant trail 1 ) , socs1 mrna levels were higher in the pancreas of animals treated with stz+trail than in animals treated with stz alone ( fig . 4 ) . effect of in vivo trail treatment on socs1 and vcam-1 rna levels in the pancreas . levels of socs1 and vcam-1 were analyzed by quantitative rt - pcr on rna extracted from pancreas of mice killed at week 6 . data are reported as means sd of quantitative rt - pcr results performed on at least five pancreata for each experimental group . in type 1 diabetes , pancreatic islet inflammation contributes to the progressive loss of insulin - producing -cells , which eventually renders the patients , many of them children or adolescents , insulin dependent for life ( 18 ) . in this respect , we have demonstrated for the first time that exogenous recombinant trail , co - injected with stz , significantly reduced the levels of islet damage with respect to animals injected with stz alone . consistently with a protective role of recombinant trail , the levels of insulinemia were significantly ( p < 0.05 ) higher in mice co - injected with stz+trail , and stz+trail - treated animals showed reduced levels of hyperglycemia throughout the whole period of the study with respect to stz - injected animals . moreover , in keeping with previous studies demonstrating that trail displays anti - inflammatory activities in vitro ( 19,20 ) , the in vivo injection of recombinant trail significantly decreased inflammatory markers , both at the systemic ( tnf- and opg ) and pancreatic ( vcam-1 ) levels . the hypothesis that trail might ameliorate stz - induced insulinitis by modulating the host immune response is consistent with another important finding of our study : the ability of trail to induce socs1 expression in both pbmcs and pancreatic islets . although we are aware that we have not formally demonstrated the role of socs1 in mediating the protective effect of recombinant trail , it is noteworthy that the socs family members play a crucial role in controlling the magnitude and duration of several cytokine signals , which have been involved in the pathogenesis of dmt1 , such as in particular tnf- and ifn- ( 10 ) . interestingly , we have also shown that recombinant trail might increase socs1 expression also at the pancreatic level . this is particularly remarkable , since increasing socs1 expression in -cells has been shown to inhibit tnf - induced fas expression in vitro and prevent the progression to diabetes in nod mice ( 2123 ) , and the overexpression of socs1 in islet grafts may inhibit or block the apoptotic pathway and prolong graft survival ( 22 ) . taking into account that 1 ) proinflammatory cytokines , such as tnf- and ifn- , significantly contribute to initiate -cell apoptosis in islets ; 2 ) treatment with recombinant trail does not impair the viability of pancreatic islets , even when overexpressed ( 6 ) ; and 3 ) pancreatic islet transplantation is an important therapeutic option to avoid the need for lifelong hormone injections in type 1 diabetes ( 24 ) , our current data suggest that systemic treatment with recombinant trail , which is currently under investigation in phase i / ii clinical trials for the treatment of several tumors and is usually well tolerated ( 1 ) , might be considered as a therapeutic option for the treatment of type 1 diabetes . this treatment would be either administered alone in the early onset of type 1 diabetes or in association with islet transplantation in the late phase of type 1 diabetes to attenuate the inflammatory and autoimmune response .

objectiveto evaluate the potential therapeutic effect of recombinant human tumor necrosis factor ( tnf)-related apoptosis - inducing ligand ( trail ) treatment in a model of type 1 diabetes.research design and methodsrecombinant trail was added in vitro to primary human and mouse peripheral blood mononuclear cells ( pbmcs ) and isolated human islets to evaluate the expression of the immunoregulatory gene socs1 . diabetes was induced by five consecutive daily injections of low - concentration ( 50 mg / kg ) streptozotocin ( stz ) in c57 black mice ( n = 24 ) . a group of these mice ( n = 12 ) was co - injected with recombinant trail ( 20 g / day ) for 5 days , and the diabetic status ( glycemia and body weight ) was followed over time . after 6 weeks , circulating levels of insulin , tnf- , and osteoprotegerin ( opg ) were measured , and animals were killed to perform the histological analysis of the pancreas.resultsthe in vitro exposure of both pbmcs and human islets to recombinant trail significantly upregulated the expression of socs1 . with respect to stz - treated animals , mice co - injected with stz+trail were characterized by 1 ) lower levels of hyperglycemia , 2 ) higher levels of body weight and insulinemia , 3 ) a partial preservation of pancreatic islets with normal morphology , and 4 ) a lower expression of both systemic ( tnf- and opg ) and pancreatic ( vascular cell adhesion molecule [ vcam]-1 ) inflammatory markers.conclusionsoverall , these data demonstrate that the administration of recombinant trail ameliorates the severity of stz - induced type 1 diabetes , and this effect was accompanied by the upregulation of socs1 expression .

RESEARCH DESIGN AND METHODS Cell cultures. RNA and protein analyses. Animal studies. Statistical analysis. RESULTS In vitro treatment with recombinant TRAIL upregulates the expression of SOCS1 in PBMCs and human islets. In vivo injection of recombinant TRAIL ameliorates the glycemic levels in diabetic mice. In vivo injection of recombinant TRAIL exhibits anti-inflammatory activity and partially preserves pancreatic islets in diabetic mice. DISCUSSION

human and mouse peripheral blood was obtained from five independent healthy blood donors and four c57 black mice , respectively . human and mouse mononuclear cells ( pbmcs ) were isolated by density gradient ( ficoll / histopaque-1077 ; sigma , st . human islets were obtained from the european consortium for islet transplantation ( ecit ) for the research distribution program ( human islet isolation facility , san raffaele institute , milano , italy ) . cell cultures were treated with recombinant trail ( 1 g / ml ) , prepared as described ( 12 ) , for 72 h. cell viability was monitored over time by trypan blue dye exclusion , and apoptosis was monitored by double staining with annexin v / propidium iodide , as previously described ( 13 ) . , by using sybr green based technology and the rt - pcr primer set for human socs1 cdna ( sabioscience , frederick , md ) or mouse socs1 and vascular cell adhesion molecule ( vcam)-1 cdnas ( qiagen , hilden , germany ) . gene expression of the target sequences was normalized in relation to the expression of endogenous controls . a total of 24 6-week - old c57 black mice were rendered diabetic by five daily intraperitoneal injections of stz ( sigma ) at a dose of 50 mg / kg . half of the mice treated with stz ( n = 12 ) were also co - injected daily with recombinant trail ( 20 g / day ) ( 12 ) . an additional group of nondiabetic mice ( n = 10 ) was treated with recombinant trail ( 20 g / day ) for 5 consecutive days . after 6 weeks , the animals were anesthetized by an intraperitoneal injection of pentobarbital sodium ( 60 mg / kg ) and killed for blood harvesting and histological examination of pancreas . insulin , tnf- , and osteoprotegerin ( opg ) were measured in serum samples using the insulin mouse ultrasensitive elisa kit ( drg instruments , marburg , germany ) and the mouse tnf- and opg elisa kits ( both purchased from r&d system , minneapolis , mn ) , according to the manufacturer 's instructions . comparisons between stz - treated animals and normal controls were performed with student t test and with test . human and mouse peripheral blood was obtained from five independent healthy blood donors and four c57 black mice , respectively . human and mouse mononuclear cells ( pbmcs ) were isolated by density gradient ( ficoll / histopaque-1077 ; sigma , st . human islets were obtained from the european consortium for islet transplantation ( ecit ) for the research distribution program ( human islet isolation facility , san raffaele institute , milano , italy ) . cell cultures were treated with recombinant trail ( 1 g / ml ) , prepared as described ( 12 ) , for 72 h. cell viability was monitored over time by trypan blue dye exclusion , and apoptosis was monitored by double staining with annexin v / propidium iodide , as previously described ( 13 ) . , by using sybr green based technology and the rt - pcr primer set for human socs1 cdna ( sabioscience , frederick , md ) or mouse socs1 and vascular cell adhesion molecule ( vcam)-1 cdnas ( qiagen , hilden , germany ) . gene expression of the target sequences was normalized in relation to the expression of endogenous controls . a total of 24 6-week - old c57 black mice were rendered diabetic by five daily intraperitoneal injections of stz ( sigma ) at a dose of 50 mg / kg . half of the mice treated with stz ( n = 12 ) were also co - injected daily with recombinant trail ( 20 g / day ) ( 12 ) . an additional group of nondiabetic mice ( n = 10 ) was treated with recombinant trail ( 20 g / day ) for 5 consecutive days . after 6 weeks , the animals were anesthetized by an intraperitoneal injection of pentobarbital sodium ( 60 mg / kg ) and killed for blood harvesting and histological examination of pancreas . insulin , tnf- , and osteoprotegerin ( opg ) were measured in serum samples using the insulin mouse ultrasensitive elisa kit ( drg instruments , marburg , germany ) and the mouse tnf- and opg elisa kits ( both purchased from r&d system , minneapolis , mn ) , according to the manufacturer 's instructions . the distribution and morphology of pancreatic islets were assessed by analyzing 4-m - thick cross - sectional serial sections after staining with hematoxylin and eosin . pancreatic islets were manually traced on the computer , and area measurements were performed , using a video - based image analysis program ( mcid ; imaging research , st . comparisons between stz - treated animals and normal controls were performed with student t test and with test . starting from preliminary cdna microarray analyses of human pbmc cultures exposed to recombinant trail , which identified socs1 as one of the most upregulated genes in response to trail , we validated this observation by quantitative rt - pcr for socs1 in both human pbmcs and mouse pbmcs ( fig . the significant upregulation of the steady - state mrna levels of socs1 was confirmed at the protein level by analyzing socs1 protein by western blot ( fig . because socs1 is known to downregulate the sensitivity of pancreatic -cells to interferon- ( ifn- ) and tnf- ( 79 ) , the effect of trail was next analyzed in human pancreatic islets . in vitro treatment with recombinant trail upregulated socs1 mrna expression also in human islets ( fig . levels of socs1 mrna and protein were analyzed by quantitative rt - pcr ( a and c ) and western blot ( b ) analyses , respectively . in a and c , after normalization to the level of the housekeeping genes , results were expressed as fold of socs1 mrna induction in trail - treated cultures with respect to the control untreated cultures . in b , the levels of socs1 protein were assessed by western blot analysis in cell lysates . after densitometric analyses , results were expressed as fold of socs1 protein induction in trail - treated cultures with respect to the control untreated cultures . having demonstrated that recombinant trail promotes the expression of socs1 in cultured human and mouse pbmcs , as well as in purified human islets , next experiments were carried out to evaluate the in vivo effect of trail in a relevant animal model of type 1 diabetes . five consecutive intraperitoneal daily injections of low - dose ( 50 mg / kg ) stz were administered to 6-week - old mice ( n = 24 ) that were monitored for 6 weeks , in comparison with a group of vehicle - treated control mice ( n = 10 ) ( fig . half of stz - injected mice ( n = 12 ) also received five intraperitoneal daily injections of recombinant trail ( 20 g / injection ) ( fig . it should be noticed that injection of recombinant trail performed in a group of nondiabetic mice ( n = 10 ) , following the same schedule of diabetic mice ( 20 g / injection per 5 days , fig . 2a ) , did not affect any of the parameters examined in this study ( glycemia , body weight , insulinemia ) . these animals were indistinguishable from vehicle - injected control mice and therefore have been omitted from the figures . effect of in vivo injection of recombinant trail on body weight and glycemia in diabetic mice . b : kinetics of blood glucose levels , reported as fold of increase with respect to time 0 , for each group of mice . c : body weight variation at 6 weeks is reported as percentage of increase with respect with time 0 . mice co - injected with stz+trail showed significantly ( p < 0.05 ) lower blood glucose levels with respect to the animals injected with stz alone , although the blood glucose levels of this group remained higher than in controls ( fig . in addition , mice co - injected with stz+trail gained significantly ( p < 0.05 ) more weight than animals treated with stz alone , although the body weight of this group of animals remained lower with respect to mock - treated animals ( fig . at the end of the experimental period ( 6 weeks ) , animals were killed for blood harvesting , to be used for the determination of the circulating levels of insulin , tnf- , and opg and for histopathological examination of the pancreas ( fig . while insulinemia was significantly ( p < 0.05 ) higher in mice co - injected with stz+trail with respect to mice injected with stz alone ( fig . 3a ) , the levels of the inflammatory markers tnf- and opg ( 8,9,1517 ) were lower ( p < 0.05 ) in szt+trail - injected animals ( fig . because repeated injections of recombinant trail attenuated hyperglycemia and increased insulinemia in the majority of diabetic mice , we next examined the total area and the morphology of pancreatic islets . 3c ) , the total area of pancreatic islets was significant higher ( p < 0.05 ) in szt+trail - injected animals than in stz - injected mice ( fig . effect of in vivo injection of recombinant trail on circulating levels of insulin and inflammatory cytokines in diabetic mice . at the end of the experimental period ( 6 weeks ) , blood was harvested for determination of the levels of insulin ( a ) and tnf- and opg ( b ) by elisa . in c , representative hematoxylin - eosin stained histological sections from pancreas of c57 black mice were treated as indicated . because the data illustrated above suggested that trail treatment decreases systemic markers of inflammation ( tnf- and opg ) , in the last group of experiments , we have analyzed the expression of vcam-1 , a well - defined marker of insulitis ( 9 ) , and socs1 at the pancreatic level . in vivo treatment with recombinant trail 1 ) , socs1 mrna levels were higher in the pancreas of animals treated with stz+trail than in animals treated with stz alone ( fig . effect of in vivo trail treatment on socs1 and vcam-1 rna levels in the pancreas . levels of socs1 and vcam-1 were analyzed by quantitative rt - pcr on rna extracted from pancreas of mice killed at week 6 . starting from preliminary cdna microarray analyses of human pbmc cultures exposed to recombinant trail , which identified socs1 as one of the most upregulated genes in response to trail , we validated this observation by quantitative rt - pcr for socs1 in both human pbmcs and mouse pbmcs ( fig . the significant upregulation of the steady - state mrna levels of socs1 was confirmed at the protein level by analyzing socs1 protein by western blot ( fig . because socs1 is known to downregulate the sensitivity of pancreatic -cells to interferon- ( ifn- ) and tnf- ( 79 ) , the effect of trail was next analyzed in human pancreatic islets . in vitro treatment with recombinant trail upregulated socs1 mrna expression also in human islets ( fig . levels of socs1 mrna and protein were analyzed by quantitative rt - pcr ( a and c ) and western blot ( b ) analyses , respectively . in a and c , after normalization to the level of the housekeeping genes , results were expressed as fold of socs1 mrna induction in trail - treated cultures with respect to the control untreated cultures . in b , the levels of socs1 protein were assessed by western blot analysis in cell lysates . after densitometric analyses , results were expressed as fold of socs1 protein induction in trail - treated cultures with respect to the control untreated cultures . having demonstrated that recombinant trail promotes the expression of socs1 in cultured human and mouse pbmcs , as well as in purified human islets , next experiments were carried out to evaluate the in vivo effect of trail in a relevant animal model of type 1 diabetes . five consecutive intraperitoneal daily injections of low - dose ( 50 mg / kg ) stz were administered to 6-week - old mice ( n = 24 ) that were monitored for 6 weeks , in comparison with a group of vehicle - treated control mice ( n = 10 ) ( fig . half of stz - injected mice ( n = 12 ) also received five intraperitoneal daily injections of recombinant trail ( 20 g / injection ) ( fig . it should be noticed that injection of recombinant trail performed in a group of nondiabetic mice ( n = 10 ) , following the same schedule of diabetic mice ( 20 g / injection per 5 days , fig . 2a ) , did not affect any of the parameters examined in this study ( glycemia , body weight , insulinemia ) . effect of in vivo injection of recombinant trail on body weight and glycemia in diabetic mice . b : kinetics of blood glucose levels , reported as fold of increase with respect to time 0 , for each group of mice . c : body weight variation at 6 weeks is reported as percentage of increase with respect with time 0 . mice co - injected with stz+trail showed significantly ( p < 0.05 ) lower blood glucose levels with respect to the animals injected with stz alone , although the blood glucose levels of this group remained higher than in controls ( fig . in addition , mice co - injected with stz+trail gained significantly ( p < 0.05 ) more weight than animals treated with stz alone , although the body weight of this group of animals remained lower with respect to mock - treated animals ( fig . at the end of the experimental period ( 6 weeks ) , animals were killed for blood harvesting , to be used for the determination of the circulating levels of insulin , tnf- , and opg and for histopathological examination of the pancreas ( fig . while insulinemia was significantly ( p < 0.05 ) higher in mice co - injected with stz+trail with respect to mice injected with stz alone ( fig . 3a ) , the levels of the inflammatory markers tnf- and opg ( 8,9,1517 ) were lower ( p < 0.05 ) in szt+trail - injected animals ( fig . because repeated injections of recombinant trail attenuated hyperglycemia and increased insulinemia in the majority of diabetic mice , we next examined the total area and the morphology of pancreatic islets . 3c ) , the total area of pancreatic islets was significant higher ( p < 0.05 ) in szt+trail - injected animals than in stz - injected mice ( fig . effect of in vivo injection of recombinant trail on circulating levels of insulin and inflammatory cytokines in diabetic mice . at the end of the experimental period ( 6 weeks ) , blood was harvested for determination of the levels of insulin ( a ) and tnf- and opg ( b ) by elisa . in c , representative hematoxylin - eosin stained histological sections from pancreas of c57 black mice were treated as indicated . in d , islet area was measured in each mice group , as described in research design and methods , by analyzing at least three independent histological pancreatic sections for each mouse ( arbitrary units [ a.u . ] in a , b , and d , horizontal bars are median , upper and lower edges of box are 75th and 25th percentiles , and lines extending from box are 10th and 90th percentiles . because the data illustrated above suggested that trail treatment decreases systemic markers of inflammation ( tnf- and opg ) , in the last group of experiments , we have analyzed the expression of vcam-1 , a well - defined marker of insulitis ( 9 ) , and socs1 at the pancreatic level . in vivo treatment with recombinant trail 1 ) , socs1 mrna levels were higher in the pancreas of animals treated with stz+trail than in animals treated with stz alone ( fig . effect of in vivo trail treatment on socs1 and vcam-1 rna levels in the pancreas . in type 1 diabetes , pancreatic islet inflammation contributes to the progressive loss of insulin - producing -cells , which eventually renders the patients , many of them children or adolescents , insulin dependent for life ( 18 ) . in this respect , we have demonstrated for the first time that exogenous recombinant trail , co - injected with stz , significantly reduced the levels of islet damage with respect to animals injected with stz alone . consistently with a protective role of recombinant trail , the levels of insulinemia were significantly ( p < 0.05 ) higher in mice co - injected with stz+trail , and stz+trail - treated animals showed reduced levels of hyperglycemia throughout the whole period of the study with respect to stz - injected animals . moreover , in keeping with previous studies demonstrating that trail displays anti - inflammatory activities in vitro ( 19,20 ) , the in vivo injection of recombinant trail significantly decreased inflammatory markers , both at the systemic ( tnf- and opg ) and pancreatic ( vcam-1 ) levels . the hypothesis that trail might ameliorate stz - induced insulinitis by modulating the host immune response is consistent with another important finding of our study : the ability of trail to induce socs1 expression in both pbmcs and pancreatic islets . although we are aware that we have not formally demonstrated the role of socs1 in mediating the protective effect of recombinant trail , it is noteworthy that the socs family members play a crucial role in controlling the magnitude and duration of several cytokine signals , which have been involved in the pathogenesis of dmt1 , such as in particular tnf- and ifn- ( 10 ) . this is particularly remarkable , since increasing socs1 expression in -cells has been shown to inhibit tnf - induced fas expression in vitro and prevent the progression to diabetes in nod mice ( 2123 ) , and the overexpression of socs1 in islet grafts may inhibit or block the apoptotic pathway and prolong graft survival ( 22 ) . taking into account that 1 ) proinflammatory cytokines , such as tnf- and ifn- , significantly contribute to initiate -cell apoptosis in islets ; 2 ) treatment with recombinant trail does not impair the viability of pancreatic islets , even when overexpressed ( 6 ) ; and 3 ) pancreatic islet transplantation is an important therapeutic option to avoid the need for lifelong hormone injections in type 1 diabetes ( 24 ) , our current data suggest that systemic treatment with recombinant trail , which is currently under investigation in phase i / ii clinical trials for the treatment of several tumors and is usually well tolerated ( 1 ) , might be considered as a therapeutic option for the treatment of type 1 diabetes . this treatment would be either administered alone in the early onset of type 1 diabetes or in association with islet transplantation in the late phase of type 1 diabetes to attenuate the inflammatory and autoimmune response .

human metcam ( humetcam ) , a cam in the immunoglobulin - like gene superfamily , is an integral membrane glycoprotein . alternative names for metcam are muc18 , cd146 , mcam , melcam , a32 , and s - endo 1 . to avoid confusion with mucins and to reflect its biological functions , we have renamed muc18 as metcam ( metastasis cam ) , which means an immunoglobulin - like cam that affects or regulates metastasis , . the humetcam has 646 aminoacids that include a n - terminal extracellular domain of 558 aminoacids , which has 28 aminoacids characteristics of a signal peptide sequence at its n - terminus , a transmembrane domain of 24 aminoacids ( amino acid number 559583 ) , and a cytoplasmic domain of 64 aminoacids at the c - terminus . humetcam has eight putative n - glycosylation sites ( asn - x - ser / thr ) , of which six are conserved , and are heavily glycosylated and sialylated resulting in an apparent molecular weight of 113,000150,000 . the extracellular domain of the protein comprises five immunoglobulin - like domains ( v - v - c2-c2-c2 ) [ 1 , 7 ] and an x domain . the cytoplasmic tail contains peptide sequences that will potentially be phosphorylated by protein kinase a ( pka ) , protein kinase c ( pkc ) , and casein kinase 2 ( ck 2 ) [ 1 , 7 , 8 ] . my lab has also cloned and sequenced the mouse metcam ( mometcam ) cdna , which contains 648 aminoacids with a 76.2% identity with humetcam , suggesting that mometcam is likely to have biochemical properties and biological functions similar to the human counterpart . the structure of the humetcam protein is depicted in figure 1 , suggesting that metcam , similar to most cams , plays an active role in mediating cell - cell and cell - extracellular interactions , crosstalk with many intracellular signaling pathways , and modulating the social behaviors of cells . humetcam is expressed in a limited number of normal tissues , such as hair follicular cells , smooth muscle cells , endothelial cells , cerebellum , normal mammary epithelial cells , basal cells of the lung , activated t cells , intermediate trophoblast , and normal nasopharyngeal epithelial cells . the protein is overly expressed in most ( 67% ) malignant melanoma cells , and in most ( more than 80% ) premalignant prostate epithelial cells ( pin ) , high - grade prostatic carcinoma cells , and metastatic lesions [ 12 , 13 ] . humetcam is also expressed in other cancers , such as gestational trophoblastic tumors , leiomyosarcoma , angiosarcoma , haemangioma , kaposi 's sarcoma , schwannoma , some lung squamous and small cell carcinomas , some breast cancer , some neuroblastoma , and also nasopharyngeal carcinoma and ovarian cancer . it is now well documented that in addition to tissue - specific signatures in different cancer types , cancers from different tissues also express some common genes [ 1517 ] . cams do not merely act as a molecular glue to hold together homotypic cells in a specific tissue or to facilitate interactions of heterotypic cells ; cams also actively govern the social behaviors of cells by affecting the adhesion status of cells and modulating cell signaling . they also actively mediate the cell - to - cell and cell - to - extracellular matrix interactions to allow cells to constantly respond to physiological fluctuations and to alter / remodel the surrounding microenvironment for survival . they do so by crosstalk with cellular surface growth factor receptors , which interact with growth factors that may be secreted from stromal cells or released from circulation and embedded in the extracellular matrix [ 18 , 19 ] . thus , an altered expression of cams affects the motility and invasiveness of many tumor cells in vitro and metastasis in vivo [ 18 , 19 ] . cams also play an important role in the favorable soil that provides a proper microenvironment at a suitable period to awaken the dormant metastatic tumor cells to enter into an aggressive growth phase . actually , the metastatic potential of a tumor cell , as documented in many carcinomas , is the consequence of a complex participation of many over- and under - expressed cams [ 18 , 19 ] . based on the above information , aberrant expression of humetcam may also affect the motility and invasiveness of many tumor cells in vitro and metastasis in vivo . it is logical to hypothesize that humetcam / muc18 should play an important role in promoting the malignant progression of many cancer types [ 7 , 18 ] . however , recently we observed an unexpected opposite function of metcam / muc18 in the malignant progression of a mouse melanoma subline and ovarian cancer cells , in which it functioned as a tumor and metastasis suppressor ( wu , unpublished results ) . in this paper , we will review its dual roles in the tumorigenesis and metastasis in different cancer types . metcam - induced tumorigenesis has been studied in melanoma , prostate cancer , breast cancer , and ovarian cancer . overexpression of metcam may have no effect , a negative effect , or a positive effect on tumorigenesis , dependent upon the cell lines used , as shown in the following . overexpression of metcam had a slight tumor suppression effect on tumorigenesis of human melanoma cells in xenograft mice , as shown in figure 2 , but it had no effect on tumorigenesis of two sublines , number 3 and number 10 , of the mouse melanoma cell line k1735 in syngeneic mice . figure 3 shows only the effect of mometcam on the tumorigenesis of k1735 - 3 . only one group showed that overexpression of metcam increased tumorigenesis of a human melanoma cell line in xenograft mice ; however , the results were questionable because only the tumorigenicity of one mouse injected with metcam - expressing clone and one mouse with control cells was determined , and thus no standard deviations were indicated and no statistical analysis was done , as shown in figure 4 . the most compelling evidence for its tumor suppressor effect is in the subline number 9 of the mouse melanoma cell line k1735 ( k9 ) in syngeneic c3h mice . overexpression of mometcam in the k9 cells decreased subcutaneous tumorigenesis in immunocompetent syngeneic c3h mice [ 22 , 23 ] , as shown in figure 5 . shih et al . showed that metcam was not expressed in mcf-7 cell line , and they showed that the overexpression of humetcam in mcf-7 cells suppressed tumor formation of the cells in scid mice , as shown in figure 6 , suggesting that metcam is a possible tumor suppressor in breast cancer . we have confirmed from their western blot and immunohistochemistry results that metcam is not expressed in mcf-7 cells ( 0% ) , very weakly expressed in sk - br-3 cells ( 5% ) , and weakly expressed , though slightly higher levels than the above two cells lines , in the human mammary cancer cell lines , mda - mb-231 ( a low metastatic cell line ) ( 16% ) , and mda - mb-468 ( a high metastatic cell line ) ( 22% ) , as shown in figure 7 . recently gene expression profiles of breast cancer cell lines have indicated that the gene expression profiles of mcf-7 and sk - br3 are more closely related to the luminal subtype of the breast cancers , whereas those of mda - mb-231 and mda - mb-468 are more closely related to the basal - like subtype [ 25 , 26 ] . it appeared that metcam is not or weakly expressed in cell lines established from luminal subtypes , but it is moderately expressed in cell lines established from basal - like subtypes , mda - mb-231 and mda - mb-468 . . found that overexpression of metcam inhibited the in vitro invasiveness of mda - mb-231 cells , supporting the notion of shih et al . on the contrary , garcia et al . and zabouo et al . supported the opposite role of metcam in the progression of human breast cancer cells in that it plays a role of tumor promoter . however , all three groups did not substantiate their claim with studies in animal models . to resolve this controversy , we recently reinvestigated the role of metcam in the tumorigenesis of human breast cancer cells in animal models and found that overexpression of metcam promoted the tumorigenesis of four human breast cancer cell lines , mcf7 , sk - br-3 , mda - mb-231 , and mda - mb-468 [ 30 , 31 ] . tumorigenesis of mcf-7 in female scid mice is shown in figure 8 , and that of sk - br-3 in female nude mice in figure 9 . thus , the tumor suppression role of metcam in tumorigenesis of human breast cancer cells is not supported by the above evidence . on the contrary , it suggests the alternative notion that metcam increased tumorigenesis and perhaps also the metastasis of human breast cancer cells . recently , both our group and another group found that metcam was upregulated in human ovarian cancer specimens , suggesting that metcam may be a marker for the poor prognosis of ovarian cancer patients [ 14 , 32 ] , and that metcam may play a positive role in the development of ovarian cancer [ 14 , 32 ] . however , preliminary animal tests ( injection of bg-1 cells in nonorthotopic , subcutaneous sites of female nude mice ) show that overexpression of metcam did not have any significant effect on the tumor formation of a human ovarian cancer cell line , bg-1 ( data not shown ) . to rule out the possibility that this effect might be an artifact because the tests were carried out in the nonorthotopic , subcutaneous sites , which did not provide a proper microenvironment for tumorigenesis , we carried out further tests of the effect of overexpression of metcam on tumorigenesis of bg-1 cells by injecting the clones in an orthotopic site , the intraperitoneal cavity of female scid mice . we found that tumorigenesis of bg-1 clones was also very poor , suggesting that estrogen supplement by subcutaneous implantation may enhance the tumorigenesis of bg-1 cells in both immunodeficient mice . nevertheless , the test of the effect of overexpression of metcam on tumorigenesis of bg-1 cells in orthotopic sites had a somewhat suppressive effect , as shown in figure 10 . we also carried out animal studies by using another human ovarian cancer cell line , sk - ov-3 and found that overexpression of metcam suppressed tumorigenesis of sk - ov-3 cells at both nonorthotopic , subcutaneous sites , as well as an orthotopic site ( the intraperitoneal cavity ) , as shown in figure 11 . overexpression of metcam significantly increases the tumor - take and promote tumorigenicity and tumorigenesis of a human prostate cancer cell line , lncap , as shown in figure 12 [ 35 , 36 ] . we found that mometcam was expressed at a higher level in a mouse angiosarcoma clone , svr , which was transfected with h - ras , than in the immortalized normal endothelial cell line control , ms-1 ( figure 13 ) . the higher level of mometcam expression appeared to correlate with the higher tumorigenicity of the svr cell line [ 7 , 37 ] , suggesting a positive role for metcam in promoting angiosarcoma . there is a negative correlation of metcam expression with the human nasopharyngeal carcinoma specimens , suggesting that metcam may also play a tumor suppressor role in the tumorigenesis of nasopharyngeal carcinoma . a tumor suppressor role of metcam may also be implicated in haemangiomas , since metcam expression was decreased during the progression of haemangiomas . metcam - induced metastasis has been studied in melanoma , prostate cancer , osteosarcoma , and ovarian cancer lines . overexpression of metcam in melanoma cells mostly have a positive effect on the metastasis of human melanoma cell lines in immunodeficient mice ( both athymic nude and scid mice ) [ 3 , 20 ] , mouse melanoma cell lines in syngeneic mice [ 7 , 21 ] , and a human prostate cancer cell line , lncap , in nude mice [ 7 , 13 , 36 ] . overexpression of metcam also has a positive effect on the metastasis of osteosarcoma cell lines . surprisingly , we have recently found that overexpression of metcam has a negative effect on the metastasis of one subline , number 9 , of mouse melanoma cell k1735 [ 22 , 23 ] and ovarian cancer cell lines [ 3032 ] . humetcam was originally found to be abundantly expressed on the cellular surface of most malignant human melanomas ; since then , it has been postulated to play a role in the progression of human melanoma . this notion is also supported by the positive correlation of mometcam expression with the metastatic ability of several mouse melanoma cells lines . definitive proof comes from the results that the stably ectopic expression of the humetcam cdna gene in three nonmetastatic human cutaneous melanoma cell lines increases the metastatic abilities of these cell lines in immune - deficient mouse models [ 3 , 20 ] . furthermore , the stable , ectopic expression of mometcam cdna in two low - metastatic mouse melanoma cell lines increases the metastatic abilities of these cell lines in immune - competent syngeneic mice . however , metcam enables melanoma cells to establish pulmonary metastasis only when the cells are injected into the tail vein ( experimental metastasis assay ) [ 3 , 20 , 21 ] , thus bypassing the initial stages of metastasis . no metastasis was found when metcam - expressing melanoma cells were injected subcutaneously ( spontaneous metastasis assay ) either in immune - deficient mouse models [ 3 , 20 ] or in immune - competent syngeneic mouse models . taken together , metcam definitely promotes the metastasis of melanoma cells , but at later stages ; thus overexpression of metcam did not initiate the metastasis of melanoma cells . this result is consistent with the recent observation that fibroblast growth factor 2 , but neither humetcam nor integrin actually initiates the malignant progression of subcutaneous melanocyte into melanoma . in contrast to these results , overexpression of mometcam in one mouse melanoma cell line k1735 subline number 9 ( k9 ) decreased pulmonary lung nodule formation when cells were injected into tail veins ( experimental metastasis test ) [ 22 , 23 ] , as shown in figure 14 . overexpression of metcam is not limited to melanoma as previously thought [ 7 , 10 ] . our group has pioneered the successful determination of humetcam expression in prostate cancer cells and tissues using our chicken polyclonal anti - humetcam and carried out extensive studies of humetcam - mediated prostate cancer metastasis . we have used molecular biological and immunological methods to study the expression of humetcam in three established prostate cancer cell lines and human prostate cancer tissues , and in immunohistochemical studies of paraffin - embedded human prostate cancer tissue sections [ 7 , 8 , 12 , 13 ] . from the results , we have suggested that humetcam may be a new diagnostic marker for the metastatic potential of human prostate cancer . this is further corroborated by results of a positive correlation of mometcam expression with the progression of mouse prostate adenocarcinoma in a transgenic mouse model , tramp . from these results , we have also suggested that humetcam may be a key determinant in promoting tumorigenesis and metastasis of human prostate cancer cells . to test this hypothesis , we determined the effect of ectopic expression of humetcam on the ability of human prostate lncap cells to form tumor in the prostate gland and to initiate metastasis in nude mice . we found that overexpression of metcam had a positive effect on the metastasis of the human prostate cancer cell , lncap , when the cells were injected at the orthotopic site ( the dorsolateral lobes of the nude mice ) . the metastatic lesions were found in multiple organs , such as seminal vesicles , ureter , kidney , and periaortic lymph nodes . different mice had metastatic lesions in one or two organs , but all of them had metastatic lesions in the lymph nodes . the parental lncap cells , which do not express any metcam , can form tumors in the prostate , but these tumors did not manifest any metastasis . but our recent preliminary results appear to show that overexpression of metcam may be able to enhance establishment of the growth of a bone - homing c42b clone of lncap cells in nude mice . , metcam can actually initiate the metastasis of lncap cells , thus affecting the progression of prostate cancer cells at the early stage of metastasis [ 7 , 36 ] . recently , one group has shown that metcam is overly expressed in two of the six human osteosarcoma cell lines . the metastasis can be blocked by a humanized antibody against metcam , suggesting metcam plays a positive role in the progression of osteosarcomas . recently we found that overexpression of metcam / muc18 suppressed metastasis and ascites formation of sk - ov-3 cells in the intraperitoneal cavity , as shown in figure 15 . decreased expression of metcam has been correlated with the progression of haemangioma , suggesting the possible negative effect of metcam on progression of haemangioma . though metcam was downregulated in nasopharyngeal carcinoma , interestingly it was upregulated again in metastatic lesions in nasopharyngeal patients , suggesting that metcam may play a positive role in the malignant progression of nasopharyngeal carcinoma after a transient suppression of tumorigenesis . taken together , we suggest that the possible tumor and metastasis suppressor role of metcam may not be limited to melanoma and ovarian cancers , and that this may be a new function of metcam yet to be explored . summary 3.5 table 1 summarizes the possible role of metcam in the tumorigenesis and metastasis of various tumors / cancers.taken together , humetcam is a tumor promoter for prostate and breast cancers , and a metastatic gene for most melanoma cell lines , prostate cancer , osteosarcoma , and perhaps , breast cancer and nasopharyngeal carcinoma . it is a tumor suppressor for a mouse melanoma subline and ovarian cancers , and perhaps , haemangioma and nasopharyngeal carcinoma ; it is a metastasis suppressor for a mouse melanoma subline , ovarian cancer , and perhaps , haemangioma . table 1 summarizes the possible role of metcam in the tumorigenesis and metastasis of various tumors / cancers.taken together , humetcam is a tumor promoter for prostate and breast cancers , and a metastatic gene for most melanoma cell lines , prostate cancer , osteosarcoma , and perhaps , breast cancer and nasopharyngeal carcinoma . it is a tumor suppressor for a mouse melanoma subline and ovarian cancers , and perhaps , haemangioma and nasopharyngeal carcinoma ; it is a metastasis suppressor for a mouse melanoma subline , ovarian cancer , and perhaps , haemangioma . how does metcam mediate or regulate tumorigenesis and metastasis of cancer cells ? by deducing knowledge learned from the tumorigenesis of other tumors [ 1519 , 42 ] and the humetcam - mediated progression of melanoma [ 4345 ] and angiogenesis [ 2 , 4651 ] , we may be able to find some common clues to begin understanding its mechanisms . first , the transcriptional expression of metcam gene may be regulated by pka / creb ( camp - responsive element binding protein ) , ap-2 [ 44 , 45 ] , and other transcription factors , such as sp-1 , c - myb , n - oct2 , ets , carg , egr-1 , and transcription factors binding to insulin - response elements , as shown in figure 16 . among these potential regulators , it is well documented that the ap-2 transcription factor plays a crucial tumor suppressor role in the progression of melanoma , prostate , and breast cancer . it has been shown that pka / creb plays a positive role in the progression of melanoma , and perhaps also applicable to breast cancer and prostate cancer , by inhibiting the expression of ap-2and increasing the expression of metcam . the roles of other transcription regulators , tissue - specific enhancers and repressors , epigenetic control , and control at the level of chromatin remodeling of the gene have still yet to be investigated . second , since the cytoplasmic tail of metcam contains consensus sequences potentially to be phosphorylated by pka , pkc , and ck2 , it may manifest its functions by crosstalk with various signaling pathways mediated by these protein kinases . for example , metcam expression in melanoma cells is reciprocally regulated by akt , in which akt up - regulates the level metcam and overexpression of metcam activates endogenous akt , which in turn inhibits apoptosis and increases survival ability . however , it is not clear if a similar mechanism is also used in breast , prostate , and other cancers . also , the detailed mechanism of how akt up - regulates the expression of metcam has not been worked out . pka , pkc , and ck2 may phosphorylate the cytoplasmic tail of metcam , which then facilitates its interaction with fak , thus promoting cytoskeleton remodeling . alternatively , after phosphorylation of its cytoplasmic tail by these protein kinases , metcam may interact with the downstream effectors of ras , activating erk and jnk , which in turn may transcriptionally activate the expression of akt or other genes that promote the proliferation and angiogenesis of tumor cells . though metcam has not been shown to be a substrate of ck2 , which has been shown to phosphorylate other cams , such as cd44 , e - cadherin , l1-cam , and vitronectin , it is also likely that ck2 may be able to phosphorylate metcam and link it to akt to affect the proliferation , survival , and other tumorigenesis - related functions of tumor cells . third , after the engagement of metcam with the ligand(s ) or extracellular matrix , it may transmit the outside - in signals into tumor cells by activating fak and the downstream - signaling components , promoting cytoskeleton remodeling and increasing tumor cell motility and invasiveness [ 2 , 7 ] . fourth , from what we know about the roles of other cams in the progression of other tumors [ 1519 , 42 ] , it is logical to postulate that metcam may affect cancer cell progression by crosstalk with signaling pathways that affect apoptosis , survival and proliferation , and angiogenesis of tumor cells [ 7 , 18 , 42 ] . thus , metcam may affect tumorigenesis and metastasis by altering the expression of various indexes in apoptosis , survival signaling , proliferation signaling , and angiogenesis . to support this notion , we have found that metcam promotes the progression of prostate cancer cells by rendering the cells with increased proliferative ability by elevating levels of ki67 and pcna , with increased survival ability by elevating the level of phosphorylated akt , and with increased angiogenic ability by elevating levels of vegf , vegfr2 , and cd31 ; but it has no effect on the process of apoptosis . in contrast to this , metcam promotes the progression of melanoma cells differently by preventing the apoptosis of melanoma cells and reciprocally affecting the expression of a survival index , phospho - akt . further systematic studies by using specific rnais to knockdown the downstream effectors one - by - one in the metcam - expressing clones may be necessary to further understand this aspect of mechanism . fifth , metcam may mediate hematogenous spreading of melanoma cells , which had been implicated by its expression in endothelial cells , as well as in malignant melanoma cells , further shown to be present in the junctions of endothelial cells [ 49 , 50 ] and essential for tumor angiogenesis in at least three tumor cell lines and human prostate cancer lncap cells . it is highly likely that metcam expression may promote hematogenous spreading of prostate cancer cells , similar to melanoma cells . similar mechanisms may also be used for the metcam - mediated hematogenous spreading of breast cancer and osteosarcoma cells . however , it is not known if metcam also plays a role in the lymphatic spread of cancer cells . recent results from one group showed that metcam is one of the lymphatic metastasis - associated genes , which is upregulated in malignant mouse hepatocarcinoma ; suggesting that metcam may also play a role in promoting lymphatic metastasis of cancer cells . labeling the cells with viable dyes and following the process in real time by using a newly developed nonintruding , but highly photo - penetrating imaging method of photoacoustic tomography ( pat ) [ 54 , 55 ] may be useful for monitoring each step in the metcam - mediated progression . for the metcam - mediated dynamic spreading of melanoma cells in vivo , the pat imaging method coupled with using hairless syngeneic mouse animal models should reveal more clearly the process in real time . sixth , metcam has been shown to express in normal mesenchymal cells ( smooth muscle , endothelium , and schwann cells ) in the tissue stroma and be a marker for the mesenchymal stem cells , metcam may play an important role in regulating tumor dormancy or awakening , driving or preventing cancer cells to premetastatic niche , and formatting a microenvironment for favorable or unfavorable tumor growth in secondary sites . seventh , metcam may affect the progression of cancer cells by interactions with the host immune system , which , however , has been shown to have a paradoxical role in tumor progression . recently , one group has shown that a subset of host b lymphocytes may control melanoma metastasis through metcam - dependent interaction . on the other hand , it is highly likely that the tumor suppression effect of metcam expression in melanoma k1735 - 9 subline may be due to the interaction of metcam - expressing cells with the host immune defense system in the immunocompetent syngeneic c3h brown mouse , since the intrinsic motility and invasiveness of mouse melanoma k1735 - 9 was increased by the metcam expression [ 22 , 23 ] . for example , the surface metcam expressed in this particular melanoma cell line may have a homophilic interaction with the nk cells , which also express metcam and enhance cytotoxic functions of nk cells . this hypothesis should be testable by studying the metcam - mediated metastasis of metcam - expressing k1735 - 9 cells in various genetically altered mice with a knockout of cd4 + t cells , cd8 + t cells , or nk cells , or mice with a combined knockout of these immune cells . eighth , malignant progression of cancer cells has been shown to associate with an abnormal glycosylation , resulting in expression of altered carbohydrate determinants . thus , the glycosylated status of metcam in different cancer types may be different from normal cells , thus manifesting positive or negative effect on the progression of different cancer types . this aspect of the metcam - mediated cancer progression has not been well studied , but is especially intriguing since metcam possesses six conserved n - glycosylation sites in the extracellular domain [ 7 , 8 ] . we should always keep in mind that mechanisms of metcam - mediated cancer progression may be slightly different in different cancer cells due to their different intrinsic properties , which provides different cofactors and/or different ligand(s ) that either positively or negatively regulate the metcam - mediated tumorigenesis and metastasis . to further understand the role of metcam in these processes , it is essential to diligently identify the cofactors and the metcam - cognate heterophilic ligand(s ) , which modulate the biological functions of metcam . the endeavor in this direction appears to be promising from our preliminary attempts that we may have successfully found a possible candidate of metcam 's heterophilic ligand in metcam - expressing human prostate cancer cells . mechanisms of metcam - mediated negative role in the progression of some cancer cells have not been studied at all . does metcam in some cancers behave like e - cadherin , which always plays a negative role in the tumorigenesis and metastasis of most epithelial cancer cells ? but even e - cadherin may function differently in different cancer cells . for example , its expression is temporally different and correlates with different stages during the progression of ovarian cancer : e - cadherin is not expressed in the ovarian surface epithelial cells , expressed in premalignant lesions and in well - differentiated tumors , and finally not expressed in late - stage invasive tumors . likewise , metcam may express and function normally in the normal nasopharyngeal epithelium , transiently reduce its expression and lose its function during the development of nasopharyngeal carcinoma , resume its expression , and function in the invasion stage of the cancer . alternatively , metcam may behave differently from e - cadherin by being modulated by different cofactors or ligands , which are expressed at different stages of the cancer . the tumor suppressor role of metcam in ovarian cancer cells may not be due to the altered intrinsic properties of the cancer cells , since the intrinsic motility and invasiveness of human ovarian cancer bg-1 and sk - ov-3 cells was not affected by the metcam expression [ 34 , 35 ] . our preliminary results appear to suggest a special mechanism that a soluble form of metcam , which is produced by mmps in the metcam - expressing cells , may mediate the suppressive effect in ovarian cancer cells , similar to the production of a soluble form of p - cadherin by the induced mmps in breast cancer cells , which then dictates , instead of suppresses , the aggressive behavior of the breast cancer cells . metcam may have a key positive function in the progression of angiosarcoma , breast cancer , osteosarcoma , prostate cancer , and most melanoma cell lines . on the other hand , it may also have a key function in suppressing the progression of a few melanoma cell lines , ovarian cancer , haemangioma , and other cancers . to further understand its mechanisms in these processes , it is crucial to define its functional domains , identify its cognate ligand(s ) and cofactor regulators , and study its crosstalk with members of various signaling pathways . these model systems may be useful for real - time observation of the dynamic process of cancer progression by using a nonintrusive and high photo - penetrating imaging system , such as the newly developed photoacoustic tomography ( pat ) , to further understanding the process in mouse models [ 54 , 55 ] . the knowledge gained would also be useful for designing effective means to decrease , or even to block the metastatic potential of these cancers . along these lines , a preclinical trial of using doxazosin , an 1-adrenergic antagonist that has been used to treat the bph patients , has been shown to be able to suppress prostate cancer metastasis in the tramp mouse model . furthermore , preclinical trials using a fully humanized anti - metcam antibody against melanoma growth and metastasis [ 65 , 66 ] and using a mouse anti - metcam monoclonal antibody against angiogenesis and tumor growth of hepatocarcinoma , leiomyosarcoma , and pancreatic cancer have been successfully demonstrated . alternatively , small soluble peptides derived from metcam may also be useful for blocking the tumor formation and tumor angiogenesis [ 52 , 67 , 68 ] .

metcam , an integral membrane cell adhesion molecule ( cam ) in the ig - like gene superfamily , is capable of performing typical functions of cams , such as mediating cell - cell and cell - extracellular interactions , crosstalk with intracellular signaling pathways , and modulating social behaviors of cells . metcam is expressed in about nine normal cells / tissues . aberrant expression of metcam has been associated with the progression of several epithelial tumors . further in vitro and in vivo studies show that metcam plays a dual role in the progression of different tumors . it can promote the malignant progression of several tumors . on the other hand , it can suppress the malignant progression of other tumors . we suggest that the role of metcam in the progression of different cancer types may be modulated by different intrinsic factors present in different cancer cells and also in different stromal microenvironment . many possible mechanisms mediated by this cam during early tumor development and metastasis are suggested .

1. Introduction 2. 3. 4. Mechanisms of 5. Conclusions and Clinical Applications

human metcam ( humetcam ) , a cam in the immunoglobulin - like gene superfamily , is an integral membrane glycoprotein . to avoid confusion with mucins and to reflect its biological functions , we have renamed muc18 as metcam ( metastasis cam ) , which means an immunoglobulin - like cam that affects or regulates metastasis , . the structure of the humetcam protein is depicted in figure 1 , suggesting that metcam , similar to most cams , plays an active role in mediating cell - cell and cell - extracellular interactions , crosstalk with many intracellular signaling pathways , and modulating the social behaviors of cells . humetcam is expressed in a limited number of normal tissues , such as hair follicular cells , smooth muscle cells , endothelial cells , cerebellum , normal mammary epithelial cells , basal cells of the lung , activated t cells , intermediate trophoblast , and normal nasopharyngeal epithelial cells . the protein is overly expressed in most ( 67% ) malignant melanoma cells , and in most ( more than 80% ) premalignant prostate epithelial cells ( pin ) , high - grade prostatic carcinoma cells , and metastatic lesions [ 12 , 13 ] . humetcam is also expressed in other cancers , such as gestational trophoblastic tumors , leiomyosarcoma , angiosarcoma , haemangioma , kaposi 's sarcoma , schwannoma , some lung squamous and small cell carcinomas , some breast cancer , some neuroblastoma , and also nasopharyngeal carcinoma and ovarian cancer . it is now well documented that in addition to tissue - specific signatures in different cancer types , cancers from different tissues also express some common genes [ 1517 ] . cams do not merely act as a molecular glue to hold together homotypic cells in a specific tissue or to facilitate interactions of heterotypic cells ; cams also actively govern the social behaviors of cells by affecting the adhesion status of cells and modulating cell signaling . they also actively mediate the cell - to - cell and cell - to - extracellular matrix interactions to allow cells to constantly respond to physiological fluctuations and to alter / remodel the surrounding microenvironment for survival . they do so by crosstalk with cellular surface growth factor receptors , which interact with growth factors that may be secreted from stromal cells or released from circulation and embedded in the extracellular matrix [ 18 , 19 ] . thus , an altered expression of cams affects the motility and invasiveness of many tumor cells in vitro and metastasis in vivo [ 18 , 19 ] . cams also play an important role in the favorable soil that provides a proper microenvironment at a suitable period to awaken the dormant metastatic tumor cells to enter into an aggressive growth phase . based on the above information , aberrant expression of humetcam may also affect the motility and invasiveness of many tumor cells in vitro and metastasis in vivo . it is logical to hypothesize that humetcam / muc18 should play an important role in promoting the malignant progression of many cancer types [ 7 , 18 ] . however , recently we observed an unexpected opposite function of metcam / muc18 in the malignant progression of a mouse melanoma subline and ovarian cancer cells , in which it functioned as a tumor and metastasis suppressor ( wu , unpublished results ) . in this paper , we will review its dual roles in the tumorigenesis and metastasis in different cancer types . showed that metcam was not expressed in mcf-7 cell line , and they showed that the overexpression of humetcam in mcf-7 cells suppressed tumor formation of the cells in scid mice , as shown in figure 6 , suggesting that metcam is a possible tumor suppressor in breast cancer . we have confirmed from their western blot and immunohistochemistry results that metcam is not expressed in mcf-7 cells ( 0% ) , very weakly expressed in sk - br-3 cells ( 5% ) , and weakly expressed , though slightly higher levels than the above two cells lines , in the human mammary cancer cell lines , mda - mb-231 ( a low metastatic cell line ) ( 16% ) , and mda - mb-468 ( a high metastatic cell line ) ( 22% ) , as shown in figure 7 . it appeared that metcam is not or weakly expressed in cell lines established from luminal subtypes , but it is moderately expressed in cell lines established from basal - like subtypes , mda - mb-231 and mda - mb-468 . found that overexpression of metcam inhibited the in vitro invasiveness of mda - mb-231 cells , supporting the notion of shih et al . supported the opposite role of metcam in the progression of human breast cancer cells in that it plays a role of tumor promoter . to resolve this controversy , we recently reinvestigated the role of metcam in the tumorigenesis of human breast cancer cells in animal models and found that overexpression of metcam promoted the tumorigenesis of four human breast cancer cell lines , mcf7 , sk - br-3 , mda - mb-231 , and mda - mb-468 [ 30 , 31 ] . thus , the tumor suppression role of metcam in tumorigenesis of human breast cancer cells is not supported by the above evidence . on the contrary , it suggests the alternative notion that metcam increased tumorigenesis and perhaps also the metastasis of human breast cancer cells . recently , both our group and another group found that metcam was upregulated in human ovarian cancer specimens , suggesting that metcam may be a marker for the poor prognosis of ovarian cancer patients [ 14 , 32 ] , and that metcam may play a positive role in the development of ovarian cancer [ 14 , 32 ] . however , preliminary animal tests ( injection of bg-1 cells in nonorthotopic , subcutaneous sites of female nude mice ) show that overexpression of metcam did not have any significant effect on the tumor formation of a human ovarian cancer cell line , bg-1 ( data not shown ) . there is a negative correlation of metcam expression with the human nasopharyngeal carcinoma specimens , suggesting that metcam may also play a tumor suppressor role in the tumorigenesis of nasopharyngeal carcinoma . a tumor suppressor role of metcam may also be implicated in haemangiomas , since metcam expression was decreased during the progression of haemangiomas . overexpression of metcam in melanoma cells mostly have a positive effect on the metastasis of human melanoma cell lines in immunodeficient mice ( both athymic nude and scid mice ) [ 3 , 20 ] , mouse melanoma cell lines in syngeneic mice [ 7 , 21 ] , and a human prostate cancer cell line , lncap , in nude mice [ 7 , 13 , 36 ] . surprisingly , we have recently found that overexpression of metcam has a negative effect on the metastasis of one subline , number 9 , of mouse melanoma cell k1735 [ 22 , 23 ] and ovarian cancer cell lines [ 3032 ] . humetcam was originally found to be abundantly expressed on the cellular surface of most malignant human melanomas ; since then , it has been postulated to play a role in the progression of human melanoma . this result is consistent with the recent observation that fibroblast growth factor 2 , but neither humetcam nor integrin actually initiates the malignant progression of subcutaneous melanocyte into melanoma . we have used molecular biological and immunological methods to study the expression of humetcam in three established prostate cancer cell lines and human prostate cancer tissues , and in immunohistochemical studies of paraffin - embedded human prostate cancer tissue sections [ 7 , 8 , 12 , 13 ] . this is further corroborated by results of a positive correlation of mometcam expression with the progression of mouse prostate adenocarcinoma in a transgenic mouse model , tramp . from these results , we have also suggested that humetcam may be a key determinant in promoting tumorigenesis and metastasis of human prostate cancer cells . to test this hypothesis , we determined the effect of ectopic expression of humetcam on the ability of human prostate lncap cells to form tumor in the prostate gland and to initiate metastasis in nude mice . the metastatic lesions were found in multiple organs , such as seminal vesicles , ureter , kidney , and periaortic lymph nodes . but our recent preliminary results appear to show that overexpression of metcam may be able to enhance establishment of the growth of a bone - homing c42b clone of lncap cells in nude mice . , metcam can actually initiate the metastasis of lncap cells , thus affecting the progression of prostate cancer cells at the early stage of metastasis [ 7 , 36 ] . recently , one group has shown that metcam is overly expressed in two of the six human osteosarcoma cell lines . the metastasis can be blocked by a humanized antibody against metcam , suggesting metcam plays a positive role in the progression of osteosarcomas . decreased expression of metcam has been correlated with the progression of haemangioma , suggesting the possible negative effect of metcam on progression of haemangioma . though metcam was downregulated in nasopharyngeal carcinoma , interestingly it was upregulated again in metastatic lesions in nasopharyngeal patients , suggesting that metcam may play a positive role in the malignant progression of nasopharyngeal carcinoma after a transient suppression of tumorigenesis . taken together , we suggest that the possible tumor and metastasis suppressor role of metcam may not be limited to melanoma and ovarian cancers , and that this may be a new function of metcam yet to be explored . summary 3.5 table 1 summarizes the possible role of metcam in the tumorigenesis and metastasis of various tumors / cancers.taken together , humetcam is a tumor promoter for prostate and breast cancers , and a metastatic gene for most melanoma cell lines , prostate cancer , osteosarcoma , and perhaps , breast cancer and nasopharyngeal carcinoma . table 1 summarizes the possible role of metcam in the tumorigenesis and metastasis of various tumors / cancers.taken together , humetcam is a tumor promoter for prostate and breast cancers , and a metastatic gene for most melanoma cell lines , prostate cancer , osteosarcoma , and perhaps , breast cancer and nasopharyngeal carcinoma . by deducing knowledge learned from the tumorigenesis of other tumors [ 1519 , 42 ] and the humetcam - mediated progression of melanoma [ 4345 ] and angiogenesis [ 2 , 4651 ] , we may be able to find some common clues to begin understanding its mechanisms . first , the transcriptional expression of metcam gene may be regulated by pka / creb ( camp - responsive element binding protein ) , ap-2 [ 44 , 45 ] , and other transcription factors , such as sp-1 , c - myb , n - oct2 , ets , carg , egr-1 , and transcription factors binding to insulin - response elements , as shown in figure 16 . among these potential regulators , it is well documented that the ap-2 transcription factor plays a crucial tumor suppressor role in the progression of melanoma , prostate , and breast cancer . it has been shown that pka / creb plays a positive role in the progression of melanoma , and perhaps also applicable to breast cancer and prostate cancer , by inhibiting the expression of ap-2and increasing the expression of metcam . second , since the cytoplasmic tail of metcam contains consensus sequences potentially to be phosphorylated by pka , pkc , and ck2 , it may manifest its functions by crosstalk with various signaling pathways mediated by these protein kinases . also , the detailed mechanism of how akt up - regulates the expression of metcam has not been worked out . pka , pkc , and ck2 may phosphorylate the cytoplasmic tail of metcam , which then facilitates its interaction with fak , thus promoting cytoskeleton remodeling . alternatively , after phosphorylation of its cytoplasmic tail by these protein kinases , metcam may interact with the downstream effectors of ras , activating erk and jnk , which in turn may transcriptionally activate the expression of akt or other genes that promote the proliferation and angiogenesis of tumor cells . though metcam has not been shown to be a substrate of ck2 , which has been shown to phosphorylate other cams , such as cd44 , e - cadherin , l1-cam , and vitronectin , it is also likely that ck2 may be able to phosphorylate metcam and link it to akt to affect the proliferation , survival , and other tumorigenesis - related functions of tumor cells . third , after the engagement of metcam with the ligand(s ) or extracellular matrix , it may transmit the outside - in signals into tumor cells by activating fak and the downstream - signaling components , promoting cytoskeleton remodeling and increasing tumor cell motility and invasiveness [ 2 , 7 ] . fourth , from what we know about the roles of other cams in the progression of other tumors [ 1519 , 42 ] , it is logical to postulate that metcam may affect cancer cell progression by crosstalk with signaling pathways that affect apoptosis , survival and proliferation , and angiogenesis of tumor cells [ 7 , 18 , 42 ] . thus , metcam may affect tumorigenesis and metastasis by altering the expression of various indexes in apoptosis , survival signaling , proliferation signaling , and angiogenesis . to support this notion , we have found that metcam promotes the progression of prostate cancer cells by rendering the cells with increased proliferative ability by elevating levels of ki67 and pcna , with increased survival ability by elevating the level of phosphorylated akt , and with increased angiogenic ability by elevating levels of vegf , vegfr2 , and cd31 ; but it has no effect on the process of apoptosis . in contrast to this , metcam promotes the progression of melanoma cells differently by preventing the apoptosis of melanoma cells and reciprocally affecting the expression of a survival index , phospho - akt . however , it is not known if metcam also plays a role in the lymphatic spread of cancer cells . recent results from one group showed that metcam is one of the lymphatic metastasis - associated genes , which is upregulated in malignant mouse hepatocarcinoma ; suggesting that metcam may also play a role in promoting lymphatic metastasis of cancer cells . sixth , metcam has been shown to express in normal mesenchymal cells ( smooth muscle , endothelium , and schwann cells ) in the tissue stroma and be a marker for the mesenchymal stem cells , metcam may play an important role in regulating tumor dormancy or awakening , driving or preventing cancer cells to premetastatic niche , and formatting a microenvironment for favorable or unfavorable tumor growth in secondary sites . seventh , metcam may affect the progression of cancer cells by interactions with the host immune system , which , however , has been shown to have a paradoxical role in tumor progression . on the other hand , it is highly likely that the tumor suppression effect of metcam expression in melanoma k1735 - 9 subline may be due to the interaction of metcam - expressing cells with the host immune defense system in the immunocompetent syngeneic c3h brown mouse , since the intrinsic motility and invasiveness of mouse melanoma k1735 - 9 was increased by the metcam expression [ 22 , 23 ] . for example , the surface metcam expressed in this particular melanoma cell line may have a homophilic interaction with the nk cells , which also express metcam and enhance cytotoxic functions of nk cells . eighth , malignant progression of cancer cells has been shown to associate with an abnormal glycosylation , resulting in expression of altered carbohydrate determinants . thus , the glycosylated status of metcam in different cancer types may be different from normal cells , thus manifesting positive or negative effect on the progression of different cancer types . we should always keep in mind that mechanisms of metcam - mediated cancer progression may be slightly different in different cancer cells due to their different intrinsic properties , which provides different cofactors and/or different ligand(s ) that either positively or negatively regulate the metcam - mediated tumorigenesis and metastasis . to further understand the role of metcam in these processes , it is essential to diligently identify the cofactors and the metcam - cognate heterophilic ligand(s ) , which modulate the biological functions of metcam . mechanisms of metcam - mediated negative role in the progression of some cancer cells have not been studied at all . does metcam in some cancers behave like e - cadherin , which always plays a negative role in the tumorigenesis and metastasis of most epithelial cancer cells ? but even e - cadherin may function differently in different cancer cells . for example , its expression is temporally different and correlates with different stages during the progression of ovarian cancer : e - cadherin is not expressed in the ovarian surface epithelial cells , expressed in premalignant lesions and in well - differentiated tumors , and finally not expressed in late - stage invasive tumors . the tumor suppressor role of metcam in ovarian cancer cells may not be due to the altered intrinsic properties of the cancer cells , since the intrinsic motility and invasiveness of human ovarian cancer bg-1 and sk - ov-3 cells was not affected by the metcam expression [ 34 , 35 ] . our preliminary results appear to suggest a special mechanism that a soluble form of metcam , which is produced by mmps in the metcam - expressing cells , may mediate the suppressive effect in ovarian cancer cells , similar to the production of a soluble form of p - cadherin by the induced mmps in breast cancer cells , which then dictates , instead of suppresses , the aggressive behavior of the breast cancer cells . metcam may have a key positive function in the progression of angiosarcoma , breast cancer , osteosarcoma , prostate cancer , and most melanoma cell lines . on the other hand , it may also have a key function in suppressing the progression of a few melanoma cell lines , ovarian cancer , haemangioma , and other cancers . to further understand its mechanisms in these processes , it is crucial to define its functional domains , identify its cognate ligand(s ) and cofactor regulators , and study its crosstalk with members of various signaling pathways . these model systems may be useful for real - time observation of the dynamic process of cancer progression by using a nonintrusive and high photo - penetrating imaging system , such as the newly developed photoacoustic tomography ( pat ) , to further understanding the process in mouse models [ 54 , 55 ] . along these lines , a preclinical trial of using doxazosin , an 1-adrenergic antagonist that has been used to treat the bph patients , has been shown to be able to suppress prostate cancer metastasis in the tramp mouse model .

mathematical models that describe electric dependencies in the receiver tested are built from discrete components . for a full description of such a model , it is required to identify the parameters xi of these elements ( see eq . 1 ) . most frequently , this identification is carried out indirectly through the measurements of electrical quantities ai on the object tested [ 5 , 8 ] . the parameters sought are determined from the mathematical relations ( 1 ) that describe the object.1\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \left\ { { \begin{array}{*{20}c } { a_{1 } = f_{1 } \left ( { y_{1 } , y_{2 } , \ldots , y_{i } , \ldots , y_{n } } \right ) } \hfill \\ { a_{2 } = f_{2 } \left ( { y_{1 } , y_{2 } , \ldots , y_{i } , \ldots , y_{n } } \right ) } \hfill \\ \vdots \hfill \\ { a_{i } = f_{i } \left ( { y_{1 } , y_{2 } , \ldots , y_{i } , \ldots , y_{n } } \right ) } \hfill \\ \vdots \hfill \\ { a_{n } = f_{n } \left ( { y_{1 } , y_{2 } , \ldots , y_{i } , \ldots , y_{n } } \right ) } \hfill \\ $ $ \end{document}where fi certain functions depending on the model , ai the values measured , yi parameters that describe the model . measurement inaccuracies that are contained in ai are transferred to parameters yi to be determined.2\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \left\ { { \begin{array}{*{20}c } { y_{1 } = g_{1 } \left ( { a_{1 } , a_{2 } , \ldots , a_{n } } \right ) } \hfill \\ { y_{2 } = g_{2 } \left ( { a_{1 } , a_{2 } , \ldots , a_{n } } \right ) } \hfill \\ \vdots \hfill \\ { y_{n } = g_{n } \left ( { a_{1 } , a_{2 } , \ldots , a_{n } } \right ) } \hfill \\ ( 2 ) are determined for the values of environment ai and which contain measurement errors . in this case , approximate solutions are sought which satisfy relation ( 3).3\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \left| { a_{i } - f_{i } \left ( { y_{1 } , y_{2 } , \ldots , y_{n } } \right ) } \right| \approx 0 $ $ \end{document}the solution will be close to coefficients ai . the purpose of the analysis is to determine current voltage dependences on the terminals of one motor phase . these relationships can be determined from the model that consists of serially connected elements : rs , ls i es ( fig . 1electric model of the motor and the power source , where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ u = u_{m } \sin \omega t,\quad e_{s } ( t ) = \left\ { { \begin{array}{*{20}c } { e_{m } \sin ( \omega t + \phi_{es } ) } \hfill & { t < t_{0 } } \hfill \\ { e_{m } e^ { - \alpha t } \sin ( \omega t + \phi_{es } ) } \hfill & { t \ge t_{0 } } \hfill \\ \end{array } } \right . , $ $ \end{document } r internal resistance of sources electric model of the motor and the power source , where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ u = u_{m } \sin \omega t,\quad e_{s } ( t ) = \left\ { { \begin{array}{*{20}c } { e_{m } \sin ( \omega t + \phi_{es } ) } \hfill & { t < t_{0 } } \hfill \\ { e_{m } e^ { - \alpha t } \sin ( \omega t + \phi_{es } ) } \hfill & { t \ge t_{0 } } \hfill \\ \end{array } } \right . , $ $ \end{document } r internal resistance of sources coefficients rs , ls , em , es , that are being sought represent many of the phenomena that occur in the motor and the system that is driven . for example , the inertia of the rotor and the system driven will affect es , and the angular velocity will exert an influence on mutual inductances , which are described with ls . when searching for the parameters of the model , the fact is also important that these factors can not be determined with the engine being stopped . this means that the rs does not reflect the winding resistance and ls does not reflect their inductance . the parameters of the model are defined for a constant load on the machine shaft and for constant rotations . when changing the load , the parameters of the model change , as well . in this situation , the parameters that are being determined can not in any way be unified . they should be determined for a specific drive train ( the motor and the machine driven ) . these parameters can vary considerably for the same engine with different mechanical properties of the system driven . the identification of the model consists in searching for em , es , rs , and ls . these parameters can be determined on the receiver [ 6 , 7 , 911 ] by making measurements in the steady state ( in the case of an induction motor : during operation with a constant load and a constant speed ) in the system as shown below ( fig . 2measuring circuit for parametric identification measuring circuit for parametric identification according to the model adopted , we know that:4\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ e_{s } = e_{s } \sqrt 2 \sin \left ( { \omega t + \phi_{es } } \right ) . $ $ \end{document}in the field of complex numbers , the following can be written:5\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \underline{e}_{s } = e_{s } e^{{j\phi_{es } } } , $ $ \end{document}6\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \underline{u } = ue^{{j\phi_{u } } } . $ $ \end{document}for one mesh , the voltage equation is as follows:7\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \underline{i}_{a } \left ( { r_{s } + r + jx_{s } } \right ) + \underline{e}_{s } - \underline{u } = 0 , $ $ \end{document}where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ x_{s } = \omega l_{s } . $ $ \end{document } next , by transforming ( 7 ) , we determine current ia:8\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \underline{i}_{a } = \frac{{\underline{u } - \underline{e}_{s } } } { { r_{s } + r + jx_{s } } } $ $ \end{document}voltmeter v measures the difference in the supply voltage and in the voltage drop across internal resistance r. thus , in the field of complex numbers , there will be the following:9\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \underline{u}_{v } = \underline{u } - \underline{i}_{a } r. $ $ \end{document}from eqs . ( 8) and ( 9 ) , one can obtain the following:10\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \underline{u}_{v } = \frac{1}{{r_{s } + r + jx_{s } } } \left [ { \underline{u } \left ( { r_{s } + jx_{s } } \right ) + \underline{e}_{s } r } \right ] . $ $ \end{document}knowing that the forces and the current are equal , respectively:\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ p_{w } = \text{re } \left ( { \underline{u}_{v } \underline{i}_{a}^ { * } } \right),\quad q_{w } = \text{im } \left ( { \underline{u}_{v } \underline{i}_{a}^ { * } } \right),\quad \underline{i}_{a}^ { * } = \frac{{\underline{u}^ { * } - \underline{e}_{s}^ { * } } } { { r_{s } + r - jx_{s } } } $ $ \end{document}we obtain the following equations:11\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \left\ { { \begin{array}{*{20}c } { i_{a } = \left| { \frac{{\underline{u } - \underline{e}_{s } } } { { r_{s } + r + jx_{s } } } } \right| } \hfill \\ { u_{v } = \left| { \frac{1}{{r_{s } + r + jx_{s } } } \left [ { \underline{u } ( r_{s } + jx_{s } ) + \underline{e}_{s } r } \right ] } \right| } \hfill \\ { p_{w } = \text{re } \left\ { { \frac{{\left [ { \underline{u } ( r_{s } + jx_{s } ) + \underline{e}_{s } r } \right ] \cdot \left [ { \underline{u}^ { * } - \underline{e}_{s}^ { * } } \right]}}{{(r_{s } + r)^{2 } + x_{s}^{2 } } } } \right\ } } \hfill \\ { q_{w } = \text{im } \left\ { { \frac{{\left [ { \underline{u } ( r_{s } + jx_{s } ) + \underline{e}_{s } r } \right ] \cdot \left [ { \underline{u}^ { * } - \underline{e}_{s}^ { * } } \right]}}{{(r_{s } + r)^{2 } + x_{s}^{2 } } } } \right\ } } \hfill \\ \end{array } } \right .. $ $ \end{document } equation ( 11 ) is consistent with ( 1 ) . the coefficients of the model of the receiver that are obtained from the above equations are not determinable for all the input parameters ( uv , ia , pw , qw ) . there are those areas that result from measurement inaccuracies where the system of eq . it was found that these coefficients can not be determined using the newton s interpolation algorithm [ 15 , 16 ] . process time constant 1/ that is being sought , and which is mainly related to the inertia of the rotor and the system driven , can be determined experimentally by observing the course of voltage versus time at the motor terminals immediately after commutation . in , the authors proved that amplitude es can be equal to amplitude u. in this paper , it was also observed that frequency es is similar to the frequency of the mains voltage . it was also noted that phase shift es is equal to 0 . in this model , it is assumed that the frequencies of both sources are identical . coefficients em , rs , and ls can be determined from eq . ( 11 ) using a neural network . the network input parameters x1 = uv [ v ] , x2 = ia [ a ] , x3 = pw [ w ] , x4 = qw [ var ] contain measurement errors . due to the nature of the adopted activation function [ 13 ] , the output neuron of the output layer must be within range \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ y \in \left ( { 0,\,1 } \right ) $ $ \end{document}. the initial values were as follows : y1 = rs [ k ] , y2 = ls [ h ] , y3 = es [ kv ] . training of the network must be for those learning vectors \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \sigma = \left [ { x_{1 } , \ldots , x_{{n_{0 } } } , \left| { , d_{1}^{\left ( l \right ) } , \ldots , d_{{n_{l } } } ^{\left ( l \right ) } } \right . } learning vectors are constructed from eqs . ( 1 ) or ( 11 ) for random values y1 , y2 , y3 that lie within the set of permissible changes , and which is limited with values a and b [ 4 , 12 ] . the test vector is built according to fig . 3 for values yi that are not contained within the training set.fig . 3construction of learning vectors construction of learning vectors the neural network was built in a vba environment in excel . 4 determines the random values : \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ d_{1 } = r_{s } \in \left\langle { 0.005 \div 0.2 } \right\rangle { \text{k}}\upomega , \quad d_{2 } = l_{s } \in \left\langle { 0.005 \div 0.5 } \right\rangle { \text{h}},\quad d_{3 } = e_{s } \in \left\langle { 0.15 \div u } \right\rangle { \text{kv } } $ $ \end{document}.fig . 4the workbook that builds learning vectors the workbook that builds learning vectors further values uv , ia , pw and qw are determined from eq . ( after tests of several neural networks , a decision was made to build a neural network with topology ( fig . 5structure of the neural network structure of the neural network individual neurons in the network are structured according to fig . 6diagram of neuron \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ n_{i}^{(k ) } $ $ \end{document } diagram of neuron \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ n_{i}^{(k ) } $ $ \end{document } in the network being built , the following indications were accepted : titeration step , t = 1 , 2 , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ y_{i}^{(k ) } ( t ) $ $ \end{document}ith output of the neuron \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ n_{i}^{(k ) } $ $ \end{document}\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ y_{i}^{\left ( l \right ) } \left ( t \right ) $ $ \end{document}ith output of the networkknetwork layer , k = 1 , , llnetwork output layer , the number of network layersineuron number in layer , i = 1 , , nk\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ x_{j}^{(k ) } ( t ) $ $ \end{document}input signal in the kth layer\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ x_{j } ( t ) = x_{j}^{(1 ) } ( t ) $ $ \end{document}input of the networkjnumber of the input signal in the kth layer , j = 1 , , nk1n0number of inputs to the networknknumber of neurons in the kth layernlnumber of neurons in the last layer\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ s_{i}^{(k ) } ( t ) $ $ \end{document}neuron membrane potential \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ n_{i}^{(k ) } $ $ \end{document } in the kth layer\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ w_{i , j}^{(k ) } ( t ) $ $ \end{document}weight of the jth input of the ith neuron \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ n_{i}^{(k ) } $ $ \end{document } in the kth layer\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ d_{i}^{(l ) } ( t ) $ $ \end{document}ith reference signal output from the learning vector\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \varepsilon_{i}^{(l ) } ( t ) $ $ \end{document}error of the ith network output , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \varepsilon_{i}^{(l ) } ( t ) = d_{i}^{(l ) } ( t ) - y_{i}^{(l ) } ( t ) $ $ \end{document}network learning rateq(t)error at the output of the network for one reference vectorq(t)error at the output of the network for the entire epoch iteration step , t = 1 , 2 , ith output of the neuron \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ n_{i}^{(k ) } $ $ \end{document } ith output of the network network layer , k = 1 , , l network output layer , the number of network layers neuron number in layer , i = 1 , , nk input signal in the kth layer number of the input signal in the kth layer , j = 1 , , nk1 number of inputs to the network number of neurons in the kth layer number of neurons in the last layer neuron membrane potential \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ n_{i}^{(k ) } $ $ \end{document } in the kth layer weight of the jth input of the ith neuron \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ n_{i}^{(k ) } $ $ \end{document } in the kth layer ith reference signal output from the learning vector error of the ith network output , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \varepsilon_{i}^{(l ) } ( t ) = d_{i}^{(l ) } ( t ) - y_{i}^{(l ) } ( t ) $ $ \end{document } network learning rate error at the output of the network for one reference vector error at the output of the network for the entire epoch the output of neuron \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ n_{i}^{(k ) } $ $ \end{document } ( fig . 6 ) at time t is described with the following relation:12\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ y_{i}^{(k ) } ( t ) = f\left ( { s_{i}^{(k ) } ( t ) } \right ) $ $ \end{document}while membrane potential \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ s_{i}^{(k ) } $ $ \end{document } is equal to:13\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ s_{i}^{(k ) } ( t ) = \sum\limits_{j = 0}^{{n_{k - 1 } } } { w_{i , j}^{(k ) } ( t ) \cdot x_{j}^{(k ) } ( t ) } $ $ \end{document } the input neuron for k = 1 layer is equal to network inputs \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ x_{j } ( t ) = x_{j}^{(1 ) } ( t ) $ $ \end{document}. each layer has one input \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ x_{0}^{(k ) } ( t ) = 1 $ $ \end{document}. other inputs are the outputs of the previous layer.14\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ x_{j}^{k } ( t ) = \left\ { { \begin{array}{*{20}c } { x_{j } ( t ) } \hfill & { { \text{for } } k = 1 } \hfill \\ { y_{j}^{k - 1 } ( t ) } \hfill & { { \text{for } } k = 2 , \ldots , l } \hfill \\ 1 \hfill & { { \text{for } } j = 0,\quad k = 1 , \ldots , l } \hfill \\ $ $ \end{document}the error at the output of the network for one learning vector is:15\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ q(t ) = \sum\limits_{i = 1}^{{n_{l } } } { ( \varepsilon_{i}^{l } ( t))^{2 } } = \sum\limits_{i = 1}^{{n_{l } } } { ( d_{i}^{l } ( t ) - y_{i}^{l } ( t))^{2 } } $ $ \end{document}the weights of the individual neuron inputs are determined from the steepest descent rule:16\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ w(t + 1 ) = w(t ) - \eta \cdot { \mathbf{g}}(w(t ) ) $ $ \end{document}where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { \mathbf{g}}(w(t ) ) = \left [ { \begin{array}{*{20}c } { \frac{\partial q\left ( t \right)}{{\partial w_{1 } \left ( t \right ) } } , } & { \frac{\partial q\left ( t \right)}{{\partial w_{2 } \left ( t \right ) } } , } & { \frac{\partial q\left ( t \right)}{{\partial w_{3 } \left ( t \right ) } } , } & { \ldots , } & { \frac{\partial q\left ( t \right)}{{\partial w_{n } \left ( t \right ) } } } \\ \end{array } } \right]^{t } $ $ \end{document } is the vector gradient . from eq . ( 16 ) , for any weight in any layer , the following is obtained:17\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ w_{i , j}^{(k ) } ( t + 1 ) = w_{i , j}^{(k ) } ( t ) - \eta \frac{\partial q(t)}{{\partial w_{i , j}^{(k ) } ( t ) } } = w_{i , j}^{(k ) } ( t ) + 2\eta \delta_{i}^{(k ) } ( t)x_{j}^{(k ) } ( t ) $ $ \end{document}parameter \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \delta_{i}^{(k ) } ( t ) $ $ \end{document } is determined differently than for the output layer and the hidden layer:18\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \delta_{i}^{(k ) } ( t ) = \left\ { { \begin{array}{*{20}c } { \varepsilon_{i}^{(l ) } ( t ) \cdot f^ { ' } \left ( { s_{i}^{(l ) } ( t ) } \right ) } \hfill & { { \text{for } } k = l } \hfill \\ { f^ { ' } \left ( { s_{i}^{(k ) } ( t ) } \right ) \cdot \sum\limits_{m = 1}^{{n_{k + 1 } } } { \delta_{m}^{(k + 1 ) } ( t)w_{i , m}^{(k + 1 ) } ( t ) } } \hfill & { { \text{for } } k \ne l } \hfill \\ $ $ \end{document}where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \varepsilon_{i}^{(l ) } ( t ) = d_{i}^{(l ) } ( t ) - y_{i}^{(l ) } ( t ) . $ $ \end{document } network training is carried out by an incremental updating of weights , that is , each time after the entry of a successive learning vector , responses are determined and the weights are modified . the simulation is continued until the total output error for entire epoch q(t ) is smaller than the accepted set qmin.19\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ q^ { * } ( t ) = \sum\limits_{m = 1}^{m } { q_{m } ( t ) \le q _ { \hbox{min } } } $ $ \end{document}where m is the number of learning vectors in the epoch . the neuron activation function was adopted as a continuous unipolar function of the signum type:20\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ f(s_{i } ( t ) ) = \frac{1}{{1 + e^ { { - \beta \cdot s_{i } ( t ) } } } } $ $ \end{document}where is the steepness factor . with low values of coefficient , the derivative of the activation function is as follows:21\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ f^ { ' } ( s_{i } ( t ) ) = \frac{{\beta \cdot e^ { { - \beta \cdot s_{i } ( t ) } } } } { { \left ( { 1 + e^ { { - \beta \cdot s_{i } ( t ) } } } \right)^{2 } } } = \beta \cdot f(s_{i } ( t ) ) \cdot ( 1 - f(s_{i } ( t ) ) ) $ $ \end{document}the calculation sheet in fig . starting of the network training produces a script written in vba that executes in a loop of a neural network algorithm according to ( 12 ) ( 21 ) and the block diagram in fig . . 8block diagram of the network learning algorithm sheet for the visualization of the network operation block diagram of the network learning algorithm the start of the algorithm is possible for the weights that are selected at random from range \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \left\langle { - 1,\,1 } \right\rangle $ $ \end{document } or the reading stored from the previous simulations ( fig . owing to the ability to read and write data , it is possible to pause the simulation and to change its parameters during operation [ 4 , 12 ] . reading of the stored data allows a continuation of the previously stopped simulation . the window in fig . 10sheet with the saved results of the simulation sheet with the saved results of the simulation the output values of the neurons ( fig . 8a ) are determined by analyzing the neurons in layers starting from the input layer ; the output layer comes last . for all the layers , the steepness factor ( 20 ) of activation function fx was assumed as equal to 0.1 . the determination of value \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \delta_{i}^{(k ) } ( t ) $ $ \end{document } ( fig . this determination takes place starting from the output layer ; the input layer comes last . . network learning factor from formula ( 17 ) was adopted on the first stage of the simulation as being constant and equal to 0.1 . 70,000 epochs , the value of target function q(t ) , which was calculated in accordance with formula ( 19 ) , began to oscillate on the level of 1.42 . a decrease in q(t ) occurred only after a reduction in network learning rate . the correct procedure for the network training should provide for an ability to change this ratio during the analysis ( fig . 11the method to reduce the coefficient of network learning the method to reduce the coefficient of network learning oscillations around the optimal solution are manifested with a momentary increase in the value of q(t).22\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ q^ { * } ( t ) \ge q^ { * } ( t - 1 ) $ $ \end{document}once the required value of q(t ) from eq . ( 19 ) has been reached , the network test is performed ( fig . the network test consists in determining the values of uv , ia , pw , and qw from relation ( 11 ) . these values are then substituted into the neural network input , whose solution is rs , ls , and es . 12 also allows a determination of the network s solution for a selected set of weights . table 1 illustrates the network test for randomly selected values of rs , ls , and es.table 1verification of the network errorrandom valuesnetwork inputnetwork outputrelative error x 1 x 2 x 3 x 4 y 1 y 2 y 3 1 2 3 r s l s e s u v i a p q r s l s e s [ ][h][v][v][a][w][var][][h][v]200.2180229.950.757653.21165.8819.7210.210168.6260.0140.0500.0630.1271510.315193229.970.204839.39525.7897147.0830.354192.8490.0260.1240.0010.1511500.09197229.960.215948.7949.18694152.1070.093197.0190.0140.0330.00010.0481470.3197229.970.188836.56323.415147.4130.352192.9760.0030.1730.0200.197470.46193229.990.243417.36853.21141.3730.409195.9460.1200.1110.0150.2461500.09193229.950.242154.70710.301151.5900.092196.8660.0110.0220.0200.053450.448190229.980.270619.01559.25641.1820.408195.7710.0850.0890.0300.2051450.4190229.970.208336.21831.353145.7730.357192.8100.0050.1080.0150.128500.03160229.731.3705309.42258.16358.6410.026159.8290.1730.1330.0010.3071500.1200229.960.195544.00569.20584152.3940.093197.1030.0160.0700.0140.1001400.317189229.960.238444.693131.755146.1330.354192.5440.0440.1170.0190.179400.317195229.980.325928.03669.50740.4160.402195.0290.0100.2680.00020.2791350.4195229.970.189631.93729.691143.7510.358192.9720.0650.1050.0100.1801450.3190229.960.231144.56828.936146.4430.353192.6360.0100.1770.0140.2011400.4190229.970.212536.37832.615145.1570.356192.7900.0370.1100.0150.162 verification of the network error the relative error for all the output neurons for the randomly adopted input vectors is:23\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \delta_{1 } = \frac{{y_{1 } - r_{s } } } { { r_{s } } } , \quad \delta_{2 } = \frac{{y_{2 } - l_{s } } } { { l_{s } } } , \quad \delta_{3 } = \frac{{y_{3 } - e_{s } } } { { e_{s } } } $ $ \end{document}the total network error for the accepted values of rs , ls , and es are:24\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \delta = \left| { \frac{{y_{1 } - r_{s } } } { { r_{s } } } } \right| + \left| { \frac{{y_{2 } - l_{s } } } { { l_{s } } } } \right| + \left| { \frac{{y_{3 } - e_{s } } } { { e_{s } } } } \right| . $ $ \end{document}the percentage error made by the network is determined from the largest error ( the top bar in the chart in fig . the large error value is shown for the input values that occur least frequently in the training set . an improved performance is possible by enlarging the training set or by reducing the range of acceptable changes of the values being sought . owing to the method presented of the selection of the electrical model parameters from the values that are measured on the receiver , it is not required to build any complex physical and electrical dependences . the engineering method of voltage , current , and power measurement allows one to determine the parameters of the model for constant electrical and mechanical conditions in the engine . the method presented is particularly useful in situations where measurement errors make it impossible to solve eq . an important advantage of this approach is the ability to build its own networks of any topology . the design loop iteration depends largely on how one defines those variables that describe the network . in the present solution , a sample definition of the variable holding the weights of neurons is:\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { \text{public}}\;{\text{w(}}1{\text { to lweights)as}}\;{\text{single } } $ $ \end{document}where lweights is the number of weights of all neurons.this solution facilitates the construction of a loop program , but special attention is to be paid to assigning the weight number with the neuron number . an alternative is to build one s own variable ( using the opportunity to build one s own type of variables ) that represents the neuron , and then group all the parameters that describe the type of the neuron in this variable . this approach will make the program more transparent , but there are problems in the construction of iterative loops .

this paper presents an algorithm for solving n - equations of n - unknowns . this algorithm allows to determine the solution in a situation where coefficients ai in equations are burdened with measurement errors . for some values of ai ( where i = 1 , , n ) , there is no inverse function of input equations . in this case , it is impossible to determine the solution of equations of classical methods .

Introduction An example of a model for identification Construction of an artificial neural network Learning of the network Network test Conclusions

measurement inaccuracies that are contained in ai are transferred to parameters yi to be determined.2\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \left\ { { \begin{array}{*{20}c } { y_{1 } = g_{1 } \left ( { a_{1 } , a_{2 } , \ldots , a_{n } } \right ) } \hfill \\ { y_{2 } = g_{2 } \left ( { a_{1 } , a_{2 } , \ldots , a_{n } } \right ) } \hfill \\ \vdots \hfill \\ { y_{n } = g_{n } \left ( { a_{1 } , a_{2 } , \ldots , a_{n } } \right ) } \hfill \\ ( 2 ) are determined for the values of environment ai and which contain measurement errors . in this case , approximate solutions are sought which satisfy relation ( 3).3\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \left| { a_{i } - f_{i } \left ( { y_{1 } , y_{2 } , \ldots , y_{n } } \right ) } \right| \approx 0 $ $ \end{document}the solution will be close to coefficients ai . in , the authors proved that amplitude es can be equal to amplitude u. in this paper , it was also observed that frequency es is similar to the frequency of the mains voltage . in this model , it is assumed that the frequencies of both sources are identical . 6diagram of neuron \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ n_{i}^{(k ) } $ $ \end{document } diagram of neuron \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ n_{i}^{(k ) } $ $ \end{document } in the network being built , the following indications were accepted : titeration step , t = 1 , 2 , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ y_{i}^{(k ) } ( t ) $ $ \end{document}ith output of the neuron \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ n_{i}^{(k ) } $ $ \end{document}\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ y_{i}^{\left ( l \right ) } \left ( t \right ) $ $ \end{document}ith output of the networkknetwork layer , k = 1 , , llnetwork output layer , the number of network layersineuron number in layer , i = 1 , , nk\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ x_{j}^{(k ) } ( t ) $ $ \end{document}input signal in the kth layer\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ x_{j } ( t ) = x_{j}^{(1 ) } ( t ) $ $ \end{document}input of the networkjnumber of the input signal in the kth layer , j = 1 , , nk1n0number of inputs to the networknknumber of neurons in the kth layernlnumber of neurons in the last layer\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ s_{i}^{(k ) } ( t ) $ $ \end{document}neuron membrane potential \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ n_{i}^{(k ) } $ $ \end{document } in the kth layer\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ w_{i , j}^{(k ) } ( t ) $ $ \end{document}weight of the jth input of the ith neuron \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ n_{i}^{(k ) } $ $ \end{document } in the kth layer\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ d_{i}^{(l ) } ( t ) $ $ \end{document}ith reference signal output from the learning vector\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \varepsilon_{i}^{(l ) } ( t ) $ $ \end{document}error of the ith network output , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \varepsilon_{i}^{(l ) } ( t ) = d_{i}^{(l ) } ( t ) - y_{i}^{(l ) } ( t ) $ $ \end{document}network learning rateq(t)error at the output of the network for one reference vectorq(t)error at the output of the network for the entire epoch iteration step , t = 1 , 2 , ith output of the neuron \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ n_{i}^{(k ) } $ $ \end{document } ith output of the network network layer , k = 1 , , l network output layer , the number of network layers neuron number in layer , i = 1 , , nk input signal in the kth layer number of the input signal in the kth layer , j = 1 , , nk1 number of inputs to the network number of neurons in the kth layer number of neurons in the last layer neuron membrane potential \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ n_{i}^{(k ) } $ $ \end{document } in the kth layer weight of the jth input of the ith neuron \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ n_{i}^{(k ) } $ $ \end{document } in the kth layer ith reference signal output from the learning vector error of the ith network output , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \varepsilon_{i}^{(l ) } ( t ) = d_{i}^{(l ) } ( t ) - y_{i}^{(l ) } ( t ) $ $ \end{document } network learning rate error at the output of the network for one reference vector error at the output of the network for the entire epoch the output of neuron \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ n_{i}^{(k ) } $ $ \end{document } ( fig . owing to the ability to read and write data , it is possible to pause the simulation and to change its parameters during operation [ 4 , 12 ] . owing to the method presented of the selection of the electrical model parameters from the values that are measured on the receiver , it is not required to build any complex physical and electrical dependences . the engineering method of voltage , current , and power measurement allows one to determine the parameters of the model for constant electrical and mechanical conditions in the engine . the method presented is particularly useful in situations where measurement errors make it impossible to solve eq .

patients with type 1 diabetes were recruited from oregon health & sciences university ( ohsu ) outpatient clinics in portland . patients who were pregnant , had uncontrolled concurrent illnesses , had physical or visual impairment preventing the issue of a continuous glucose monitoring system , or needed uninterrupted acetaminophen use were excluded . the research protocol was approved by the ohsu institutional review board , and all subjects provided written informed consent . the subjects were a mean age of 37.4 3.3 years , with a duration of diabetes of 22.4 2.9 years . mean hba1c was 7.9 0.2% , and mean bmi was 25.9 1.0 kg / m . two sensors were placed to the right of the umbilicus and two to the left . the pair on the right was positioned 20 cm apart from the pair on the left . each pair was placed at a predetermined distance on the surface of the abdominal skin , with the goal of achieving a fixed distance between the tips of the glucose sensors in the subcutaneous tissue . the study was designed so that the intersensor distances in the subcutaneous tissue would be approximately 2 , 10 , 20 , and 30 mm . subjects wore the four sensor receivers in a small pack to keep them within 5 feet of the transmitters and were instructed to calibrate the sensors 2 h and 8 h after insertion . the following morning , subjects were admitted to the oregon clinical & translational research institute at ohsu . the forearm was warmed with a heating pad to arterialize the venous blood . at the beginning of the 9-h study , the mean of two simultaneous measured venous blood glucose values was used to calibrate the four sensors . venous glucose was subsequently measured every 15 min for 9 h using a hemocue glucose 201 analyzer . subjects were fed two standardized meals and given premeal insulin based on their typical insulin - to - carbohydrate ratio along with additional insulin for correction of hyperglycemia according to their usual outpatient regimen . subjects were treated with fruit juice for hypoglycemia if the venous glucose value fell below 70 mg / dl . at the end of each study , coned - down oblique x - ray images of the sensor sites were taken to measure the actual distance between the tips of the glucose sensors . magnification was taken into account by multiplying the distance between the x - ray source to the sensor divided by distance between the x - ray source and the x - ray plate . data were analyzed using generalized estimating equations , which took into account correlated data and repeated measures . the measured distance between the sensor tips was used in place of the expected distance . is a continuous glucose - error grid analysis ( cg - ega ) , an outcome metric that addresses the problem of evaluating the temporal characteristics of the continuous glucose sensor process by considering pairs of reference and sensor readings as a process in time that takes into account inherent physiologic time lags ( 9 ) . the cg - ega classifies errors in three clinically relevant categories accurate readings ( region a ) , benign errors ( region b ) , and erroneous readings ( regions c , d , and e)with clinically relevant classification cutoffs based on the potential error in treatment given the sensor inaccuracy . principal component analysis ( pca ) is a multivariate data analysis method that was used here to flag and remove potentially aberrant sensor values to assess if removing these values would improve overall sensor accuracy ( 10,11 ) . in the case of two sensors , when three or more sensor measurements are available , pca can be used to detect inconsistencies and identify the inaccurate or faulty sensor from the group . in practice , our data set at each time step consisted of three or more variables ( sensor sources ) , each with a set of observations , the number of which depended on the frequency of available sensor measurements and the predetermined length of the measurement history , set to 15 and 75 min , respectively , in this case . performing pca on these data reveals a new set of uncorrelated variables called principle components ( pcs ) , with weights assigned to each of the original sensor sources to indicate the contribution of each original sensor source to the pc . the most influential sensor signal carries the highest weighting coefficient in the first pc , whereas a sensor signal that is considered an outlier will carry the highest weighting coefficient in the last pc . this information , in conjunction with the percent of variability of the original data set that is captured by the first pc , was used retrospectively to identify an inaccurate or faulty sensor ; in practice , the method is designed to be used in real - time . the sensor chosen as the inaccurate sensor updates at each time step . in the case of missing data , for example , if two of three sensors are available , then the two - sensor implementation of the pca is used . two sensors were placed to the right of the umbilicus and two to the left . the pair on the right was positioned 20 cm apart from the pair on the left . each pair was placed at a predetermined distance on the surface of the abdominal skin , with the goal of achieving a fixed distance between the tips of the glucose sensors in the subcutaneous tissue . the study was designed so that the intersensor distances in the subcutaneous tissue would be approximately 2 , 10 , 20 , and 30 mm . subjects wore the four sensor receivers in a small pack to keep them within 5 feet of the transmitters and were instructed to calibrate the sensors 2 h and 8 h after insertion . the following morning , the forearm was warmed with a heating pad to arterialize the venous blood . at the beginning of the 9-h study , the mean of two simultaneous measured venous blood glucose values was used to calibrate the four sensors . venous glucose was subsequently measured every 15 min for 9 h using a hemocue glucose 201 analyzer . subjects were fed two standardized meals and given premeal insulin based on their typical insulin - to - carbohydrate ratio along with additional insulin for correction of hyperglycemia according to their usual outpatient regimen . subjects were treated with fruit juice for hypoglycemia if the venous glucose value fell below 70 mg / dl . at the end of each study , coned - down oblique x - ray images of the sensor sites were taken to measure the actual distance between the tips of the glucose sensors . magnification was taken into account by multiplying the distance between the x - ray source to the sensor divided by distance between the x - ray source and the x - ray plate . data were analyzed using generalized estimating equations , which took into account correlated data and repeated measures . the measured distance between the sensor tips was used in place of the expected distance . is a continuous glucose - error grid analysis ( cg - ega ) , an outcome metric that addresses the problem of evaluating the temporal characteristics of the continuous glucose sensor process by considering pairs of reference and sensor readings as a process in time that takes into account inherent physiologic time lags ( 9 ) . the cg - ega classifies errors in three clinically relevant categories accurate readings ( region a ) , benign errors ( region b ) , and erroneous readings ( regions c , d , and e)with clinically relevant classification cutoffs based on the potential error in treatment given the sensor inaccuracy . principal component analysis ( pca ) is a multivariate data analysis method that was used here to flag and remove potentially aberrant sensor values to assess if removing these values would improve overall sensor accuracy ( 10,11 ) . in the case of two sensors , the methodology can be used to detect a potential inconsistency between the sensor signals . when three or more sensor measurements are available , pca can be used to detect inconsistencies and identify the inaccurate or faulty sensor from the group . in practice , our data set at each time step consisted of three or more variables ( sensor sources ) , each with a set of observations , the number of which depended on the frequency of available sensor measurements and the predetermined length of the measurement history , set to 15 and 75 min , respectively , in this case . performing pca on these data reveals a new set of uncorrelated variables called principle components ( pcs ) , with weights assigned to each of the original sensor sources to indicate the contribution of each original sensor source to the pc . the most influential sensor signal carries the highest weighting coefficient in the first pc , whereas a sensor signal that is considered an outlier will carry the highest weighting coefficient in the last pc . this information , in conjunction with the percent of variability of the original data set that is captured by the first pc , was used retrospectively to identify an inaccurate or faulty sensor ; in practice , the method is designed to be used in real - time . the sensor chosen as the inaccurate sensor updates at each time step . in the case of missing data , for example , if two of three sensors are available , then the two - sensor implementation of the pca is used . eleven women and 8 men with type 1 diabetes participated in 36 studies , each lasting 9 h. the data from one study were excluded due to a diluted venous blood glucose value that caused incorrect calibration of the sensors , leaving 35 studies for inclusion in the data analysis . the benefit of multiple sensors was analyzed by comparing how often the mean and median of all four sensor values deviated 25% or more and 50% or more from the reference blood glucose compared with the values of each single sensor . errors of these degrees may cause inappropriate treatment decisions , and in particular , errors of 50% or higher might cause harm if these sensor values were used as an input into an artificial pancreas algorithm . large errors , defined as the sensor value being 25% above or below the reference venous blood glucose values , were significantly reduced by using the mean values ( 9.5% of values [ 95% ci 5.815.4 ] ) or median values ( 10.0% [ 6.216.1 ] ) from four sensors compared with one sensor ( 17.5% [ 13.323.0 ] , p < 0.001 ) . very large errors , defined as the sensor value being 50% or more above or below the reference blood glucose value , were also significantly reduced by using the mean ( 0.4% [ 0.11.2 ] ) or median ( 0.5% [ 0.21.2 ] ) of four sensor values compared with one sensor ( 2.6% [ 1.64.0 ] , p < 0.001 ) . in many cases , there were no large errors . however , in those experiments with a substantial number of very large errors , which reached as high as 61% of values , use of the mean of four sensors dramatically reduced the frequency of very large errors ( fig . summary of very large errors , defined as sensor values 50% away from the reference venous blood glucose . the percentages of very large errors when the four sensors are averaged are shown by black open circles , and values for each single sensor are shown by gray xs . the mean absolute relative difference ( ard ) was significantly improved by use of the mean ( 11.6 1.0% ) or median ( 11.8 1.0% ) of four sensors compared with one sensor ( 14.8 1.0% , p < 0.001 ) . there was no advantage to selecting the sensor with the lowest ard at the time of calibration at the study start ( mean ard 15.0 1.4% , p = ns ) . the mean absolute difference , the metric for reference venous blood glucose values < 75 mg / dl , also improved significantly by use of the mean of four sensors compared with a single sensor ( mean absolute difference 9.6 1.0 vs. 14.1 1.3 mg / dl , p < 0.001 ) . large errors , as defined above , were also significantly reduced by using the mean of the sensor pairs ( 12.9% [ 95% ci 9.919.2 ] ) compared with a single sensor ( 17.5% [ 13.323.0 ] , p < 0.01 ) . very large errors were reduced by a mean of 62% by the use of sensor pair means versus one sensor ( 1.0% [ 0.51.9 ] vs. 2.6% [ 1.64.0 ] , p < 0.001 ) . there was a reduction in the mean ard when comparing the mean of the sensor pairs with a single sensor ( 13.5 1.1 vs. 14.8 1.0% , p < 0.01 ) . there was a trend in improvement in sensor accuracy with using the mean of three sensors compared with two sensors ( 0.9 percentage point ard reduction , p < 0.06 ) , with a clear added benefit of four ( 1.6 percentage point ard reduction , p < 0.01 ) . a summary of errors for one , two , and four sensors , categorized by glucose levels , is presented in table 1 . summary of sensor errors categorized by degree of error and venous blood glucose levels in the euglycemic range ( 70180 mg / dl ) , the percentage of erroneous readings decreased significantly with additional sensor signal information ( 1.1 , 0.8 , and 0.5% of readings in the cg - error grids c , d , and e zones for the mean of 1 , 2 , and 4 sensors , respectively ) . the finding was similar in the hyperglycemic range above 180 mg / dl ( 3.5 , 2.8 , and 1.7% in c , d , and e zones for a mean of 1 , 2 , and 4 sensors ) . the number of accurate readings also significantly increased in the hyperglycemic range with increasing number of sensors ( 90.2 , 91.7 , and 94.7% ) . there were no significant differences in readings in the a region in the euglycemic range . there were also no significant differences in erroneous or accurate readings in the limited number of values in the hypoglycemic range . there was a significant increase in the a zone of the clarke error grid using multiple sensors ( 68.4 , 70.7 , and 75.4% for 1 , 2 , and 4 sensors , respectively ) . there were more values in the b zone with use of a single sensor ( 23.6 , 18.6 , and 11.5% ) . there was nonsignificant trend toward reducing the percentage of the values in the c , d , and e zones ( 2.4 , 1.3 , and 0.4% ) . voting schemes may improve upon using sensor value averaging alone , because if one sensor is highly accurate and the second is highly inaccurate , the mean is worse than use of the single highly accurate sensor . there was a significant reduction in the mean ard of the mean of three sensors in the case when data flagged by the pca was removed compared with all data ( mean ard 12.0 1.1 vs. 12.3 1.1% , p = 0.04 ) . as expected , the mean ard of the flagged data ( 16.5 2.4% ) was higher compared with nonflagged data alone ( p = 0.02 ) and also compared with all data ( p = 0.01 ) . in the case of two sensors , removing values flagged by pca did not significantly change the mean ard versus all data ( 12.9 0.9 vs. 13.0 0.9% , p = ns ) , and there was a nonsignificant increase in the mean ard of the flagged data ( 15.5 1.5 , p = 0.06 compared with all data ) . the intersensor distances as measured by x - ray imaging were 7 1 , 13 1 , 21 1 , and 28 2 mm ( fig . note that the sensors are positioned very closely on the right side of the subject s abdomen and are much farther apart on the left side . if positioning sensor pairs closely together caused entrainment , then values from both sensors would drift in the same direction at similar rates of change . in such a case , one would expect a correlation between the intersensor distance and the difference between the signed differences ( bias ) of each sensor in the pair compared with the reference blood glucose value . in other words , if sensors positioned very closely to one another were entrained , they would have similar readings and minimal differences in bias values . however , no such correlation was found between the intersensor distance and the difference between the signed differences ( r = 0.004 , fig . , there was no significant relationship between distance between sensor pairs and sensor accuracy measured by mean ard ( p = ns ) . graph of intersensor distance versus the difference between the signed differences of each sensor in a pair compared with the reference blood glucose . if close proximity caused sensor entrainment , one would expect the value on the y axis to be low . note that there is no significant correlation between intersensor distance and the difference between the signed differences of each sensor in a pair . the benefit of multiple sensors was analyzed by comparing how often the mean and median of all four sensor values deviated 25% or more and 50% or more from the reference blood glucose compared with the values of each single sensor . errors of these degrees may cause inappropriate treatment decisions , and in particular , errors of 50% or higher might cause harm if these sensor values were used as an input into an artificial pancreas algorithm . large errors , defined as the sensor value being 25% above or below the reference venous blood glucose values , were significantly reduced by using the mean values ( 9.5% of values [ 95% ci 5.815.4 ] ) or median values ( 10.0% [ 6.216.1 ] ) from four sensors compared with one sensor ( 17.5% [ 13.323.0 ] , p < 0.001 ) . very large errors , defined as the sensor value being 50% or more above or below the reference blood glucose value , were also significantly reduced by using the mean ( 0.4% [ 0.11.2 ] ) or median ( 0.5% [ 0.21.2 ] ) of four sensor values compared with one sensor ( 2.6% [ 1.64.0 ] , p < 0.001 ) . in many cases , there were no large errors . however , in those experiments with a substantial number of very large errors , which reached as high as 61% of values , use of the mean of four sensors dramatically reduced the frequency of very large errors ( fig . summary of very large errors , defined as sensor values 50% away from the reference venous blood glucose . the percentages of very large errors when the four sensors are averaged are shown by black open circles , and values for each single sensor are shown by gray xs . the mean absolute relative difference ( ard ) was significantly improved by use of the mean ( 11.6 1.0% ) or median ( 11.8 1.0% ) of four sensors compared with one sensor ( 14.8 1.0% , p < 0.001 ) . there was no advantage to selecting the sensor with the lowest ard at the time of calibration at the study start ( mean ard 15.0 1.4% , p = ns ) . the mean absolute difference , the metric for reference venous blood glucose values < 75 mg / dl , also improved significantly by use of the mean of four sensors compared with a single sensor ( mean absolute difference 9.6 1.0 vs. 14.1 1.3 mg / dl , p < 0.001 ) . large errors , as defined above , were also significantly reduced by using the mean of the sensor pairs ( 12.9% [ 95% ci 9.919.2 ] ) compared with a single sensor ( 17.5% [ 13.323.0 ] , p < 0.01 ) . very large errors were reduced by a mean of 62% by the use of sensor pair means versus one sensor ( 1.0% [ 0.51.9 ] vs. 2.6% [ 1.64.0 ] , p < 0.001 ) . there was a reduction in the mean ard when comparing the mean of the sensor pairs with a single sensor ( 13.5 1.1 vs. 14.8 1.0% , p < 0.01 ) . there was a trend in improvement in sensor accuracy with using the mean of three sensors compared with two sensors ( 0.9 percentage point ard reduction , p < 0.06 ) , with a clear added benefit of four ( 1.6 percentage point ard reduction , p < 0.01 ) . a summary of errors for one , two , and four sensors , categorized by glucose levels , is presented in table 1 . in the euglycemic range ( 70180 mg / dl ) , the percentage of erroneous readings decreased significantly with additional sensor signal information ( 1.1 , 0.8 , and 0.5% of readings in the cg - error grids c , d , and e zones for the mean of 1 , 2 , and 4 sensors , respectively ) . the finding was similar in the hyperglycemic range above 180 mg / dl ( 3.5 , 2.8 , and 1.7% in c , d , and e zones for a mean of 1 , 2 , and 4 sensors ) . the number of accurate readings also significantly increased in the hyperglycemic range with increasing number of sensors ( 90.2 , 91.7 , and 94.7% ) . there were no significant differences in readings in the a region in the euglycemic range . there were also no significant differences in erroneous or accurate readings in the limited number of values in the hypoglycemic range . there was a significant increase in the a zone of the clarke error grid using multiple sensors ( 68.4 , 70.7 , and 75.4% for 1 , 2 , and 4 sensors , respectively ) . there were more values in the b zone with use of a single sensor ( 23.6 , 18.6 , and 11.5% ) . there was nonsignificant trend toward reducing the percentage of the values in the c , d , and e zones ( 2.4 , 1.3 , and 0.4% ) . voting schemes may improve upon using sensor value averaging alone , because if one sensor is highly accurate and the second is highly inaccurate , the mean is worse than use of the single highly accurate sensor . there was a significant reduction in the mean ard of the mean of three sensors in the case when data flagged by the pca was removed compared with all data ( mean ard 12.0 1.1 vs. 12.3 1.1% , p = 0.04 ) . as expected , the mean ard of the flagged data ( 16.5 2.4% ) was higher compared with nonflagged data alone ( p = 0.02 ) and also compared with all data ( p = 0.01 ) . in the case of two sensors , removing values flagged by pca did not significantly change the mean ard versus all data ( 12.9 0.9 vs. 13.0 0.9% , p = ns ) , and there was a nonsignificant increase in the mean ard of the flagged data ( 15.5 1.5 , p = 0.06 compared with all data ) . the intersensor distances as measured by x - ray imaging were 7 1 , 13 1 , 21 1 , and 28 2 mm ( fig . note that the sensors are positioned very closely on the right side of the subject s abdomen and are much farther apart on the left side . if positioning sensor pairs closely together caused entrainment , then values from both sensors would drift in the same direction at similar rates of change . in such a case , one would expect a correlation between the intersensor distance and the difference between the signed differences ( bias ) of each sensor in the pair compared with the reference blood glucose value . in other words , if sensors positioned very closely to one another were entrained , they would have similar readings and minimal differences in bias values . however , no such correlation was found between the intersensor distance and the difference between the signed differences ( r = 0.004 , fig . furthermore , there was no significant relationship between distance between sensor pairs and sensor accuracy measured by mean ard ( p = ns ) . graph of intersensor distance versus the difference between the signed differences of each sensor in a pair compared with the reference blood glucose . if close proximity caused sensor entrainment , one would expect the value on the y axis to be low . note that there is no significant correlation between intersensor distance and the difference between the signed differences of each sensor in a pair . the concept of redundancy is well established and is commonly used when errors may lead to unacceptable consequences , such as on nasa spacecraft ( 12 ) . the use of two or more sensors in a closed - loop system is appealing for multiple reasons . first , the presence of redundant sensors provides a reserve for instances of sensor or telemetry failure . currently available sensors need time for signal stabilization , so if the only sensor in place fails , hours elapse before the newly placed sensor is ready for use . in addition to signal averaging , there are several other potential ways of using data from more than one sensor . another option is to compare the two sensor values and disregard sensor data when the two values are discrepant beyond a specified criterion ( 13 ) . in an artificial pancreas setting , this method becomes problematic when no sensor readings are available for anything but a very short time period . sensor signals that are quite similar to others in the array are usually more accurate than outliers , and the values that are discrepant can be voted out . although wearing three or more sensors is impractical with current technology , sensor arrays with multiple sensing units contained in one device may be available in the future . here we demonstrated that a voting scheme based on pca is effective , when using three sensors , in detecting potential sensor errors and providing an alert flag indicating that the sensor mean may be an inaccurate estimate of the patient s blood glucose level . the pca methodology can be used in various settings . in conjunction with a multisensor filtering scheme , an inconsistent sensor measurement detected through pca is removed from the computation of the filtered sensor output . in another setting , the pca methodology may simply provide an alert to the user that a finger stick measurement is recommended before treatment and to provide a calibration point to resolve inconsistencies in sensor signals . this voting scheme did not significantly improve upon the mean of two sensors , likely due to insufficient data to determine which values to flag . four sensors provide sufficient data , but pca may not have improved upon averaging because there are enough values that the mean is not as greatly affected by an erroneous signal as when there are only three sensors . the 75-min measurement history for the pca methodology other methods to improve sensor accuracy by our group have included mathematical correction of background current , which is the current detected by a sensor in the absence of glucose ( 14 ) . others have proposed using models to predict glucose levels , which could assist in identifying sensor errors when the sensor values stray greatly from the predicted glucose ( 15 ) . one limitation of this study was that sensors could not be placed closer than 7 mm because of the size of the housing around the sensors . it is unknown whether placement of sensors closer than 7 mm apart would have decreased the benefit of redundancy . sensors are not approved to be worn in magnetic resonance imaging machines because of potential safety concerns , including heating of the surrounding tissue and possible migration of the sensor , and therefore were imaged by x - ray . this study was done only with dexcom seven plus sensors , and so the applicability to other types of sensors is unknown . medtronic guardian real - time glucose sensors are not readily apparent on x - ray images and thus were not used . freestyle navigator sensors were not available for purchase in our area when the study was conducted . we conclude that when four sensors are used simultaneously , there is an accuracy benefit compared with use of one sensor . there is also a benefit of using the mean of three or even two sensors . the benefit of redundancy is present even when sensors are positioned very close together , as close as 7 mm . these findings will be useful in the design of a small , integrated artificial pancreas device and suggest that sensors in such a device can be positioned very closely to one another .

objectiveto improve glucose sensor accuracy in subjects with type 1 diabetes by using multiple sensors and to assess whether the benefit of redundancy is affected by intersensor distance.research design and methodsnineteen adults with type 1 diabetes wore four dexcom seven plus subcutaneous glucose sensors during two 9-h studies . one pair of sensors was worn on each side of the abdomen , with each sensor pair placed at a predetermined distance apart and 20 cm away from the opposite pair . arterialized venous blood glucose levels were measured every 15 min , and sensor glucose values were recorded every 5 min . sensors were calibrated once at the beginning of the study.resultsthe use of four sensors significantly reduced very large errors compared with one sensor ( 0.4 vs. 2.6% of errors 50% from reference glucose , p < 0.001 ) and also improved overall accuracy ( mean absolute relative difference , 11.6 vs. 14.8% , p < 0.001 ) . using only two sensors also significantly improved very large errors and accuracy . intersensor distance did not affect the function of sensor pairs.conclusionssensor accuracy is significantly improved with the use of multiple sensors compared with the use of a single sensor . the benefit of redundancy is present even when sensors are positioned very closely together ( 7 mm ) . these findings are relevant to the design of an artificial pancreas device .

RESEARCH DESIGN AND METHODS Study procedures Statistical analysis RESULTS Benefit of four sensors Benefit of two sensors CG-EGA Clarke Error Grid Voting scheme using PCA Intersensor distances for sensor pairs The effect of intersensor distance CONCLUSIONS

patients with type 1 diabetes were recruited from oregon health & sciences university ( ohsu ) outpatient clinics in portland . two sensors were placed to the right of the umbilicus and two to the left . the pair on the right was positioned 20 cm apart from the pair on the left . each pair was placed at a predetermined distance on the surface of the abdominal skin , with the goal of achieving a fixed distance between the tips of the glucose sensors in the subcutaneous tissue . at the beginning of the 9-h study , the mean of two simultaneous measured venous blood glucose values was used to calibrate the four sensors . venous glucose was subsequently measured every 15 min for 9 h using a hemocue glucose 201 analyzer . at the end of each study , coned - down oblique x - ray images of the sensor sites were taken to measure the actual distance between the tips of the glucose sensors . is a continuous glucose - error grid analysis ( cg - ega ) , an outcome metric that addresses the problem of evaluating the temporal characteristics of the continuous glucose sensor process by considering pairs of reference and sensor readings as a process in time that takes into account inherent physiologic time lags ( 9 ) . principal component analysis ( pca ) is a multivariate data analysis method that was used here to flag and remove potentially aberrant sensor values to assess if removing these values would improve overall sensor accuracy ( 10,11 ) . performing pca on these data reveals a new set of uncorrelated variables called principle components ( pcs ) , with weights assigned to each of the original sensor sources to indicate the contribution of each original sensor source to the pc . this information , in conjunction with the percent of variability of the original data set that is captured by the first pc , was used retrospectively to identify an inaccurate or faulty sensor ; in practice , the method is designed to be used in real - time . two sensors were placed to the right of the umbilicus and two to the left . each pair was placed at a predetermined distance on the surface of the abdominal skin , with the goal of achieving a fixed distance between the tips of the glucose sensors in the subcutaneous tissue . at the beginning of the 9-h study , the mean of two simultaneous measured venous blood glucose values was used to calibrate the four sensors . venous glucose was subsequently measured every 15 min for 9 h using a hemocue glucose 201 analyzer . at the end of each study , coned - down oblique x - ray images of the sensor sites were taken to measure the actual distance between the tips of the glucose sensors . is a continuous glucose - error grid analysis ( cg - ega ) , an outcome metric that addresses the problem of evaluating the temporal characteristics of the continuous glucose sensor process by considering pairs of reference and sensor readings as a process in time that takes into account inherent physiologic time lags ( 9 ) . principal component analysis ( pca ) is a multivariate data analysis method that was used here to flag and remove potentially aberrant sensor values to assess if removing these values would improve overall sensor accuracy ( 10,11 ) . in practice , our data set at each time step consisted of three or more variables ( sensor sources ) , each with a set of observations , the number of which depended on the frequency of available sensor measurements and the predetermined length of the measurement history , set to 15 and 75 min , respectively , in this case . performing pca on these data reveals a new set of uncorrelated variables called principle components ( pcs ) , with weights assigned to each of the original sensor sources to indicate the contribution of each original sensor source to the pc . in the case of missing data , for example , if two of three sensors are available , then the two - sensor implementation of the pca is used . eleven women and 8 men with type 1 diabetes participated in 36 studies , each lasting 9 h. the data from one study were excluded due to a diluted venous blood glucose value that caused incorrect calibration of the sensors , leaving 35 studies for inclusion in the data analysis . the benefit of multiple sensors was analyzed by comparing how often the mean and median of all four sensor values deviated 25% or more and 50% or more from the reference blood glucose compared with the values of each single sensor . errors of these degrees may cause inappropriate treatment decisions , and in particular , errors of 50% or higher might cause harm if these sensor values were used as an input into an artificial pancreas algorithm . large errors , defined as the sensor value being 25% above or below the reference venous blood glucose values , were significantly reduced by using the mean values ( 9.5% of values [ 95% ci 5.815.4 ] ) or median values ( 10.0% [ 6.216.1 ] ) from four sensors compared with one sensor ( 17.5% [ 13.323.0 ] , p < 0.001 ) . very large errors , defined as the sensor value being 50% or more above or below the reference blood glucose value , were also significantly reduced by using the mean ( 0.4% [ 0.11.2 ] ) or median ( 0.5% [ 0.21.2 ] ) of four sensor values compared with one sensor ( 2.6% [ 1.64.0 ] , p < 0.001 ) . however , in those experiments with a substantial number of very large errors , which reached as high as 61% of values , use of the mean of four sensors dramatically reduced the frequency of very large errors ( fig . summary of very large errors , defined as sensor values 50% away from the reference venous blood glucose . the percentages of very large errors when the four sensors are averaged are shown by black open circles , and values for each single sensor are shown by gray xs . the mean absolute relative difference ( ard ) was significantly improved by use of the mean ( 11.6 1.0% ) or median ( 11.8 1.0% ) of four sensors compared with one sensor ( 14.8 1.0% , p < 0.001 ) . there was no advantage to selecting the sensor with the lowest ard at the time of calibration at the study start ( mean ard 15.0 1.4% , p = ns ) . the mean absolute difference , the metric for reference venous blood glucose values < 75 mg / dl , also improved significantly by use of the mean of four sensors compared with a single sensor ( mean absolute difference 9.6 1.0 vs. 14.1 1.3 mg / dl , p < 0.001 ) . large errors , as defined above , were also significantly reduced by using the mean of the sensor pairs ( 12.9% [ 95% ci 9.919.2 ] ) compared with a single sensor ( 17.5% [ 13.323.0 ] , p < 0.01 ) . very large errors were reduced by a mean of 62% by the use of sensor pair means versus one sensor ( 1.0% [ 0.51.9 ] vs. 2.6% [ 1.64.0 ] , p < 0.001 ) . there was a reduction in the mean ard when comparing the mean of the sensor pairs with a single sensor ( 13.5 1.1 vs. 14.8 1.0% , p < 0.01 ) . there was a trend in improvement in sensor accuracy with using the mean of three sensors compared with two sensors ( 0.9 percentage point ard reduction , p < 0.06 ) , with a clear added benefit of four ( 1.6 percentage point ard reduction , p < 0.01 ) . a summary of errors for one , two , and four sensors , categorized by glucose levels , is presented in table 1 . summary of sensor errors categorized by degree of error and venous blood glucose levels in the euglycemic range ( 70180 mg / dl ) , the percentage of erroneous readings decreased significantly with additional sensor signal information ( 1.1 , 0.8 , and 0.5% of readings in the cg - error grids c , d , and e zones for the mean of 1 , 2 , and 4 sensors , respectively ) . the number of accurate readings also significantly increased in the hyperglycemic range with increasing number of sensors ( 90.2 , 91.7 , and 94.7% ) . there was a significant increase in the a zone of the clarke error grid using multiple sensors ( 68.4 , 70.7 , and 75.4% for 1 , 2 , and 4 sensors , respectively ) . there were more values in the b zone with use of a single sensor ( 23.6 , 18.6 , and 11.5% ) . there was nonsignificant trend toward reducing the percentage of the values in the c , d , and e zones ( 2.4 , 1.3 , and 0.4% ) . voting schemes may improve upon using sensor value averaging alone , because if one sensor is highly accurate and the second is highly inaccurate , the mean is worse than use of the single highly accurate sensor . there was a significant reduction in the mean ard of the mean of three sensors in the case when data flagged by the pca was removed compared with all data ( mean ard 12.0 1.1 vs. 12.3 1.1% , p = 0.04 ) . as expected , the mean ard of the flagged data ( 16.5 2.4% ) was higher compared with nonflagged data alone ( p = 0.02 ) and also compared with all data ( p = 0.01 ) . in the case of two sensors , removing values flagged by pca did not significantly change the mean ard versus all data ( 12.9 0.9 vs. 13.0 0.9% , p = ns ) , and there was a nonsignificant increase in the mean ard of the flagged data ( 15.5 1.5 , p = 0.06 compared with all data ) . note that the sensors are positioned very closely on the right side of the subject s abdomen and are much farther apart on the left side . if positioning sensor pairs closely together caused entrainment , then values from both sensors would drift in the same direction at similar rates of change . in such a case , one would expect a correlation between the intersensor distance and the difference between the signed differences ( bias ) of each sensor in the pair compared with the reference blood glucose value . in other words , if sensors positioned very closely to one another were entrained , they would have similar readings and minimal differences in bias values . , there was no significant relationship between distance between sensor pairs and sensor accuracy measured by mean ard ( p = ns ) . graph of intersensor distance versus the difference between the signed differences of each sensor in a pair compared with the reference blood glucose . note that there is no significant correlation between intersensor distance and the difference between the signed differences of each sensor in a pair . the benefit of multiple sensors was analyzed by comparing how often the mean and median of all four sensor values deviated 25% or more and 50% or more from the reference blood glucose compared with the values of each single sensor . errors of these degrees may cause inappropriate treatment decisions , and in particular , errors of 50% or higher might cause harm if these sensor values were used as an input into an artificial pancreas algorithm . large errors , defined as the sensor value being 25% above or below the reference venous blood glucose values , were significantly reduced by using the mean values ( 9.5% of values [ 95% ci 5.815.4 ] ) or median values ( 10.0% [ 6.216.1 ] ) from four sensors compared with one sensor ( 17.5% [ 13.323.0 ] , p < 0.001 ) . very large errors , defined as the sensor value being 50% or more above or below the reference blood glucose value , were also significantly reduced by using the mean ( 0.4% [ 0.11.2 ] ) or median ( 0.5% [ 0.21.2 ] ) of four sensor values compared with one sensor ( 2.6% [ 1.64.0 ] , p < 0.001 ) . however , in those experiments with a substantial number of very large errors , which reached as high as 61% of values , use of the mean of four sensors dramatically reduced the frequency of very large errors ( fig . summary of very large errors , defined as sensor values 50% away from the reference venous blood glucose . the percentages of very large errors when the four sensors are averaged are shown by black open circles , and values for each single sensor are shown by gray xs . the mean absolute relative difference ( ard ) was significantly improved by use of the mean ( 11.6 1.0% ) or median ( 11.8 1.0% ) of four sensors compared with one sensor ( 14.8 1.0% , p < 0.001 ) . there was no advantage to selecting the sensor with the lowest ard at the time of calibration at the study start ( mean ard 15.0 1.4% , p = ns ) . the mean absolute difference , the metric for reference venous blood glucose values < 75 mg / dl , also improved significantly by use of the mean of four sensors compared with a single sensor ( mean absolute difference 9.6 1.0 vs. 14.1 1.3 mg / dl , p < 0.001 ) . large errors , as defined above , were also significantly reduced by using the mean of the sensor pairs ( 12.9% [ 95% ci 9.919.2 ] ) compared with a single sensor ( 17.5% [ 13.323.0 ] , p < 0.01 ) . very large errors were reduced by a mean of 62% by the use of sensor pair means versus one sensor ( 1.0% [ 0.51.9 ] vs. 2.6% [ 1.64.0 ] , p < 0.001 ) . there was a reduction in the mean ard when comparing the mean of the sensor pairs with a single sensor ( 13.5 1.1 vs. 14.8 1.0% , p < 0.01 ) . there was a trend in improvement in sensor accuracy with using the mean of three sensors compared with two sensors ( 0.9 percentage point ard reduction , p < 0.06 ) , with a clear added benefit of four ( 1.6 percentage point ard reduction , p < 0.01 ) . a summary of errors for one , two , and four sensors , categorized by glucose levels , is presented in table 1 . the number of accurate readings also significantly increased in the hyperglycemic range with increasing number of sensors ( 90.2 , 91.7 , and 94.7% ) . there was a significant increase in the a zone of the clarke error grid using multiple sensors ( 68.4 , 70.7 , and 75.4% for 1 , 2 , and 4 sensors , respectively ) . there were more values in the b zone with use of a single sensor ( 23.6 , 18.6 , and 11.5% ) . there was nonsignificant trend toward reducing the percentage of the values in the c , d , and e zones ( 2.4 , 1.3 , and 0.4% ) . voting schemes may improve upon using sensor value averaging alone , because if one sensor is highly accurate and the second is highly inaccurate , the mean is worse than use of the single highly accurate sensor . there was a significant reduction in the mean ard of the mean of three sensors in the case when data flagged by the pca was removed compared with all data ( mean ard 12.0 1.1 vs. 12.3 1.1% , p = 0.04 ) . as expected , the mean ard of the flagged data ( 16.5 2.4% ) was higher compared with nonflagged data alone ( p = 0.02 ) and also compared with all data ( p = 0.01 ) . in the case of two sensors , removing values flagged by pca did not significantly change the mean ard versus all data ( 12.9 0.9 vs. 13.0 0.9% , p = ns ) , and there was a nonsignificant increase in the mean ard of the flagged data ( 15.5 1.5 , p = 0.06 compared with all data ) . note that the sensors are positioned very closely on the right side of the subject s abdomen and are much farther apart on the left side . if positioning sensor pairs closely together caused entrainment , then values from both sensors would drift in the same direction at similar rates of change . in such a case , one would expect a correlation between the intersensor distance and the difference between the signed differences ( bias ) of each sensor in the pair compared with the reference blood glucose value . in other words , if sensors positioned very closely to one another were entrained , they would have similar readings and minimal differences in bias values . furthermore , there was no significant relationship between distance between sensor pairs and sensor accuracy measured by mean ard ( p = ns ) . graph of intersensor distance versus the difference between the signed differences of each sensor in a pair compared with the reference blood glucose . note that there is no significant correlation between intersensor distance and the difference between the signed differences of each sensor in a pair . the use of two or more sensors in a closed - loop system is appealing for multiple reasons . in an artificial pancreas setting , this method becomes problematic when no sensor readings are available for anything but a very short time period . here we demonstrated that a voting scheme based on pca is effective , when using three sensors , in detecting potential sensor errors and providing an alert flag indicating that the sensor mean may be an inaccurate estimate of the patient s blood glucose level . in conjunction with a multisensor filtering scheme , an inconsistent sensor measurement detected through pca is removed from the computation of the filtered sensor output . in another setting , the pca methodology may simply provide an alert to the user that a finger stick measurement is recommended before treatment and to provide a calibration point to resolve inconsistencies in sensor signals . this voting scheme did not significantly improve upon the mean of two sensors , likely due to insufficient data to determine which values to flag . four sensors provide sufficient data , but pca may not have improved upon averaging because there are enough values that the mean is not as greatly affected by an erroneous signal as when there are only three sensors . others have proposed using models to predict glucose levels , which could assist in identifying sensor errors when the sensor values stray greatly from the predicted glucose ( 15 ) . it is unknown whether placement of sensors closer than 7 mm apart would have decreased the benefit of redundancy . sensors are not approved to be worn in magnetic resonance imaging machines because of potential safety concerns , including heating of the surrounding tissue and possible migration of the sensor , and therefore were imaged by x - ray . this study was done only with dexcom seven plus sensors , and so the applicability to other types of sensors is unknown . freestyle navigator sensors were not available for purchase in our area when the study was conducted . we conclude that when four sensors are used simultaneously , there is an accuracy benefit compared with use of one sensor . there is also a benefit of using the mean of three or even two sensors . the benefit of redundancy is present even when sensors are positioned very close together , as close as 7 mm . these findings will be useful in the design of a small , integrated artificial pancreas device and suggest that sensors in such a device can be positioned very closely to one another .

tuberculosis ( tb ) is one of the most common infections worldwide , and in 2012 , an estimated 8.6 million people developed tb and 1.3 million died from the disease ( including 320,000 deaths among hiv - positive people ) [ 1 , 2 ] . mycobacterium tuberculosis ( mtb ) is an intracellular pathogen capable of infecting and surviving within the host s mononuclear cells particularly macrophages . elimination of the microorganism is through a combination of various killing mechanisms including apoptosis of host macrophages . these responses are orchestrated by t helper 1-type ( th1 ) pro - inflammatory cytokines , which are synthesized by phagocytes upon recognition of pathogen - associated molecular patterns ( pamps ) on mtb by pattern recognition receptors ( prrs ) . mtb is usually transmitted via aerosols and establishes a stable infectious state in the respiratory system . there , mtb is engulfed by macrophages and dendritic cells ( dcs ) , which serve as host cells for mtb survival and propagation . binding of mtb ligands to tlr-2 , -4 and -9 initiates release of inflammatory mediators , expression of adhesion molecules and further recruitment of macrophages , dcs and pmn to the mtb infected area . although the host s innate immune response to mtb infection is critical for the initial defense against bacteria , the adaptive immune response is ultimately required for containment of the infection in the chronic stage of disease . adaptive immunity to mtb infection is characterized by the appearance of antigen specific cd4 + t - cells that secrete ifn- , which , in turn , activates macrophages and other antigen presenting cells ( apc ) to kill intracellular bacteria . cd8 + t - cells are also important cells for controlling mtb during the chronic phase of infection [ 7 , 8 ] . in addition , th17 cells and il-17 have been reported to be involved in the pathogenesis of mtb . il-17 is a proinflammatory cytokine produced by th17 cells and by airway structural cells , which provides ifn--dependent or ifn--independent protection to mtb infection ( see figs . 1 and 2 ) [ 1012].fig . 1schematic diagram indicating the role of specific cell types and mediators on the induction of ifn by mtb and the subsequent killing of macrophage - resident bacteria . abbreviations : clr c - type lectin receptors , mtb mycobacterium tuberculosis , tlrs toll like receptors , nlrs nod - like receptorsfig . 2the putative role of tlr / inflammasome signaling on the regulation of mtb in the cells . abbreviation : tlr toll like receptor , mtb mycobacterium tuberculosis schematic diagram indicating the role of specific cell types and mediators on the induction of ifn by mtb and the subsequent killing of macrophage - resident bacteria . abbreviations : clr c - type lectin receptors , mtb mycobacterium tuberculosis , tlrs toll like receptors , nlrs nod - like receptors the putative role of tlr / inflammasome signaling on the regulation of mtb in the cells . abbreviation : tlr toll like receptor , mtb mycobacterium tuberculosis indeed , in models of mtb infection , il-17 and th17 cells were first implicated in the protective immune response to rapidly growing extracellular bacteria in the lung and gut mucosal surfaces through efficient induction of neutrophil recruitment and tissue repair [ 1315 ] . il-17 and th17 cells are important during the initial stages of infection and act upon hematopoietic and non - hematopoietic cells to promote the secretion of antimicrobial peptides such as g - csf and cxc chemokines . as a consequence of this , dcs migrate to the local lymph nodes and induce the differentiation of both th1 and th17 cells . the increased levels of chemokines in the infected lung also promote recruitment of other protective cells such as macrophages and pmn and the formation of mononuclear granulomas . moreover , an accumulation cytokines such as il-6 and il-23 in the lungs can further induce the differentiation and activation of th17 cells and accelerate the pathogenesis of tb . in this review we focus on the role of signal transduction pathways which have an impact on the pathogenesis of tb . among these , the generation of ros and the later activation of pprs including tlrs and of the inflammasome are highlighted . reactive oxygen species ( ros ) and reactive nitrogen species ( rns ) are considered to play important role in the pathogenesis of various inflammatory diseases [ 17 , 18 ] . under physiologic conditions ; ros are generated as byproducts of oxygen metabolism . ros are found in all biological systems and originate from the metabolism of molecular oxygen ( o2 ) . under physiological conditions o2 undergoes reduction by accepting four electrons which results in the formation of water . during this process , reactive intermediates such as the superoxide anion ( o2 ) , hydrogen peroxide ( h2o2 ) and hydroxyl ( oh ) radicals are formed . activated macrophages express two major enzymes , phagocyte oxidase ( nox2/gp91 ) and inducible nitric oxide synthase ( nos2 ) , which are able to generate reactive oxygen intermediates ( roi ) and reactive nitrogen intermediates ( rni ) , respectively . upon phagocytosis , the preformed nox2 subunits assemble into an enzymatically active enzyme complex that transfers electrons across the membrane from cytosolic nadph to molecular oxygen . this produces o2 which dismutate into hydrogen peroxide ( h2o2 ) and thus generate oh radicals which are toxic to mtb . following inhalation of mtb , alveolar macrophages engulf the bacilli and initiate their killing using a number of mechanisms including the generation of roi and rni [ 21 , 22 ] . the rapid generation of ros is critical in host defense against many bacteria and fungi , and ros has broad signaling functions . for example , the nadph oxidase protein complexes generate the superoxide anion and downstream ros . thus , nadph oxidase has an important role in host defense against mtb and any patients with a loss of function mutation in genes encoding components of this enzyme complex could be deficient in killing bacilli . indeed , mutations in the cybb gene encoding the gp91 ( phox ) subunit of the phagocyte nadph oxidase is associated with mtb . in addition , a hemizygous splice mutation in intron 5 of cybb was linked to the concomitant occurrence of chronic granulomatous disease ( cgd ) with mtb . ifn- induces nos2 and its product nitric oxide ( no ) which in turn can be broken down to nitrite and nitrate . under acidic conditions , such as within the phagosomes of ifn- activated macrophages , nitrite forms nitrous acid , which dismutates to no and the toxic radical , nitrogen dioxide . no can synergize with superoxide , produced by the macrophage or generated as byproduct of respiratory metabolism by the pathogen , to form the highly poisonous peroxynitrite ( onoo ) radical . these roi and rni react with a wide range of molecules , including nucleic acids , proteins , lipids and carbohydrates , resulting in the killing of mtb . to counteract these actions , mtb uses a variety of molecules to either detoxify roi and rni before they can inflict damage or to repair the damage they cause . in particular , the presence of mtb results in glucose-6-phosphate ( g6p ) being oxidised by nadp - dependent and f420-dependent ( fgd1 ) dehydrogenases to generate nadph , an important source of electrons , and thereby overcome oxidative stress . in addition , mtb uses a combination of its cell surface alpha - ketoacid dehydrogenase complexes to form a nadh - dependent peroxidase and peroxynitrite reductase . pattern recognition receptors ( prrs ) are a group of receptors which sense the presence of bacteria , fungi and viruses . prrs are also responsible for recognizing endogenous molecules released from damaged cells , which are named damage associated molecular patterns ( damps ) [ 30 , 31 ] . to date these families include transmembrane proteins such as the tlrs and c - type lectin receptors ( ctlrs ) , as well as cytoplasmic proteins such as the retinoic acid - inducible gene ( rig)-i - like receptors ( rlrs ) and nod - like receptors ( nlrs ) . tlrs are a family of single membrane - spanning receptors of which 10 have been characterized in man and 13 in mouse [ 3436 ] . tlrs play a critical role in both innate resistance and the initiation of adaptive immunity to infectious agents [ 3740 ] . they act by recognizing pathogen - associated molecular patterns ( pamps ) or endogenous inflammation - associated molecules [ 36 , 41 , 42 ] . these are distinct molecular structures on microbes and different sets of tlrs have been associated with the response to different classes of microorganisms e.g. recognition of viruses by tlr3 , tlr7 , tlr8 and tlr9 [ 36 , 41 , 4346 ] . bacterial dna which contains unmethylated cpg oligonucleotides ( odn ) motifs also acts as important regulators of human neutrophil functions via tlr9 . for example , stimulation of the tlr9 pathway by cpgodn induces cxcl8 production by human neutrophils via the generation of onoo [ 47 , 48 ] . tlr - ligand binding can induce two signaling pathways , the myeloid differentiation primary response gene 88 ( myd88)-dependent and myd88-independent pathways , which induce the production of both pro - inflammatory cytokines and type i ifns [ 36 , 49 , 50 ] . these two distinct responses are mediated via the selective use of adaptor molecules recruited to the toll / il-1 receptor ( tir ) domains of tlrs after ligand binding . four adaptor molecules have been identified to date : myd88 , tir - associated protein ( tirap ) , tir domain - containing adaptor protein - inducing ifn- ( trif ) and trif related adaptor molecules ( tram ) . myd88 and tirap are responsible for the induction of pro - inflammatory genes , and trif and tram induce ifns . in myd88-dependent signaling , myd88 is recruited to , and associates with , the cytoplasmic domain of the tlrs upon ligand binding . then il-1r - associated kinase 4 ( irak-4 ) and irak- 1 are subsequently recruited and activated by phosphorylation . activated irak-4 phosphorylates irak-1 , which then , in turn , associates with tumor necrosis factor receptor ( tnfr)-associated factor 6 ( traf6 ) . traf6 activates transforming growth factor ( tgf ) activating kinase 1 ( tak1 ) , which , in turn , phosphorylates ikk- and mitogen - activated protein kinase ( mapk ) kinase6 ( mkk6 ) , leading to degradation of i-b , nuclear translocation of nf-b and induction of inflammatory genes . as a result tlr activation upregulates the transcription of proinflammatory cytokines including il-1 , tnf- and il-6 which are essential for the recruitment of immune cells to the site of infection and controlling mtb infection [ 5355 ] . activation of the myd88-dependent pathway also results in the activation of mitogen - activated protein ( map ) kinases ( mapk ) such as p38 and jnk , which leads to the activation of ap-1 . during myd88-independent signaling tlr4 activation triggers the induction of a type 1 ifn response , leading to the induction of ifn- and ifn - inducible genes . the tlrs known to be involved in recognition of mtb are tlr2 , tlr4 , tlr9 , and possibly tlr8 [ 36 , 41 , 4346 ] . four primary immunodeficiencies ( pids ) involving mutations in myd88 , irak4 , nemo and ikba are associated with altered susceptibility to m. tuberculosis [ 5759 ] . these heterodimers have been implicated in the recognition of mycobacterial cell wall glycolipids including lipoarabinomannan ( lam ) , lipomannan ( lm ) , 38-kda and 19-kd mycobacterial glycoproteins , phosphatidylinositol mannoside ( pim ) , triacylated ( tlr2/tlr1 ) or diacylated ( tlr2/tlr6 ) lipoproteins [ 49 , 60 , 61 ] . tlr2 and tlr1 act together to mediate responses to m. tuberculosis [ 62 , 63 ] and the role of tlr1/2 gene variants in the predisposition to tuberculosis has been investigated . most studies have focused on tlr2 variants and only weak and non - replicated associations have been reported to date [ 62 , 63 ] . tlr2 is believed to be important in the initiation of the innate host defense against mtb [ 61 , 64 ] . in addition , il-1 production is dependent upon tlr2 and tlr6 , but not tlr4 or tlr9 , stimulation . tlr2 mice show defective granuloma formation following mtb infection and have a greatly enhanced susceptibility to infection compared to the wt mice [ 53 , 67 ] . in addition , tlr2 mice are unable to control chronic infection with mtb [ 67 , 68 ] . mice lacking tlr9 also succumb earlier to mtb infection than wild - type animals [ 61 , 66 , 6972 ] . the role of other tlrs , such as tlr4 and tlr9 in the pathogenesis of mtb has not studied in such detail [ 65 , 73 , 74 ] . mice deficient in the tlr / il-1r family receptor adaptor molecule myd88 have been shown to be highly susceptible to infection with mtb , which suggests a major role for this pathway in the innate defense against the mtb [ 68 , 7586 ] . in addition , tlr2-induced ros production plays a crucial role in the expression of cxcl8 and ccl2 in human monocytes requiring the activation of both p38 and erk1/2 mapk pathways . overexpression of both tlr2 and tlr4 are important for viable mtb infection in human cell lines . other studies in mice with inactivated tlr genes indicated that tlr2 is important in controlling and surviving mtb infection [ 68 , 73 ] . however , other studies suggested that tlr4 is critical for surviving mtb infection [ 80 , 87 ] . the importance of tlr4 may depend on the dose of mtb used for challenge or the mouse strain used . human studies show that polymorphisms in both tlr2 and tlr4 are associated with increased susceptibility to microbial infections possibly by changing the th1/th2 response [ 8285 ] . reported that the expression of tlrs in tb lung granulomas related to the presence or absence of immunohistologically detectable il-4 . changes in tlr expression and/or their down - stream activation state might represent useful markers of the immunological status of tb patients and their contacts . the tlr distribution in tb granulomas lesions indicates that tlr1 , tlr2 , and tlr4 are expressed in both immune cells and non - immune cells ; however tlr9 is only detectable in immune cells . furthermore , in an animal model of tb , tlr8-deficient mice succumb more rapidly to infection with m. tuberculosis , despite efficiently controlling the number of viable bacilli in different organs . although no changes in cd4 + and cd8 + t - cells were observed there were increases in lung neutrophils and macrophages . exaggerated mortality was due to massive liver necrosis and was reversed by a combination of blocking antibodies to il-1 and tnf-. thus , in this model of mtb infection , tlr8 plays a key role in dampening inflammation and tissue damage . overall , recognition of mtb by tlrs triggers various intracellular signaling cascades ultimately resulting in the production of cytokines , chemokines and antimicrobial molecules [ 91 , 92 ] . in humans , different polymorphisms in the human tlr2 gene were reported to associate with increased susceptibility to tb in some studies [ 9397 ] but not others [ 98101 ] . furthermore , a mal / tirap functional variant , affecting signaling through tlr2 , was shown to be protective in tb . genetic polymorphisms in tlr4 were linked to an increased susceptibility and severity of pulmonary tb in an asian population in india but not in indian or chinese tb patients in gambia [ 101 , 102 , 104 ] . this discrepancy might be due to a dynamic host - pathogen interplay between genetic and pathogen phenotypes . pentraxin 3 ( ptx3 ) , or tnf - stimulated gene 14 ( tsg-14 ) , is a 42-kda soluble pattern - recognition receptor produced by phagocytes and non - immune cells at sites of inflammation or injury and plays an important role in female fertility and vascular biology . ptx3 shows up to 28 % sequence identity to human c - reactive protein ( crp ) and serum amyloid p - component ( sap ) [ 104 , 105 ] . it is a member of the pentraxin family which are involved in the acute phase response to injury , trauma and infection . ptx3 is rapidly secreted into the serum of mice and humans from extra - hepatic sources after lps , il-1 or tnf- stimulation . ptx3 binds to the complement component c1q and to microorganisms , including pseudomonas aeruginosa , salmonella typhimurium and aspergillus fumigates to induce innate immune responses [ 109 , 110 ] and to drive a protective adaptive immunity . since whole mycobacteria and mycobacterial lipoarabinomannan strongly induce ptx3 production by human mononuclear phagocytes a role for ptx3 in the immunobiology of mycobacterial infection has been inferred . interestingly , ptx3 receptor gene variants are associated with an increased risk of pulmonary tuberculosis in west africans . furthermore , ptx3 , levels are significantly correlated with the severity of clinical presentation at diagnosis and of lung involvement in disease and may represent a good biomarker for inflammation and disease activity during mtb infection . there are two classes of innate immune receptors described : a ) tlrs , located on cell membranes or intracellularly , and b ) nlrs located in the cytoplasm [ 114118 ] . both classes of receptors are programmed to recognize microbial pamps and danger - associated molecular patterns ( damps ) and switch cells for activation to releasing of proinflammatory and chemokines . the importance of two receptors in pathogenesis of chronic lung disease has elicited much attention [ 119 , 120 ] . in the next section , we describe the regulation of inflammasome signaling and discuss whether abnormalities in nlrp3 inflammasome function may be associated with mtb . the inflammasome consists of a multimeric cytosolic complex comprising the adaptor protein apoptosis associated speck - like protein containing a caspase recruiting domain ( asc ) , a sensor protein such as nrlp3 together with the effector proteins caspase-1 and caspase 5 . several nlrs function in immunity through the formation of a multi - protein complex known as an inflammasome which play critical roles in the 0 pathogenesis of chronic disorders [ 119121 ] . nlrs exist in three families ; the nods , the nlrps and the ( ipafs ) . stimulation of cells with pamps , or by damps , leads to increased expression of il-1 and other il-1 cytokine family members , such as il-18 and il-33 . proinflammatory cytokines of the il-1 family may play an important role in anti - mycobacterial host defense mechanisms . moreover , mtb stimulates inflammatory cells to release il-1 through pathways involving tlr2/tlr6 and nod2 receptors . recognition of mtb by tlr and nod2 leads to increased transcription of pro - il-1 through mechanisms involving erk , p38 and rip2 , but not jnk . interestingly , although caspase-1 is necessary for the processing of pro - il-1 , activation of caspase-1 is not dependent on the stimulation of cells by mtb . in human thp-1 macrophages , mtb activation results in secretion of il-1 in an asc / nlrp3-dependent manner . in addition , mycobacterium marinum activates il-1 production in an nlrp3- and caspase-1-dependent manner in vitro highlighting the potential importance of inflammasome signaling in the pathogenesis of mbt [ 125 , 126 ] . inflammasome - mediated il-1 secretion is triggered by a combination of signal transduction pathways activated via tlrs and purinergic ( p2x7 ) receptors . in turn , il-1 induces the release of gm - csf which leads to the activation and increased survival of monocytes / macrophages and enhanced oxidative burst in the lungs , thus maintaining and prolonging inflammatory reactions . the purinergic p2x7 receptor is the key driver of atp - mediated inflammasome maturation and release of il-1 [ 122 , 128 , 129 ] . pro - inflammatory cytokine regulation by the inflammasome may be critical to long - term survival of mtb infection since experiments in il-1/ , il-1r and il-18 knockout ( ko ) mice have shown that these cytokines play a role in limiting bacterial burden in the lung , in regulating the subsequent expression of other cytokines , in controlling no production and in the formation of granulomas [ 122 , 128 , 129 ] . caspase-1 independent il-1 production may also be critical for host resistance to mtb and this occurs independently of tlr signaling in vivo . furthermore , although il-1 induction by mtb in vitro depends on tlr triggering and the inflammasome , both triggers are dispensable for il-1 production in mice infected with the pathogen in vivo . thus , although recent data established that il-1 plays a critical component in innate resistance to mtb , the pathways involved in the expression and regulation of il-1 induction following mtb infection in vivo are complex and may involve mechanisms that do not fit the classical paradigms of tlr recognition and inflammasome - mediated caspase-1 processing seen with other infections or in the response to mtb observed in vitro . mtb - induced il-1 secretion in human and mouse macrophages in vitro and this process was dependent on asc , caspase-1 , and nlrp3 , but not nlcr4 . in vivo , murine asc helps protect the host from death during chronic mtb infection whilst the effects of casp-1 and nlp3 were negligible . the inability of asc ko mice to form organized granulomas and the reduced presence of lung dendritic cells indicates a breakdown in host defense against mtb . thus , asc was identified as a critical protein involved in the host response to mtb infection in an inflammasome - independent manner . other cytokines activated by the inflammasome have also been reported to play a role in the pathogenesis of mtb . thus , il-12 and il-18 produced by dendritic cells and macrophages induce nk - cell activity and skew the immune response towards an ifn--dependent th1 response , which is considered critical for protection against mtb . data in myd88-deficient mice which are highly susceptible to mtb and succumb very rapidly to infection supports a role for myd88 in regulating mtb infection [ 75 , 131 ] . myd88 , however , plays a role in both inflammasome and tlr signaling and this raises the possibility that a lack of il-1 or il-18 is responsible for the heightened susceptibility of myd88 ko mice to mtb infection . reported that il-1r - signalling is important for protection against mtb whilst il-18r - signalling is not . in contrast , schneider et al has reported a similar degree of susceptibility to mtb infection to that observed in myd88 ko mice in il-18 ko mice . il-18 ko mice succumbed much more readily to experimental mtb infection than wt or tlr-2/-4 double ko ( tlr-2/-4 dko ) mice . in the absence of il-18 , immunity to mtb was hampered by decreased th1 responses and pmn - dominated lung immunopathology concomitant with unrestrained growth of the tubercle bacilli . thus , some controversy still remains as to the precise role of il-18 in the protective immunity against mtb infection . tb remains one of the leading causes of death from a single infectious agent worldwide . in order to generate better protective strategies we need to further define the pathological mechanisms underlying the immune response to mtb . whilst inflammasome and tlr cross talk does not seem to be essential for the primary control of mtb infection , recent data suggests a critical role of these pathways in the persistence of mtb . activation of these pathways results in the release of inflammatory mediators that recruit protective cells to the infected area . however , there is a down side to this effect . excessive production of il-23 and il-17 causes pathology due to excessive recruitment and phenotypic changes in inflammatory cells . hence , there is a fine balance between th1 and inflammasome / tlrs responses that is central in defining the outcome of mtb infection . the role and mechanisms underpinning ptx3 and other pprs in the immune response to mtb still requires further elucidation however . in addition , it is critical to further define the mechanisms associated with the cross talk between tlrs and the inflammasome and to use this knowledge to generate rational protective strategies that promote a balanced acquired immune response with minimal collateral damage . determination of key nodes within the pathways involved in the pathogenesis of mtb may provide new therapeutic targets to prevent the persistence of disease .

tuberculosis ( tb ) is considered a major worldwide health problem with 10 million new cases diagnosed each year . our understanding of tb immunology has become greater and more refined since the identification of mycobacterium tuberculosis ( mtb ) as an etiologic agent and the recognition of new signaling pathways modulating infection . understanding the mechanisms through which the cells of the immune system recognize mtb can be an important step in designing novel therapeutic approaches , as well as improving the limited success of current vaccination strategies . a great challenge in chronic disease is to understand the complexities , mechanisms , and consequences of host interactions with pathogens . innate immune responses along with the involvement of distinct inflammatory mediators and cells play an important role in the host defense against the mtb . several classes of pattern recognition receptors ( prrs ) are involved in the recognition of mtb including toll - like receptors ( tlrs ) , c - type lectin receptors ( clrs ) and nod - like receptors ( nlrs ) linked to inflammasome activation . among the tlr family , tlr1 , tlr2 , tlr4 , and tlr9 and their down - stream signaling proteins play critical roles in the initiation of the immune response in the pathogenesis of tb . the inflammasome pathway is associated with the coordinated release of cytokines such as il-1 and il-18 which also play a role in the pathogenesis of tb . understanding the cross - talk between these signaling pathways will impact on the design of novel therapeutic strategies and in the development of vaccines and immunotherapy regimes . abnormalities in prr signaling pathways regulated by tb will affect disease pathogenesis and need to be elucidated . in this review we provide an update on prr signaling during m. tuberculosis infection and indicate how greater knowledge of these pathways may lead to new therapeutic opportunities .

Introduction Role of Reactive Oxygen Specious (ROS) in Pathogenesis of TB Role of Toll Like Receptors (TLRs) in Pathogenesis of MTB Role of Pentraxin 3 (PTX3) in Pathogenesis of MTB Role of Inflammasome Signaling in MTB Conclusion and Future Outlook

tuberculosis ( tb ) is one of the most common infections worldwide , and in 2012 , an estimated 8.6 million people developed tb and 1.3 million died from the disease ( including 320,000 deaths among hiv - positive people ) [ 1 , 2 ] . mycobacterium tuberculosis ( mtb ) is an intracellular pathogen capable of infecting and surviving within the host s mononuclear cells particularly macrophages . elimination of the microorganism is through a combination of various killing mechanisms including apoptosis of host macrophages . these responses are orchestrated by t helper 1-type ( th1 ) pro - inflammatory cytokines , which are synthesized by phagocytes upon recognition of pathogen - associated molecular patterns ( pamps ) on mtb by pattern recognition receptors ( prrs ) . there , mtb is engulfed by macrophages and dendritic cells ( dcs ) , which serve as host cells for mtb survival and propagation . binding of mtb ligands to tlr-2 , -4 and -9 initiates release of inflammatory mediators , expression of adhesion molecules and further recruitment of macrophages , dcs and pmn to the mtb infected area . although the host s innate immune response to mtb infection is critical for the initial defense against bacteria , the adaptive immune response is ultimately required for containment of the infection in the chronic stage of disease . in addition , th17 cells and il-17 have been reported to be involved in the pathogenesis of mtb . 1schematic diagram indicating the role of specific cell types and mediators on the induction of ifn by mtb and the subsequent killing of macrophage - resident bacteria . abbreviations : clr c - type lectin receptors , mtb mycobacterium tuberculosis , tlrs toll like receptors , nlrs nod - like receptorsfig . 2the putative role of tlr / inflammasome signaling on the regulation of mtb in the cells . abbreviation : tlr toll like receptor , mtb mycobacterium tuberculosis schematic diagram indicating the role of specific cell types and mediators on the induction of ifn by mtb and the subsequent killing of macrophage - resident bacteria . abbreviations : clr c - type lectin receptors , mtb mycobacterium tuberculosis , tlrs toll like receptors , nlrs nod - like receptors the putative role of tlr / inflammasome signaling on the regulation of mtb in the cells . abbreviation : tlr toll like receptor , mtb mycobacterium tuberculosis indeed , in models of mtb infection , il-17 and th17 cells were first implicated in the protective immune response to rapidly growing extracellular bacteria in the lung and gut mucosal surfaces through efficient induction of neutrophil recruitment and tissue repair [ 1315 ] . il-17 and th17 cells are important during the initial stages of infection and act upon hematopoietic and non - hematopoietic cells to promote the secretion of antimicrobial peptides such as g - csf and cxc chemokines . the increased levels of chemokines in the infected lung also promote recruitment of other protective cells such as macrophages and pmn and the formation of mononuclear granulomas . moreover , an accumulation cytokines such as il-6 and il-23 in the lungs can further induce the differentiation and activation of th17 cells and accelerate the pathogenesis of tb . in this review we focus on the role of signal transduction pathways which have an impact on the pathogenesis of tb . among these , the generation of ros and the later activation of pprs including tlrs and of the inflammasome are highlighted . reactive oxygen species ( ros ) and reactive nitrogen species ( rns ) are considered to play important role in the pathogenesis of various inflammatory diseases [ 17 , 18 ] . under physiological conditions o2 undergoes reduction by accepting four electrons which results in the formation of water . during this process , reactive intermediates such as the superoxide anion ( o2 ) , hydrogen peroxide ( h2o2 ) and hydroxyl ( oh ) radicals are formed . activated macrophages express two major enzymes , phagocyte oxidase ( nox2/gp91 ) and inducible nitric oxide synthase ( nos2 ) , which are able to generate reactive oxygen intermediates ( roi ) and reactive nitrogen intermediates ( rni ) , respectively . this produces o2 which dismutate into hydrogen peroxide ( h2o2 ) and thus generate oh radicals which are toxic to mtb . following inhalation of mtb , alveolar macrophages engulf the bacilli and initiate their killing using a number of mechanisms including the generation of roi and rni [ 21 , 22 ] . the rapid generation of ros is critical in host defense against many bacteria and fungi , and ros has broad signaling functions . thus , nadph oxidase has an important role in host defense against mtb and any patients with a loss of function mutation in genes encoding components of this enzyme complex could be deficient in killing bacilli . indeed , mutations in the cybb gene encoding the gp91 ( phox ) subunit of the phagocyte nadph oxidase is associated with mtb . in addition , a hemizygous splice mutation in intron 5 of cybb was linked to the concomitant occurrence of chronic granulomatous disease ( cgd ) with mtb . ifn- induces nos2 and its product nitric oxide ( no ) which in turn can be broken down to nitrite and nitrate . under acidic conditions , such as within the phagosomes of ifn- activated macrophages , nitrite forms nitrous acid , which dismutates to no and the toxic radical , nitrogen dioxide . these roi and rni react with a wide range of molecules , including nucleic acids , proteins , lipids and carbohydrates , resulting in the killing of mtb . in particular , the presence of mtb results in glucose-6-phosphate ( g6p ) being oxidised by nadp - dependent and f420-dependent ( fgd1 ) dehydrogenases to generate nadph , an important source of electrons , and thereby overcome oxidative stress . pattern recognition receptors ( prrs ) are a group of receptors which sense the presence of bacteria , fungi and viruses . to date these families include transmembrane proteins such as the tlrs and c - type lectin receptors ( ctlrs ) , as well as cytoplasmic proteins such as the retinoic acid - inducible gene ( rig)-i - like receptors ( rlrs ) and nod - like receptors ( nlrs ) . tlrs play a critical role in both innate resistance and the initiation of adaptive immunity to infectious agents [ 3740 ] . these are distinct molecular structures on microbes and different sets of tlrs have been associated with the response to different classes of microorganisms e.g. recognition of viruses by tlr3 , tlr7 , tlr8 and tlr9 [ 36 , 41 , 4346 ] . four adaptor molecules have been identified to date : myd88 , tir - associated protein ( tirap ) , tir domain - containing adaptor protein - inducing ifn- ( trif ) and trif related adaptor molecules ( tram ) . myd88 and tirap are responsible for the induction of pro - inflammatory genes , and trif and tram induce ifns . in myd88-dependent signaling , myd88 is recruited to , and associates with , the cytoplasmic domain of the tlrs upon ligand binding . traf6 activates transforming growth factor ( tgf ) activating kinase 1 ( tak1 ) , which , in turn , phosphorylates ikk- and mitogen - activated protein kinase ( mapk ) kinase6 ( mkk6 ) , leading to degradation of i-b , nuclear translocation of nf-b and induction of inflammatory genes . activation of the myd88-dependent pathway also results in the activation of mitogen - activated protein ( map ) kinases ( mapk ) such as p38 and jnk , which leads to the activation of ap-1 . the tlrs known to be involved in recognition of mtb are tlr2 , tlr4 , tlr9 , and possibly tlr8 [ 36 , 41 , 4346 ] . four primary immunodeficiencies ( pids ) involving mutations in myd88 , irak4 , nemo and ikba are associated with altered susceptibility to m. tuberculosis [ 5759 ] . these heterodimers have been implicated in the recognition of mycobacterial cell wall glycolipids including lipoarabinomannan ( lam ) , lipomannan ( lm ) , 38-kda and 19-kd mycobacterial glycoproteins , phosphatidylinositol mannoside ( pim ) , triacylated ( tlr2/tlr1 ) or diacylated ( tlr2/tlr6 ) lipoproteins [ 49 , 60 , 61 ] . tlr2 and tlr1 act together to mediate responses to m. tuberculosis [ 62 , 63 ] and the role of tlr1/2 gene variants in the predisposition to tuberculosis has been investigated . tlr2 is believed to be important in the initiation of the innate host defense against mtb [ 61 , 64 ] . tlr2 mice show defective granuloma formation following mtb infection and have a greatly enhanced susceptibility to infection compared to the wt mice [ 53 , 67 ] . in addition , tlr2 mice are unable to control chronic infection with mtb [ 67 , 68 ] . the role of other tlrs , such as tlr4 and tlr9 in the pathogenesis of mtb has not studied in such detail [ 65 , 73 , 74 ] . mice deficient in the tlr / il-1r family receptor adaptor molecule myd88 have been shown to be highly susceptible to infection with mtb , which suggests a major role for this pathway in the innate defense against the mtb [ 68 , 7586 ] . in addition , tlr2-induced ros production plays a crucial role in the expression of cxcl8 and ccl2 in human monocytes requiring the activation of both p38 and erk1/2 mapk pathways . the importance of tlr4 may depend on the dose of mtb used for challenge or the mouse strain used . changes in tlr expression and/or their down - stream activation state might represent useful markers of the immunological status of tb patients and their contacts . the tlr distribution in tb granulomas lesions indicates that tlr1 , tlr2 , and tlr4 are expressed in both immune cells and non - immune cells ; however tlr9 is only detectable in immune cells . furthermore , in an animal model of tb , tlr8-deficient mice succumb more rapidly to infection with m. tuberculosis , despite efficiently controlling the number of viable bacilli in different organs . exaggerated mortality was due to massive liver necrosis and was reversed by a combination of blocking antibodies to il-1 and tnf-. thus , in this model of mtb infection , tlr8 plays a key role in dampening inflammation and tissue damage . overall , recognition of mtb by tlrs triggers various intracellular signaling cascades ultimately resulting in the production of cytokines , chemokines and antimicrobial molecules [ 91 , 92 ] . in humans , different polymorphisms in the human tlr2 gene were reported to associate with increased susceptibility to tb in some studies [ 9397 ] but not others [ 98101 ] . furthermore , a mal / tirap functional variant , affecting signaling through tlr2 , was shown to be protective in tb . pentraxin 3 ( ptx3 ) , or tnf - stimulated gene 14 ( tsg-14 ) , is a 42-kda soluble pattern - recognition receptor produced by phagocytes and non - immune cells at sites of inflammation or injury and plays an important role in female fertility and vascular biology . ptx3 shows up to 28 % sequence identity to human c - reactive protein ( crp ) and serum amyloid p - component ( sap ) [ 104 , 105 ] . it is a member of the pentraxin family which are involved in the acute phase response to injury , trauma and infection . ptx3 binds to the complement component c1q and to microorganisms , including pseudomonas aeruginosa , salmonella typhimurium and aspergillus fumigates to induce innate immune responses [ 109 , 110 ] and to drive a protective adaptive immunity . since whole mycobacteria and mycobacterial lipoarabinomannan strongly induce ptx3 production by human mononuclear phagocytes a role for ptx3 in the immunobiology of mycobacterial infection has been inferred . interestingly , ptx3 receptor gene variants are associated with an increased risk of pulmonary tuberculosis in west africans . furthermore , ptx3 , levels are significantly correlated with the severity of clinical presentation at diagnosis and of lung involvement in disease and may represent a good biomarker for inflammation and disease activity during mtb infection . there are two classes of innate immune receptors described : a ) tlrs , located on cell membranes or intracellularly , and b ) nlrs located in the cytoplasm [ 114118 ] . both classes of receptors are programmed to recognize microbial pamps and danger - associated molecular patterns ( damps ) and switch cells for activation to releasing of proinflammatory and chemokines . the importance of two receptors in pathogenesis of chronic lung disease has elicited much attention [ 119 , 120 ] . in the next section , we describe the regulation of inflammasome signaling and discuss whether abnormalities in nlrp3 inflammasome function may be associated with mtb . the inflammasome consists of a multimeric cytosolic complex comprising the adaptor protein apoptosis associated speck - like protein containing a caspase recruiting domain ( asc ) , a sensor protein such as nrlp3 together with the effector proteins caspase-1 and caspase 5 . several nlrs function in immunity through the formation of a multi - protein complex known as an inflammasome which play critical roles in the 0 pathogenesis of chronic disorders [ 119121 ] . nlrs exist in three families ; the nods , the nlrps and the ( ipafs ) . stimulation of cells with pamps , or by damps , leads to increased expression of il-1 and other il-1 cytokine family members , such as il-18 and il-33 . proinflammatory cytokines of the il-1 family may play an important role in anti - mycobacterial host defense mechanisms . recognition of mtb by tlr and nod2 leads to increased transcription of pro - il-1 through mechanisms involving erk , p38 and rip2 , but not jnk . interestingly , although caspase-1 is necessary for the processing of pro - il-1 , activation of caspase-1 is not dependent on the stimulation of cells by mtb . in addition , mycobacterium marinum activates il-1 production in an nlrp3- and caspase-1-dependent manner in vitro highlighting the potential importance of inflammasome signaling in the pathogenesis of mbt [ 125 , 126 ] . in turn , il-1 induces the release of gm - csf which leads to the activation and increased survival of monocytes / macrophages and enhanced oxidative burst in the lungs , thus maintaining and prolonging inflammatory reactions . pro - inflammatory cytokine regulation by the inflammasome may be critical to long - term survival of mtb infection since experiments in il-1/ , il-1r and il-18 knockout ( ko ) mice have shown that these cytokines play a role in limiting bacterial burden in the lung , in regulating the subsequent expression of other cytokines , in controlling no production and in the formation of granulomas [ 122 , 128 , 129 ] . furthermore , although il-1 induction by mtb in vitro depends on tlr triggering and the inflammasome , both triggers are dispensable for il-1 production in mice infected with the pathogen in vivo . thus , although recent data established that il-1 plays a critical component in innate resistance to mtb , the pathways involved in the expression and regulation of il-1 induction following mtb infection in vivo are complex and may involve mechanisms that do not fit the classical paradigms of tlr recognition and inflammasome - mediated caspase-1 processing seen with other infections or in the response to mtb observed in vitro . mtb - induced il-1 secretion in human and mouse macrophages in vitro and this process was dependent on asc , caspase-1 , and nlrp3 , but not nlcr4 . the inability of asc ko mice to form organized granulomas and the reduced presence of lung dendritic cells indicates a breakdown in host defense against mtb . thus , asc was identified as a critical protein involved in the host response to mtb infection in an inflammasome - independent manner . other cytokines activated by the inflammasome have also been reported to play a role in the pathogenesis of mtb . thus , il-12 and il-18 produced by dendritic cells and macrophages induce nk - cell activity and skew the immune response towards an ifn--dependent th1 response , which is considered critical for protection against mtb . data in myd88-deficient mice which are highly susceptible to mtb and succumb very rapidly to infection supports a role for myd88 in regulating mtb infection [ 75 , 131 ] . myd88 , however , plays a role in both inflammasome and tlr signaling and this raises the possibility that a lack of il-1 or il-18 is responsible for the heightened susceptibility of myd88 ko mice to mtb infection . in the absence of il-18 , immunity to mtb was hampered by decreased th1 responses and pmn - dominated lung immunopathology concomitant with unrestrained growth of the tubercle bacilli . thus , some controversy still remains as to the precise role of il-18 in the protective immunity against mtb infection . in order to generate better protective strategies we need to further define the pathological mechanisms underlying the immune response to mtb . whilst inflammasome and tlr cross talk does not seem to be essential for the primary control of mtb infection , recent data suggests a critical role of these pathways in the persistence of mtb . activation of these pathways results in the release of inflammatory mediators that recruit protective cells to the infected area . the role and mechanisms underpinning ptx3 and other pprs in the immune response to mtb still requires further elucidation however . in addition , it is critical to further define the mechanisms associated with the cross talk between tlrs and the inflammasome and to use this knowledge to generate rational protective strategies that promote a balanced acquired immune response with minimal collateral damage . determination of key nodes within the pathways involved in the pathogenesis of mtb may provide new therapeutic targets to prevent the persistence of disease .

between 1983 and 1989 , 1,441 participants with t1 dm , 1339 years of age , were enrolled in the dcct . the dcct consisted of two cohorts : the primary prevention cohort had diabetes for 15 years , no retinopathy , and urinary albumin excretion < 40 mg/24 h , and the secondary intervention cohort had diabetes for 115 years , very mild to moderate nonproliferative retinopathy , and urinary albumin excretion 200 mg/24 h at baseline ( 18 ) . approximately one - half of the subjects ( n = 711 ) were randomly assigned to intensive therapy , and the remainder ( n = 730 ) were assigned to conventional therapy . the treatment groups maintained a separation of median hba1c levels of 2 percentage points ( 7.1 vs. 9.0% ; 54.1 vs. 74.9 mmol / mol ) during the 6.5-year average dcct follow - up ( 18 ) . since it had been shown to be highly effective in reducing diabetic microvascular complications , intensive therapy was recommended for all participants when the dcct ended in 1993 ( 18,19 ) . participants were then returned to their own health care providers for diabetes care . in 1994 , 1,375 ( 96% ) of the 1,428 surviving members of dcct volunteered to participate in edic for annual observational follow - up ( 19 ) . this report incorporates data through edic year 17 ( 2010 ) , representing an average of 23.5 years of follow - up from randomization into dcct . by edic year 17 , 1,287 subjects continued to participate in edic , and of these , 1,177 ( 91% ) completed the dqol survey . ninety - five participants had died by the end of edic year 17 . for purposes of these analyses , two subjects with acute , temporary renal failure unrelated to diabetes were excluded . nonparticipants , including those who died , did not differ from participants in most characteristics at dcct baseline including sex , age , education , blood pressure , and cholesterol . nonparticipants had significantly higher hba1c levels and a higher frequency of current cigarette smokers ( supplementary table 1 ) . furthermore , 48% of participants were in the conventional treatment group compared with 61% of nonparticipants . the dqol is a self - administered multiple - choice 46-item assessment that has been described in detail ( 1517 ) . the dqol has four primary subscales ( satisfaction , impact , diabetes worry , and social / vocational worry ) that assess different aspects of quality of life . the scoring system yields scale scores that range from 0 ( lowest quality of life ) to 100 ( highest quality of life ) , identical to the procedure for scoring the medical outcome survey 36-item short - form health survey ( sf-36 ) quality - of - life measure ( 2123 ) . total dqol scores are presented in the results section unless otherwise stated , and scores on the four subscales are presented in supplementary table 2 . psychometric studies have indicated that the overall dqol measure has excellent internal consistency ( cronbach , 0.830.92 ) for both adults and adolescents ( 1517 ) . test - retest reliability over an average period of 9 days was 0.92 for the overall measure ( 17 ) . the dqol has been shown to have convergent validity with conceptually relevant measures of well - being , psychiatric symptoms , and adjustment to illness ( 17 ) . in addition , the dqol discriminates between patients with different numbers of clinically evident complications ( 17 ) and is sensitive to different therapies for t2 dm ( 17,24,25 ) and to a change in therapy for t1 dm ( i.e. , pancreatic transplantation ) ( 25 ) . for this article , past research has suggested that a difference of five points on the total dqol score represents a clinically meaningful difference in hrqol ( 1517,20,24,25 ) . consequently , this parameter was used in our longitudinal analyses of the effects of time - dependent predictors on decrease in quality of life . specifically , an event was defined as a drop from baseline 5 points on two consecutive evaluations . we used a sustained decrease in dqol score to confirm that a change occurred over a substantial time frame and was not transitory . we have used this same approach for other outcomes such as nephropathy , where a measure like albumin excretion rate ( aer ) can change up and down over time ( 26 ) . the methods and scheduling of physical examinations , outcomes assessments , and laboratory measurements have been previously described in detail and remained consistent throughout dcct and edic ( 18,19,2628 ) . during the dcct ( quarterly ) and edic ( annually ) , glycated hemoglobin values were measured in a central laboratory by high - performance liquid chromatography ( 18,19 ) . retinopathy , assessed during edic years 1114 by 7-field stereoscopic fundus photography according to the dcct / edic protocol ( 26 ) , was defined for these analyses as the presence of proliferative diabetic retinopathy ( pdr ) or worse , and/or a history of panretinal scatter - photocoagulation ( laser ) therapy . in addition , visual acuity ( va ) was assessed to determine best corrected vision in the best and worst eye . nephropathy was defined in this study as having any aer 300 mg/24 h through edic year 16 or end - stage renal disease ( esrd ) , defined as treatment with dialysis or transplantation for chronic renal failure ( 18,19,26 ) . in edic years 13 to 14 , board - certified neurologists and electromyographers conducted neurological evaluations and electrodiagnostic studies on all willing participants using the same protocol as was used in dcct to determine the presence of clinical neuropathy ( 27,28 ) . the occurrence of severe hypoglycemia was documented quarterly during the dcct and within 3 months of the annual visit during edic . it was defined in two ways : 1 ) any event requiring the assistance of another person , with either a blood glucose < 50 mg / dl ( 2.78 mmol / l ) and/or subsequent reversal of symptoms with oral carbohydrate , subcutaneous glucagon , or intravenous glucose ; or 2 ) the same criteria plus unconsciousness , seizure , or coma ( 18,29 ) . twenty - seven percent of severe hypoglycemic episodes involved coma or seizure ( 30 ) . in this article , the episodes of severe hypoglycemia requiring assistance and those also resulting in seizure or coma were analyzed separately . symptomatic data and changes since the last annual visit were self - reported each year during edic for the following symptoms : chest pain , decreased vision , paresthesias in hands / feet , recurrent urinary incontinence , and impotence . information about intercurrent psychosocial events and psychiatric history was reported quarterly during the dcct and annually during edic based on subject interviews . information about education level , marital status , psychiatric treatment , psychiatric hospitalization , and suicide attempts was documented . a psychiatric event was defined as at least one occurrence in edic accompanied by inpatient or outpatient treatment for any psychiatric event during the same year . demographic and clinical characteristics were compared using the wilcoxon rank - sum test to evaluate treatment group differences for ordinal and numeric variables ( 31 ) . the contingency test was used for categorical variables ; when the sample size was small , the fisher exact test was used ( 31 ) . cox proportional hazard models were used to determine the effects of demographic and biomedical assessments on the risk of a sustained five - point drop in total dqol over two consecutive assessments during dcct and edic . each characteristic was modeled separately as a time - dependent covariate adjusting for its baseline value as well as sex , baseline age , and baseline mean years of education . since qol was only measured during odd years in edic , each model was stratified for calendar year of randomization , allowing subjects who entered into the study during the same calendar year to have the same visit sequence . updated mean values are time - weighted , running means up to each study visit in the dcct and edic . separate ancova models were used to assess the relationship between total dqol and both complication status and functional symptoms by year 17 of edic . adjustments were made for sex , baseline age , baseline mean years of education , and baseline dqol score . least square means and ses were compared for participants with and without the characteristic or complication of interest . statistical analyses were performed using the sas v 9.2 statistical analysis software ( sas institute , cary , nc ) . between 1983 and 1989 , 1,441 participants with t1 dm , 1339 years of age , were enrolled in the dcct . the dcct consisted of two cohorts : the primary prevention cohort had diabetes for 15 years , no retinopathy , and urinary albumin excretion < 40 mg/24 h , and the secondary intervention cohort had diabetes for 115 years , very mild to moderate nonproliferative retinopathy , and urinary albumin excretion 200 mg/24 h at baseline ( 18 ) . approximately one - half of the subjects ( n = 711 ) were randomly assigned to intensive therapy , and the remainder ( n = 730 ) were assigned to conventional therapy . the treatment groups maintained a separation of median hba1c levels of 2 percentage points ( 7.1 vs. 9.0% ; 54.1 vs. 74.9 mmol / mol ) during the 6.5-year average dcct follow - up ( 18 ) . since it had been shown to be highly effective in reducing diabetic microvascular complications , intensive therapy was recommended for all participants when the dcct ended in 1993 ( 18,19 ) . participants were then returned to their own health care providers for diabetes care . in 1994 , 1,375 ( 96% ) of the 1,428 surviving members of dcct volunteered to participate in edic for annual observational follow - up ( 19 ) . this report incorporates data through edic year 17 ( 2010 ) , representing an average of 23.5 years of follow - up from randomization into dcct . by edic year 17 , 1,287 subjects continued to participate in edic , and of these , 1,177 ( 91% ) completed the dqol survey . ninety - five participants had died by the end of edic year 17 . for purposes of these analyses , two subjects with acute , temporary renal failure unrelated to diabetes were excluded . nonparticipants , including those who died , did not differ from participants in most characteristics at dcct baseline including sex , age , education , blood pressure , and cholesterol . nonparticipants had significantly higher hba1c levels and a higher frequency of current cigarette smokers ( supplementary table 1 ) . furthermore , 48% of participants were in the conventional treatment group compared with 61% of nonparticipants . the dqol is a self - administered multiple - choice 46-item assessment that has been described in detail ( 1517 ) . the dqol has four primary subscales ( satisfaction , impact , diabetes worry , and social / vocational worry ) that assess different aspects of quality of life . the scoring system yields scale scores that range from 0 ( lowest quality of life ) to 100 ( highest quality of life ) , identical to the procedure for scoring the medical outcome survey 36-item short - form health survey ( sf-36 ) quality - of - life measure ( 2123 ) . total dqol scores are presented in the results section unless otherwise stated , and scores on the four subscales are presented in supplementary table 2 . psychometric studies have indicated that the overall dqol measure has excellent internal consistency ( cronbach , 0.830.92 ) for both adults and adolescents ( 1517 ) . test - retest reliability over an average period of 9 days was 0.92 for the overall measure ( 17 ) . the dqol has been shown to have convergent validity with conceptually relevant measures of well - being , psychiatric symptoms , and adjustment to illness ( 17 ) . in addition , the dqol discriminates between patients with different numbers of clinically evident complications ( 17 ) and is sensitive to different therapies for t2 dm ( 17,24,25 ) and to a change in therapy for t1 dm ( i.e. , pancreatic transplantation ) ( 25 ) . past research has suggested that a difference of five points on the total dqol score represents a clinically meaningful difference in hrqol ( 1517,20,24,25 ) . consequently , this parameter was used in our longitudinal analyses of the effects of time - dependent predictors on decrease in quality of life . specifically , an event was defined as a drop from baseline 5 points on two consecutive evaluations . we used a sustained decrease in dqol score to confirm that a change occurred over a substantial time frame and was not transitory . we have used this same approach for other outcomes such as nephropathy , where a measure like albumin excretion rate ( aer ) can change up and down over time ( 26 ) . the methods and scheduling of physical examinations , outcomes assessments , and laboratory measurements have been previously described in detail and remained consistent throughout dcct and edic ( 18,19,2628 ) . during the dcct ( quarterly ) and edic ( annually ) , glycated hemoglobin values were measured in a central laboratory by high - performance liquid chromatography ( 18,19 ) . retinopathy , assessed during edic years 1114 by 7-field stereoscopic fundus photography according to the dcct / edic protocol ( 26 ) , was defined for these analyses as the presence of proliferative diabetic retinopathy ( pdr ) or worse , and/or a history of panretinal scatter - photocoagulation ( laser ) therapy . in addition , visual acuity ( va ) was assessed to determine best corrected vision in the best and worst eye . nephropathy was defined in this study as having any aer 300 mg/24 h through edic year 16 or end - stage renal disease ( esrd ) , defined as treatment with dialysis or transplantation for chronic renal failure ( 18,19,26 ) . in edic years 13 to 14 , board - certified neurologists and electromyographers conducted neurological evaluations and electrodiagnostic studies on all willing participants using the same protocol as was used in dcct to determine the presence of clinical neuropathy ( 27,28 ) . the occurrence of severe hypoglycemia was documented quarterly during the dcct and within 3 months of the annual visit during edic . it was defined in two ways : 1 ) any event requiring the assistance of another person , with either a blood glucose < 50 mg / dl ( 2.78 mmol / l ) and/or subsequent reversal of symptoms with oral carbohydrate , subcutaneous glucagon , or intravenous glucose ; or 2 ) the same criteria plus unconsciousness , seizure , or coma ( 18,29 ) . twenty - seven percent of severe hypoglycemic episodes involved coma or seizure ( 30 ) . in this article , the episodes of severe hypoglycemia requiring assistance and those also resulting in seizure or coma were analyzed separately . symptomatic data and changes since the last annual visit were self - reported each year during edic for the following symptoms : chest pain , decreased vision , paresthesias in hands / feet , recurrent urinary incontinence , and impotence . information about intercurrent psychosocial events and psychiatric history was reported quarterly during the dcct and annually during edic based on subject interviews . information about education level , marital status , psychiatric treatment , psychiatric hospitalization , and suicide attempts was documented . a psychiatric event was defined as at least one occurrence in edic accompanied by inpatient or outpatient treatment for any psychiatric event during the same year . demographic and clinical characteristics were compared using the wilcoxon rank - sum test to evaluate treatment group differences for ordinal and numeric variables ( 31 ) . the contingency test was used for categorical variables ; when the sample size was small , the fisher exact test was used ( 31 ) . cox proportional hazard models were used to determine the effects of demographic and biomedical assessments on the risk of a sustained five - point drop in total dqol over two consecutive assessments during dcct and edic . each characteristic was modeled separately as a time - dependent covariate adjusting for its baseline value as well as sex , baseline age , and baseline mean years of education . since qol was only measured during odd years in edic , each model was stratified for calendar year of randomization , allowing subjects who entered into the study during the same calendar year to have the same visit sequence . updated mean values are time - weighted , running means up to each study visit in the dcct and edic . separate ancova models were used to assess the relationship between total dqol and both complication status and functional symptoms by year 17 of edic . adjustments were made for sex , baseline age , baseline mean years of education , and baseline dqol score . least square means and ses were compared for participants with and without the characteristic or complication of interest . statistical analyses were performed using the sas v 9.2 statistical analysis software ( sas institute , cary , nc ) . there were no significant differences between treatment groups in demographic or clinical characteristics at baseline , other than a clinically insignificant difference in systolic blood pressure ( table 1 ) . at the 17-year edic follow - up , after an average of 23.5 years , 66% of all dcct / edic participants had developed hypertension , 65% hypercholesterolemia , and 19% proliferative diabetic retinopathy or worse . the lower prevalence of retinopathy and nephropathy in the former intensive treatment group at edic year 17 reflects the previously published salutary effects of intensive therapy ( 19 ) . characteristics of participants who completed the dqol in edic year 17 no differences in total dqol or dqol subscale scores were found between the intensive and conventional groups at any time points . mean total dqol scores were not significantly different between the former dcct intensive and conventional treatment groups at baseline ( 78 8 vs. 78 9 ) , dcct closeout ( 78.1 8.8 vs. 78.2 9.4 ) , or edic year 17 ( 75 11 vs. 74 11 ) . the group as a whole did not show a significant trend for decreasing dqol scores over time ( table 1 and supplementary table 2 ) . there were 755 ( 52% ) dcct / edic participants who had a dqol event ( defined as a decrease in dqol from baseline of 5 points on two consecutive evaluations ) by edic year 17 , with 293 ( 39% ) occurring during dcct and 462 ( 61% ) during edic . there were no significant treatment group differences ( p = 0.9339 ) . compared with females , males had a 13.8% lower risk of reaching a dqol end point ( hazard ratio 0.862 [ 95% ci 0.7440.999 ] ; p = 0.0484 ) . higher values of hba1c , log(aer ) , mean blood pressure , and bmi were all associated with a sustained drop of 5 points in dqol score , before and after adjustment for sex , age , and mean years of education at dcct baseline ( table 2 ) . subjects who experienced severe hypoglycemic events had a 36% higher risk of having a 5-point decrease in dqol than those without such events . analyses using the more stringent definition of severe events that included those that led to unconsciousness , coma , or seizure did not reveal an impact on dqol scores . the effects of hypoglycemia , hba1c , and bmi were significant even after adjustments of other covariates including log(aer ) and mean blood pressure ( table 2 ) . association of time - dependent covariates with risk of a sustained drop of 5 points in total dqol since dcct baseline development of advanced stages of retinopathy ( p = 0.0196 ) , nephropathy ( p = 0.0019 ) , and confirmed clinical neuropathy ( p < 0.0001 ) were associated with lower total dqol scores at year 17 of edic ( an average of 23.5 years of follow - up ) , after adjusting for sex , age , and mean years of education at baseline ( table 3 ) . there was a significant effect of the development of any complications ( none vs. one or more ) ( p = 0.0009 ) , with a linear trend showing that those subjects with all three complications had the lowest dqol score of 71.6 0.7 compared with no complications ( p < 0.0001 ) ( table 3 ) . by edic year 17 , 13 subjects had chronic renal failure due to diabetes and were treated with either dialysis or transplantation . medical complications by total dqol score at edic year 17 after an average of 23.5 years of follow - up self - reported symptoms associated with these complications at year 17 were also linked to decreased dqol scores ( table 4 ) . specifically , chest pain ( p = 0.0004 ) , decreased vision in both eyes ( p = 0.0005 ) , paresthesias in hands or feet ( p < 0.0001 ) , urinary incontinence ( p = 0.0001 ) , and male erectile dysfunction ( p < 0.0001 ) were associated with lower edic year 17 dqol scores , after adjusting for sex , baseline age , baseline mean years of education , and baseline dqol score . among both men and women , increasing number of symptoms was associated with a lower dqol score ( men , p = 0.0091 ; women , p = 0.0028 ) . because the principal dcct end point was progressive retinal disease , we also compared dqol scores for participants at different levels of va . the 14 participants with va worse than 20/100 in the worse eye had a mean total dqol score of 68.5 7.9 compared with 74.9 10.5 , 74.6 10.7 , and 76.0 12.5 for va better or equal to 20/20 , 20/20 to 20/40 , and 20/40 to 20/100 , respectively ( p = 0.0661 ) . self - reported functional status and history of psychiatric events occurring in edic by total dqol score at edic year 17 after an average of 23.5 years of follow - up self - reported history of treated anxiety ( p < 0.0001 ) and treated depression ( p < two hundred subjects reported both anxiety and depression , and their mean adjusted quality - of - life score was 69.2 0.7 . only a small number of participants were ever hospitalized for a psychiatric illness or reported suicidal attempts ( 27 attempts in 18 individuals ) , and both were associated with decreased dqol scores ( p < 0.0001 ) . an increasing number of psychiatric events was reflected in a decreased dqol score ( p < 0.0001 ) . there were no significant differences between treatment groups in demographic or clinical characteristics at baseline , other than a clinically insignificant difference in systolic blood pressure ( table 1 ) . at the 17-year edic follow - up , after an average of 23.5 years , 66% of all dcct / edic participants had developed hypertension , 65% hypercholesterolemia , and 19% proliferative diabetic retinopathy or worse . the lower prevalence of retinopathy and nephropathy in the former intensive treatment group at edic year 17 reflects the previously published salutary effects of intensive therapy ( 19 ) . characteristics of participants who completed the dqol in edic year 17 no differences in total dqol or dqol subscale scores were found between the intensive and conventional groups at any time points . mean total dqol scores were not significantly different between the former dcct intensive and conventional treatment groups at baseline ( 78 8 vs. 78 9 ) , dcct closeout ( 78.1 8.8 vs. 78.2 9.4 ) , or edic year 17 ( 75 11 vs. 74 11 ) . the group as a whole did not show a significant trend for decreasing dqol scores over time ( table 1 and supplementary table 2 ) . there were 755 ( 52% ) dcct / edic participants who had a dqol event ( defined as a decrease in dqol from baseline of 5 points on two consecutive evaluations ) by edic year 17 , with 293 ( 39% ) occurring during dcct and 462 ( 61% ) during edic . there were no significant treatment group differences ( p = 0.9339 ) . compared with females , males had a 13.8% lower risk of reaching a dqol end point ( hazard ratio 0.862 [ 95% ci 0.7440.999 ] ; p = 0.0484 ) . higher values of hba1c , log(aer ) , mean blood pressure , and bmi were all associated with a sustained drop of 5 points in dqol score , before and after adjustment for sex , age , and mean years of education at dcct baseline ( table 2 ) . subjects who experienced severe hypoglycemic events had a 36% higher risk of having a 5-point decrease in dqol than those without such events . analyses using the more stringent definition of severe events that included those that led to unconsciousness , coma , or seizure did not reveal an impact on dqol scores . the effects of hypoglycemia , hba1c , and bmi were significant even after adjustments of other covariates including log(aer ) and mean blood pressure ( table 2 ) . association of time - dependent covariates with risk of a sustained drop of 5 points in total dqol since dcct baseline development of advanced stages of retinopathy ( p = 0.0196 ) , nephropathy ( p = 0.0019 ) , and confirmed clinical neuropathy ( p < 0.0001 ) were associated with lower total dqol scores at year 17 of edic ( an average of 23.5 years of follow - up ) , after adjusting for sex , age , and mean years of education at baseline ( table 3 ) . there was a significant effect of the development of any complications ( none vs. one or more ) ( p = 0.0009 ) , with a linear trend showing that those subjects with all three complications had the lowest dqol score of 71.6 0.7 compared with no complications ( p < 0.0001 ) ( table 3 ) . by edic year 17 , 13 subjects had chronic renal failure due to diabetes and were treated with either dialysis or transplantation . medical complications by total dqol score at edic year 17 after an average of 23.5 years of follow - up self - reported symptoms associated with these complications at year 17 were also linked to decreased dqol scores ( table 4 ) . specifically , chest pain ( p = 0.0004 ) , decreased vision in both eyes ( p = 0.0005 ) , paresthesias in hands or feet ( p < 0.0001 ) , urinary incontinence ( p = 0.0001 ) , and male erectile dysfunction ( p < 0.0001 ) were associated with lower edic year 17 dqol scores , after adjusting for sex , baseline age , baseline mean years of education , and baseline dqol score . among both men and women , increasing number of symptoms was associated with a lower dqol score ( men , p = 0.0091 ; women , p = 0.0028 ) . because the principal dcct end point was progressive retinal disease , the 14 participants with va worse than 20/100 in the worse eye had a mean total dqol score of 68.5 7.9 compared with 74.9 10.5 , 74.6 10.7 , and 76.0 12.5 for va better or equal to 20/20 , 20/20 to 20/40 , and 20/40 to 20/100 , respectively ( p = 0.0661 ) . self - reported functional status and history of psychiatric events occurring in edic by total dqol score at edic year 17 after an average of 23.5 years of follow - up self - reported history of treated anxiety ( p < 0.0001 ) and treated depression ( p < two hundred subjects reported both anxiety and depression , and their mean adjusted quality - of - life score was 69.2 0.7 . only a small number of participants were ever hospitalized for a psychiatric illness or reported suicidal attempts ( 27 attempts in 18 individuals ) , and both were associated with decreased dqol scores ( p < 0.0001 ) . an increasing number of psychiatric events was reflected in a decreased dqol score ( p < 0.0001 ) . we present the longest prospective follow - up study of hrqol in individuals with t1 dm and describe their course of quality - of - life experiences over 25 years . the therapy applied to the intensive treatment group during dcct and encouraged for the entire cohort during the 17 years of edic follow - up has resulted in relatively low rates of severe diabetes complications . our findings support a salutary effect of the prevention and delay of complications on hrqol ( 18,2628,30,32 ) . decreases in hrqol are particularly likely among patients with a higher prevalence of severe complications . with regard to our first research question and consistent with our previous report at the end of the dcct , there are no apparent adverse effects of intensive therapy on hrqol ( 2 ) . with regard to our second research question and in contrast to our initial hrqol report based on an average of 6.5 years of treatment during dcct ( 2 ) , after an additional 17 years of follow - up , we now find an association between elevated hba1c levels and severe hypoglycemia requiring assistance and a sustained decrease in dqol scores . with regard to our third research question , the development of advanced complications and the symptoms resulting from these complications , such as decreased va , erectile dysfunction , and incontinence , were associated with decreases in hrqol . each of the three complications assessed ( retinopathy , neuropathy , and nephropathy ) were individually associated with lower dqol scores in comparison with those without the complication ; moreover , using a composite index , increasing numbers of advanced complications led to lower quality of life . consistent with the assessment of complications , functional symptoms were also associated with lower quality of life . it is important to note that while almost all patients developed some degree of retinopathy ( 88% prior intensive group ; 96% prior conventional group ) and 19% required laser treatment or vitreoretinal surgery in at least one eye , only 4.8% ( n = 52 ) had best corrected vision worse than 20/40 in one eye , and only 13 had best corrected vision worse than 20/100 in one eye . patients with best corrected vision less than 20/100 in their worse eye had lower hrqol than those with better va . finally , the development of common psychiatric syndromes like depression is linked to hrqol ( 5 ) . this relationship was seen in the association of psychiatric conditions and their treatment on lower dqol scores after an average of 23.5 years of follow - up . the current study has unique strengths compared with previous studies of t1 dm , including its long - term , consistent follow - up of a large cohort ; detailed demographic and clinical information collected prospectively using standardized methods from relatively early in the course of illness ; repeated measurement of hrqol ; the diversity of clinical outcomes ; and the wide range of ages represented during the study period ( 1365 years of age ) . these include the selection and self - selection of participants to enroll in a randomized clinical trial that required careful screening and adherence to a complex regimen over an extended period of time . moreover , potential participants with psychosocial problems and limited social support would have been screened out . the dcct / edic cohort had relatively high average socioeconomic status and education level of the group and was predominantly caucasian . during dcct , there was a high degree of assistance provided by the study nurse - coordinators and physicians . such factors would tend to lessen the impact on hrqol , so these findings may understate the effects in typical clinical populations , who also may have higher rates of severe complications . although our cohort , and in particular the intensive treatment group , received an unusual level of attention and support during the 6.5 years of the dcct , during 17 of the average of 23.5 years represented by this study , the participants have been followed in typical practice settings with less support from the study . the return to usual care settings may increase the representativeness of this group of t1 dm patients . our ability to examine the effects of treatment assignment , exposure to varying levels of glycemia , including severe hypoglycemia , and the development of medical complications provide a benchmark for comparison with other research studies and with patients followed in typical clinical settings . we have constructed a quantitative picture of the impact of different interventions , levels of metabolic control , and complications on patient experiences of illness . smaller studies that have explored in greater depth the experiences of illness using qualitative methods augment these findings while evaluating specific aspects of illness ( 33 ) . other studies , typically cross - sectional or of shorter duration , but with broader representation of patient types , have provided information that is reasonably consistent with our findings . moreover , as in our study , intensification of treatment using multiple daily injections and insulin pumps does not lead to decreased quality of life ( 1,7,11,12,13 ) . indeed , intensive treatment and the subsequent reduction of symptomatic complications helps maintain the long - term quality of life of patients with diabetes . the information derived from studies like this one can be useful for clinicians as the initiate newly diagnosed patients into treatment and work with patients collaboratively over time . indeed , such personal experiences are increasingly viewed as critical for patients in making their own therapeutic decisions ( 34,35 ) and engaging their clinical care team in dealing with the rigors of long - term diabetes . moreover , symptoms related to sexual and urologic functions may be embarrassing for patients to discuss , but have distinct impact on patient experience of illness and represent an important area for inquiry by treating health care professionals .

objectiveto examine the long - term effects of type 1 diabetes treatment , metabolic control , and complications on health - related quality of life ( hrqol).research design and methodsa total of 1,441 participants , initially 1339 years of age , were followed for an average of 23.5 years as part of the diabetes control and complications trial ( dcct ) and the epidemiology of diabetes interventions and complications ( edic ) follow - up study . the diabetes quality - of - life questionnaire ( dqol ) was administered annually during dcct and every other year during edic . biomedical data , including hba1c levels , exposure to severe hypoglycemia , intercurrent psychiatric events , and development of diabetes complications were collected at regular intervals throughout the follow-up.resultsmean total dqol scores were not significantly different between the former dcct intensive and conventional treatment groups ( dcct baseline , 78 8 vs. 78 9 ; edic year 17 , 75 11 vs. 74 11 ) . over the course of the study , a drop of 5 points in dqol score from dcct baseline maintained on two successive visits occurred in 755 individuals and was associated with increased hba1c , albumin excretion rate , mean blood pressure , bmi , and occurrence of hypoglycemic events requiring assistance . lower dqol scores after 23.5 years of follow - up were associated with prior development of retinopathy ( p = 0.0196 ) , nephropathy ( p = 0.0019 ) , and neuropathy ( p < 0.0001 ) as well as self - reported chest pain ( p = 0.0004 ) , decreased vision in both eyes ( p = 0.0005 ) , painful paresthesias ( p < 0.0001 ) , recurrent urinary incontinence ( p = 0.0001 ) , erectile dysfunction ( p < 0.0001 ) , and history of psychiatric events ( p < 0.0001).conclusionsamong dcct / edic participants , worsening metabolic control , serious diabetes complications and their associated symptoms , and development of psychiatric conditions led to decreased hrqol .

RESEARCH DESIGN AND METHODS Study sample Quality-of-life assessment Biomedical evaluations and assessment of diabetes complications Intercurrent psychosocial events and psychiatric symptoms Statistical analyses RESULTS Clinical characteristics of the participants Impact of prior treatment group assignment on HRQOL Impact of glycemic control and hypoglycemic episodes on HRQOL Impact of advanced complications on HRQOL Relationship of self-reported symptoms and HRQOL Relationship of psychiatric events and HRQOL CONCLUSIONS

between 1983 and 1989 , 1,441 participants with t1 dm , 1339 years of age , were enrolled in the dcct . the treatment groups maintained a separation of median hba1c levels of 2 percentage points ( 7.1 vs. 9.0% ; 54.1 vs. 74.9 mmol / mol ) during the 6.5-year average dcct follow - up ( 18 ) . this report incorporates data through edic year 17 ( 2010 ) , representing an average of 23.5 years of follow - up from randomization into dcct . by edic year 17 , 1,287 subjects continued to participate in edic , and of these , 1,177 ( 91% ) completed the dqol survey . nonparticipants , including those who died , did not differ from participants in most characteristics at dcct baseline including sex , age , education , blood pressure , and cholesterol . the scoring system yields scale scores that range from 0 ( lowest quality of life ) to 100 ( highest quality of life ) , identical to the procedure for scoring the medical outcome survey 36-item short - form health survey ( sf-36 ) quality - of - life measure ( 2123 ) . total dqol scores are presented in the results section unless otherwise stated , and scores on the four subscales are presented in supplementary table 2 . consequently , this parameter was used in our longitudinal analyses of the effects of time - dependent predictors on decrease in quality of life . specifically , an event was defined as a drop from baseline 5 points on two consecutive evaluations . we used a sustained decrease in dqol score to confirm that a change occurred over a substantial time frame and was not transitory . retinopathy , assessed during edic years 1114 by 7-field stereoscopic fundus photography according to the dcct / edic protocol ( 26 ) , was defined for these analyses as the presence of proliferative diabetic retinopathy ( pdr ) or worse , and/or a history of panretinal scatter - photocoagulation ( laser ) therapy . the occurrence of severe hypoglycemia was documented quarterly during the dcct and within 3 months of the annual visit during edic . symptomatic data and changes since the last annual visit were self - reported each year during edic for the following symptoms : chest pain , decreased vision , paresthesias in hands / feet , recurrent urinary incontinence , and impotence . information about intercurrent psychosocial events and psychiatric history was reported quarterly during the dcct and annually during edic based on subject interviews . cox proportional hazard models were used to determine the effects of demographic and biomedical assessments on the risk of a sustained five - point drop in total dqol over two consecutive assessments during dcct and edic . each characteristic was modeled separately as a time - dependent covariate adjusting for its baseline value as well as sex , baseline age , and baseline mean years of education . adjustments were made for sex , baseline age , baseline mean years of education , and baseline dqol score . between 1983 and 1989 , 1,441 participants with t1 dm , 1339 years of age , were enrolled in the dcct . approximately one - half of the subjects ( n = 711 ) were randomly assigned to intensive therapy , and the remainder ( n = 730 ) were assigned to conventional therapy . the treatment groups maintained a separation of median hba1c levels of 2 percentage points ( 7.1 vs. 9.0% ; 54.1 vs. 74.9 mmol / mol ) during the 6.5-year average dcct follow - up ( 18 ) . in 1994 , 1,375 ( 96% ) of the 1,428 surviving members of dcct volunteered to participate in edic for annual observational follow - up ( 19 ) . this report incorporates data through edic year 17 ( 2010 ) , representing an average of 23.5 years of follow - up from randomization into dcct . by edic year 17 , 1,287 subjects continued to participate in edic , and of these , 1,177 ( 91% ) completed the dqol survey . nonparticipants , including those who died , did not differ from participants in most characteristics at dcct baseline including sex , age , education , blood pressure , and cholesterol . the dqol has four primary subscales ( satisfaction , impact , diabetes worry , and social / vocational worry ) that assess different aspects of quality of life . the scoring system yields scale scores that range from 0 ( lowest quality of life ) to 100 ( highest quality of life ) , identical to the procedure for scoring the medical outcome survey 36-item short - form health survey ( sf-36 ) quality - of - life measure ( 2123 ) . total dqol scores are presented in the results section unless otherwise stated , and scores on the four subscales are presented in supplementary table 2 . consequently , this parameter was used in our longitudinal analyses of the effects of time - dependent predictors on decrease in quality of life . we used a sustained decrease in dqol score to confirm that a change occurred over a substantial time frame and was not transitory . retinopathy , assessed during edic years 1114 by 7-field stereoscopic fundus photography according to the dcct / edic protocol ( 26 ) , was defined for these analyses as the presence of proliferative diabetic retinopathy ( pdr ) or worse , and/or a history of panretinal scatter - photocoagulation ( laser ) therapy . the occurrence of severe hypoglycemia was documented quarterly during the dcct and within 3 months of the annual visit during edic . symptomatic data and changes since the last annual visit were self - reported each year during edic for the following symptoms : chest pain , decreased vision , paresthesias in hands / feet , recurrent urinary incontinence , and impotence . information about intercurrent psychosocial events and psychiatric history was reported quarterly during the dcct and annually during edic based on subject interviews . cox proportional hazard models were used to determine the effects of demographic and biomedical assessments on the risk of a sustained five - point drop in total dqol over two consecutive assessments during dcct and edic . each characteristic was modeled separately as a time - dependent covariate adjusting for its baseline value as well as sex , baseline age , and baseline mean years of education . adjustments were made for sex , baseline age , baseline mean years of education , and baseline dqol score . there were no significant differences between treatment groups in demographic or clinical characteristics at baseline , other than a clinically insignificant difference in systolic blood pressure ( table 1 ) . at the 17-year edic follow - up , after an average of 23.5 years , 66% of all dcct / edic participants had developed hypertension , 65% hypercholesterolemia , and 19% proliferative diabetic retinopathy or worse . the lower prevalence of retinopathy and nephropathy in the former intensive treatment group at edic year 17 reflects the previously published salutary effects of intensive therapy ( 19 ) . characteristics of participants who completed the dqol in edic year 17 no differences in total dqol or dqol subscale scores were found between the intensive and conventional groups at any time points . mean total dqol scores were not significantly different between the former dcct intensive and conventional treatment groups at baseline ( 78 8 vs. 78 9 ) , dcct closeout ( 78.1 8.8 vs. 78.2 9.4 ) , or edic year 17 ( 75 11 vs. 74 11 ) . there were 755 ( 52% ) dcct / edic participants who had a dqol event ( defined as a decrease in dqol from baseline of 5 points on two consecutive evaluations ) by edic year 17 , with 293 ( 39% ) occurring during dcct and 462 ( 61% ) during edic . higher values of hba1c , log(aer ) , mean blood pressure , and bmi were all associated with a sustained drop of 5 points in dqol score , before and after adjustment for sex , age , and mean years of education at dcct baseline ( table 2 ) . the effects of hypoglycemia , hba1c , and bmi were significant even after adjustments of other covariates including log(aer ) and mean blood pressure ( table 2 ) . association of time - dependent covariates with risk of a sustained drop of 5 points in total dqol since dcct baseline development of advanced stages of retinopathy ( p = 0.0196 ) , nephropathy ( p = 0.0019 ) , and confirmed clinical neuropathy ( p < 0.0001 ) were associated with lower total dqol scores at year 17 of edic ( an average of 23.5 years of follow - up ) , after adjusting for sex , age , and mean years of education at baseline ( table 3 ) . there was a significant effect of the development of any complications ( none vs. one or more ) ( p = 0.0009 ) , with a linear trend showing that those subjects with all three complications had the lowest dqol score of 71.6 0.7 compared with no complications ( p < 0.0001 ) ( table 3 ) . medical complications by total dqol score at edic year 17 after an average of 23.5 years of follow - up self - reported symptoms associated with these complications at year 17 were also linked to decreased dqol scores ( table 4 ) . specifically , chest pain ( p = 0.0004 ) , decreased vision in both eyes ( p = 0.0005 ) , paresthesias in hands or feet ( p < 0.0001 ) , urinary incontinence ( p = 0.0001 ) , and male erectile dysfunction ( p < 0.0001 ) were associated with lower edic year 17 dqol scores , after adjusting for sex , baseline age , baseline mean years of education , and baseline dqol score . among both men and women , increasing number of symptoms was associated with a lower dqol score ( men , p = 0.0091 ; women , p = 0.0028 ) . the 14 participants with va worse than 20/100 in the worse eye had a mean total dqol score of 68.5 7.9 compared with 74.9 10.5 , 74.6 10.7 , and 76.0 12.5 for va better or equal to 20/20 , 20/20 to 20/40 , and 20/40 to 20/100 , respectively ( p = 0.0661 ) . self - reported functional status and history of psychiatric events occurring in edic by total dqol score at edic year 17 after an average of 23.5 years of follow - up self - reported history of treated anxiety ( p < 0.0001 ) and treated depression ( p < two hundred subjects reported both anxiety and depression , and their mean adjusted quality - of - life score was 69.2 0.7 . only a small number of participants were ever hospitalized for a psychiatric illness or reported suicidal attempts ( 27 attempts in 18 individuals ) , and both were associated with decreased dqol scores ( p < 0.0001 ) . an increasing number of psychiatric events was reflected in a decreased dqol score ( p < 0.0001 ) . at the 17-year edic follow - up , after an average of 23.5 years , 66% of all dcct / edic participants had developed hypertension , 65% hypercholesterolemia , and 19% proliferative diabetic retinopathy or worse . the lower prevalence of retinopathy and nephropathy in the former intensive treatment group at edic year 17 reflects the previously published salutary effects of intensive therapy ( 19 ) . characteristics of participants who completed the dqol in edic year 17 no differences in total dqol or dqol subscale scores were found between the intensive and conventional groups at any time points . mean total dqol scores were not significantly different between the former dcct intensive and conventional treatment groups at baseline ( 78 8 vs. 78 9 ) , dcct closeout ( 78.1 8.8 vs. 78.2 9.4 ) , or edic year 17 ( 75 11 vs. 74 11 ) . there were 755 ( 52% ) dcct / edic participants who had a dqol event ( defined as a decrease in dqol from baseline of 5 points on two consecutive evaluations ) by edic year 17 , with 293 ( 39% ) occurring during dcct and 462 ( 61% ) during edic . there were no significant treatment group differences ( p = 0.9339 ) . higher values of hba1c , log(aer ) , mean blood pressure , and bmi were all associated with a sustained drop of 5 points in dqol score , before and after adjustment for sex , age , and mean years of education at dcct baseline ( table 2 ) . the effects of hypoglycemia , hba1c , and bmi were significant even after adjustments of other covariates including log(aer ) and mean blood pressure ( table 2 ) . association of time - dependent covariates with risk of a sustained drop of 5 points in total dqol since dcct baseline development of advanced stages of retinopathy ( p = 0.0196 ) , nephropathy ( p = 0.0019 ) , and confirmed clinical neuropathy ( p < 0.0001 ) were associated with lower total dqol scores at year 17 of edic ( an average of 23.5 years of follow - up ) , after adjusting for sex , age , and mean years of education at baseline ( table 3 ) . there was a significant effect of the development of any complications ( none vs. one or more ) ( p = 0.0009 ) , with a linear trend showing that those subjects with all three complications had the lowest dqol score of 71.6 0.7 compared with no complications ( p < 0.0001 ) ( table 3 ) . medical complications by total dqol score at edic year 17 after an average of 23.5 years of follow - up self - reported symptoms associated with these complications at year 17 were also linked to decreased dqol scores ( table 4 ) . specifically , chest pain ( p = 0.0004 ) , decreased vision in both eyes ( p = 0.0005 ) , paresthesias in hands or feet ( p < 0.0001 ) , urinary incontinence ( p = 0.0001 ) , and male erectile dysfunction ( p < 0.0001 ) were associated with lower edic year 17 dqol scores , after adjusting for sex , baseline age , baseline mean years of education , and baseline dqol score . among both men and women , increasing number of symptoms was associated with a lower dqol score ( men , p = 0.0091 ; women , p = 0.0028 ) . because the principal dcct end point was progressive retinal disease , the 14 participants with va worse than 20/100 in the worse eye had a mean total dqol score of 68.5 7.9 compared with 74.9 10.5 , 74.6 10.7 , and 76.0 12.5 for va better or equal to 20/20 , 20/20 to 20/40 , and 20/40 to 20/100 , respectively ( p = 0.0661 ) . self - reported functional status and history of psychiatric events occurring in edic by total dqol score at edic year 17 after an average of 23.5 years of follow - up self - reported history of treated anxiety ( p < 0.0001 ) and treated depression ( p < two hundred subjects reported both anxiety and depression , and their mean adjusted quality - of - life score was 69.2 0.7 . only a small number of participants were ever hospitalized for a psychiatric illness or reported suicidal attempts ( 27 attempts in 18 individuals ) , and both were associated with decreased dqol scores ( p < 0.0001 ) . an increasing number of psychiatric events was reflected in a decreased dqol score ( p < 0.0001 ) . we present the longest prospective follow - up study of hrqol in individuals with t1 dm and describe their course of quality - of - life experiences over 25 years . the therapy applied to the intensive treatment group during dcct and encouraged for the entire cohort during the 17 years of edic follow - up has resulted in relatively low rates of severe diabetes complications . with regard to our second research question and in contrast to our initial hrqol report based on an average of 6.5 years of treatment during dcct ( 2 ) , after an additional 17 years of follow - up , we now find an association between elevated hba1c levels and severe hypoglycemia requiring assistance and a sustained decrease in dqol scores . with regard to our third research question , the development of advanced complications and the symptoms resulting from these complications , such as decreased va , erectile dysfunction , and incontinence , were associated with decreases in hrqol . each of the three complications assessed ( retinopathy , neuropathy , and nephropathy ) were individually associated with lower dqol scores in comparison with those without the complication ; moreover , using a composite index , increasing numbers of advanced complications led to lower quality of life . it is important to note that while almost all patients developed some degree of retinopathy ( 88% prior intensive group ; 96% prior conventional group ) and 19% required laser treatment or vitreoretinal surgery in at least one eye , only 4.8% ( n = 52 ) had best corrected vision worse than 20/40 in one eye , and only 13 had best corrected vision worse than 20/100 in one eye . this relationship was seen in the association of psychiatric conditions and their treatment on lower dqol scores after an average of 23.5 years of follow - up . the current study has unique strengths compared with previous studies of t1 dm , including its long - term , consistent follow - up of a large cohort ; detailed demographic and clinical information collected prospectively using standardized methods from relatively early in the course of illness ; repeated measurement of hrqol ; the diversity of clinical outcomes ; and the wide range of ages represented during the study period ( 1365 years of age ) . the dcct / edic cohort had relatively high average socioeconomic status and education level of the group and was predominantly caucasian . although our cohort , and in particular the intensive treatment group , received an unusual level of attention and support during the 6.5 years of the dcct , during 17 of the average of 23.5 years represented by this study , the participants have been followed in typical practice settings with less support from the study . our ability to examine the effects of treatment assignment , exposure to varying levels of glycemia , including severe hypoglycemia , and the development of medical complications provide a benchmark for comparison with other research studies and with patients followed in typical clinical settings . we have constructed a quantitative picture of the impact of different interventions , levels of metabolic control , and complications on patient experiences of illness . moreover , as in our study , intensification of treatment using multiple daily injections and insulin pumps does not lead to decreased quality of life ( 1,7,11,12,13 ) . indeed , intensive treatment and the subsequent reduction of symptomatic complications helps maintain the long - term quality of life of patients with diabetes . indeed , such personal experiences are increasingly viewed as critical for patients in making their own therapeutic decisions ( 34,35 ) and engaging their clinical care team in dealing with the rigors of long - term diabetes .

one of the current evolutionary models put forward to explain antagonistic coexistence is the continual arms race between bacterial hosts and their viral parasites . in this model , mutations occur in the host that confer resistance to viruses and thus prevent infection and decimation of the host population . subsequently , mutations occur in the virus that enables it to re - infect the recently emergent resistant population . hence the coexistence of host and parasite is maintained through repeated cycles of host mutation and viral counter - mutation . under this model selection is directional with a continuous increase in the breadth of resistance and infectivity of host and parasite , respectively , resulting in generalist hosts and viruses . despite this directionality , these antagonists often remain in the same evolutionary position relative to each other in a red queen - like manner . however , it is important to point out that red queen dynamics do not necessarily lead to directional selection and therefore the terms arms race and red queen dynamics are not interchangeable . despite the wide recognition of the arms race model in the scientific community , most coevolutionary experimental studies indicate that the arms race between bacteria and viruses does not continue indefinitely , often being limited to no more than a few cycles . the first is due to genetic constraints on the phage for counter - mutation , whereby a resistant host emerges yet no subsequent phage acquires a mutation enabling it to reinfect this resistant strain . in such situations resistant bacteria would come to dominate the population with a concomitant decline in virus numbers that could even lead to viral extinction . the second factor likely to limit the arms race is related to metabolic constraints on the host that are associated with resistance . this can result , for example , if host nutrient acquisition proteins at the surface of the host also serve as viral receptors . resistance - conferring mutations in such proteins would prevent viral attachment to the cell surface , but are also likely to impair nutrient uptake and utilization by the host ( see winter et al . and refs . within ) . this metabolic limitation is a cost of resistance that is manifested as a reduction in growth rate and thus lowers fitness of the mutants , making the resistant host less competitive than previous hosts . in light of the limitations described above , an alternative evolutionary model has been suggested to explain antagonistic coexistence between bacterial hosts and viral parasites , that of density dependent fluctuating selection . in this model , mutants emerge that lead to diverse bacterial and viral populations . different to the arms race model , however , mutations do not need to be continuously produced and the emergent bacteria and viruses have different rather than greater resistance and infectivity ranges , respectively . furthermore , while a metabolic cost of resistance is considered to limit the extent of the arms race , such a cost is a basic assumption of fluctuating selection . this cost of resistance prevents competitive exclusion of susceptible hosts within which viral populations propagate . to illustrate the fluctuating nature of population dynamics in this model we will describe a simplified situation of a susceptible and a resistant host and a single parasite . as the abundance of the fast - growing susceptible host rises to dominate the population , so does the contact rate with its abundant parasite , resulting in increased host mortality and a subsequent decline in the susceptible host population . resistant mutants are unaffected by the abundant virus and will subsequently increase in numbers despite their slower growth rate . concomitantly , the abundance of the parasite decreases due to the obligatory requirement of susceptible hosts for parasite propagation . the ensuing low parasite abundance eliminates the advantage for resistant cells , and the faster growing susceptible hosts will now out - compete the resistant ones , and the cycle can begin again . in this manner neither resistant nor susceptible strains are driven to extinction , and the latter allow viral populations to also persist . thus , abundances of resistant cells , susceptible hosts and parasites oscillate in the community over time due to the negative density dependent selection driven by alternating selection pressures : viral selection for resistant mutants and competitive selection for faster growing hosts . hypothesis is an ecological model similar to that of fluctuating selection that relates to oceanic communities . similar to fluctuating selection , kill the winner assumes a metabolic cost for resistance , and is linked to resource utilization . the outcome of both models is fluctuations in population composition resulting in a diverse community . however , some other assumptions differ between the two models . for example , kill the winner assumes that a particular host can be infected by only one phage and that one phage can infect only a single host . other major differences between these models relate to the ecological vs. evolutionary perspectives used to build them . for example the kill the winner hypothesis relates to abiotic conditions , particularly nutrient availability , together with differences in uptake rates by diverse bacteria and the ensuing competition that results . these ecological and evolutionary models of fluctuating selection can not be easily reconciled with existing experimental data . first , they assume a tradeoff between viral resistance and fitness and generally relate this tradeoff to competition for resources that negatively impact the growth rate of the resistant bacterium . while resistance is often accompanied by a decrease in growth rate relative to the susceptible cells from which they were derived , it is also quite common that a growth rate cost of resistance is not detected . therefore , it is generally assumed that the conditions under which a reduction in growth rate occur were simply not found . furthermore , while mutations conferring resistance to infection are sometimes found in nutrient uptake associated genes , some are associated with lipopolysaccharide and cell - wall biosynthesis genes . such mutants are not necessarily competitively inferior in their ability to utilize resources ( unless they show a growth rate cost which would indirectly affect their capacity for resource utilization ) . additionally , even when a growth rate cost of resistance does appear , compensatory mutations can evolve and thus decrease , if not eliminate the cost . therefore the question remains : if a cell can develop resistance without paying a growth rate cost what prevents it from taking over the community ? model predicts that under viral predation the bacterial population will be highly diverse and that the dominant species in the marine pelagic system will not exceed a density threshold of ~10,000 cells per ml . this is in direct contrast to the high densities of dominant bacterial groups present in the marine environment . for example , single ecotypes of the dominant photosynthetic cyanobacterium , prochlorococcus , reach 200,000 cells per ml and that of the heterotrophic bacterium , pelagibacter , can reach 125,000 cells per ml . therefore other factors must be taken into consideration if we are to adequately explain long - term antagonistic coexistence . at the genetic level , two models of infection - resistance specificity between hosts and parasites are commonly considered . the gene - for - gene model relates to the recognition between a specific host receptor and a matching pathogen elicitor molecule that leads to host resistance . a lack of a match between these molecules leads to infection . as in the arms race however , at the population level , this would lead to the fixation of the generalist , and seeing as this does nt occur in nature , these models invoke a metabolic cost associated with generalism that would lead to fluctuating selection . the matching - alleles model assumes that an exact match between a host and parasite is needed for infection and therefore each pathogen is specific to , and can only infect , a single host . furthermore , in this model if mutations occur they cause a change in the range of resistance and infectivity , but a single parasite still only infects a single host ( similar to the assumption used in the kill the winner hypothesis ) . therefore this model does not permit universal infectivity or resistance by assuming a genetic constraint on directional selection of an arms race . such populations are controlled by fluctuating selection between different genotypes and population diversity is maintained without a metabolic cost associated with resistance . experimental data suggest that the gene - for - gene model is found in plant - pathogen systems and that matching - alleles - like interactions occur at some level in vertebrate immunity systems . these genetic models may actually be at either ends of a specificity continuum with specificity more realistically lying somewhere in the middle . while this is a purely theoretical combination of these two models , it predicts that a single parasite can both efficiently infect a specific host ( as in the matching - alleles model ) as well as infect other hosts with lower efficiency ( as in the gene - for - gene model ) in an imperfect lock and key manner . the above suggestion that genetic diversity within populations can be maintained without a metabolic cost of resistance is seldom incorporated into models of fluctuating selection ( but see refs . 4 and 22 ) and has not been shown for systems other than vertebrate immunity . below we describe our recent findings of hyperdiversity in viral susceptibility regions of cyanobacterial genomes and argue that such genetic microdiversity , together with mutations that lead to changes in infection dynamics , facilitates fluctuations at the subpopulation level , providing experimental evidence in line with the ideas outlined in agrawal and lively . we investigated bacterial resistance to viral infection using the cyanobacterium prochlorococcus and its viruses as a model system , as it exemplifies a microbe that coexists with antagonistic viruses at high abundances in the marine environment . using four ancestral laboratory strains belonging to two high - light adapted prochlorococcus ecotypes , we isolated 77 substrains , each experimentally selected for resistance to one of 10 different viruses . the genomic characterization of these resistant substrains revealed mutations in a variety of predicted cell - surface genes . only one of these 24 genes is known to encode for a protein involved in resource utilization a subunit for phosphorus uptake . the majority of mutations were in genes that appear to be involved in lipopolysaccharide or cell wall biosynthesis and modification or are of unknown function . the resistance conferring mutations were in strain specific genes that localized primarily to a single hypervariable region of the genome , known as genomic island 4 . this region is highly variable in terms of gene content and gene sequence diversity when comparing among strains from the same ecotype . this is in stark contrast to the high degree of gene synteny and gene sequence identity in most other regions of the genome . the strain specific nature of the genes in this genomic region , suggests that viral selection has promoted the loss of susceptibility genes accompanied by the lateral gain of new genes with similar cell - surface functions , but with different viral recognition determinants . the high diversity of this genome region in natural populations of prochlorococcus suggests that it is constantly losing and gaining genes and accumulating other mutations . therefore this genome region can be considered a viral susceptibility region that is prone to enhanced diversification , that has developed over long evolutionary scales , and that is continuing to develop today in response to viral selection pressure . the hypervariability resulting from this viral selection pressure has given rise to prochlorococcus populations in the oceans that are composed of various subpopulations with diverse viral susceptibility regions . since each subpopulation is susceptible to a different set of viruses no single viral type can cause the collapse of the entire prochlorococcus population . we suggest that fluctuating selection acts at this subpopulation level facilitating long - term host - virus coexistence despite high densities of the overall host population . metagenomic analyses revealed genomic islands enriched with uncommon cell - surface genes in other marine bacterial and archaeal genomes . thus this genome mechanism of diversification of viral susceptibility regions , both through nucleotide polymorphisms as well as loss and gain of cell - surface genes , is potentially widespread among a variety of host - virus systems . therefore , when addressing antagonistic host - virus coexistence in abundant populations in the environment , the microdiversity in viral susceptibility regions of these populations needs to betaken into consideration . indeed this microdiversity has been addressed in recent discussions of subpopulation oscillations in aquatic environments . the challenge now will be to determine what constitutes a discrete subpopulation and whether these can be discerned from the standpoint of host - virus interactions . many of the mutants in our study exhibited a reduced growth rate , including those with a mutation in the phosphorus uptake gene and others with mutations in genes involved in lipopolysaccharide and cell wall biosynthesis and modification . however , as discussed above , many other mutants displayed no detectable growth rate cost associated with resistance . we found that a number of these strains exhibited a novel type of cost of resistance , that of enhanced susceptibility to other viruses . in these strains , the exact same mutations that conferred resistance to one set of viruses brought about enhanced infection dynamics by other viruses . in at least two cases this was due to more rapid adsorption by these other viruses to the resistant mutant relative to the ancestral strain . additional situations were also observed , whereby resistant strains were infected more slowly by other viruses . moreover , studies by others have shown that mutations that conferred resistance to one virus enabled infection by another virus that was incapable of infecting the ancestral bacterium from which the resistant strain was derived . therefore , a bacterial mutation can be under varying levels of selection pressures due to different degrees of infectivity by distinct viruses , including those belonging to different viral families . these findings indicate that infection is not a binary off or on trait , but rather that infectivity can be considered a continuum that ranges from complete resistance , through varying infection efficiencies , to highly infective . this was also suggested by holmfeldt et al . who showed drastic differences in infection efficiency by the same virus for different strains of flavobacteria isolated from the marine environment . based on these findings , we propose that the enhanced infection cost of resistance can serve as the tradeoff between viral resistance and fitness and drive fluctuating selection . this novel cost of resistance can , therefore , explain antagonistic coexistence , even when there is no metabolic cost associated with it . in this scenario , oscillations in population sizes are still density dependent , but the selective force is always viral pressure , alternating between pressure from one virus and pressure from another virus rather than between viral pressure and competitive selection . however , host resistance or susceptibility and the degree of that susceptibility in any given interaction , will vary depending on the virus encountered . the continuum of infection efficiencies resulting from enhanced infection , that would lead to the generation of fluctuating selection between diverse genotypes without the requirement for a metabolic cost of resistance , provides empirical evidence for the combined theoretical model proposed by agrawal and lively ( described above ) . it should be noted that fluctuations between host subpopulations can be driven by viruses even when new mutations conferring resistance have neither type of cost , as long as a particular bacterium can be infected by more than one virus and that each resistance mutation affects the ability of only one of them to infect the host . however , these fluctuations will be much more common and considerably more pronounced when resistance is accompanied with the enhanced infection cost ( fig . 1 ) . this is because mutation - induced enhanced infection and susceptibility to a changing set of viruses dramatically increases the probability for infection relative to mutations that confer resistance alone . this is due both to a greater chance for the mutant to meet infectious viruses ( fig . 2 ) and the rapidity of the ensuing infection once these viruses are encountered . furthermore , enhanced infection would prevent the development of generalist hosts with a wide range of resistance to co - occurring viruses , and would thus preclude the directional selection expected from a continuous arms race ( fig . 2 ) . enhanced infection can , however , support red queen dynamics , in which mutations serve to enhance diversity within host and virus populations without any directionality of selection . schematic representation of bacterial population dynamics with and without enhanced infection . in this simplified depiction , the ancestral host is susceptible to viruses a , b and d each of which infect the host with equal efficiency . this ancestral host is not initially susceptible to virus c. in the absence of enhanced infection , declines in the abundance of the host ( red line ) result from contact with viruses a , b and d but not with virus c and reduce the population to the same abundance . with the existence of enhanced infection , declines in the abundance of the host ( blue line ) result from interactions with all 4 viruses and are to different lower bounds . in this latter scenario , the mutation conferring resistance to virus a increased the infection efficiency of virus b for the emergent resistant strain , and the mutation conferring resistance to virus a or b made it susceptible to virus c. this depiction assumes that there is no growth rate cost to mutations and no virus counter - mutations occur . enhanced infection leads to shifts in the phages exerting population control . in a given environment a host population ( large oval shape ) encounters diverse viral types ( small geometric symbols ) with different capacities for infecting the host . non - infective viruses are positioned at the top corner , viruses with high infectivity ( rapid ) are at the bottom left corner and viruses with low infectivity ( slow ) are at the bottom right corner of each infectivity triangle . over time the host acquires mutations that change the ability of viruses to infect it , thus changing their position in the infectivity triangle . to aid traceability the color of the viruses represents their initial capacity for infection in the t0 triangle while their position at subsequent time points ( t1 and t2 ) represents their current capacity for infection . in the left panel host mutations conferring resistance to viruses are not associated with enhanced infection by other viruses and therefore viral shifts in infection capacity are unidirectional . in this case , the accumulation of resistance mutations leads to a generalist bacterium that , in the absence of a metabolic cost of resistance or a counter - mutation in the virus , would come to dominate the population . in the right panel host mutations conferring resistance to viruses are associated with enhanced infection leading to bidirectional changes in infection capabilities by other viruses . in this case , the accumulation of resistance mutations results in shifts in the viruses capable of infecting the host rather than a reduction in the number of viruses that can infect it . therefore enhanced infection prevents a single bacterial strain from taking over the population by continuous shifts in the virus exerting control , and thus helps maintain bacterial diversity . therefore resistance costs , whether caused by a metabolic or enhanced infection cost , can drive fluctuating selection . while metabolic costs are linked to environmental conditions through competition for resource availability , the enhanced infection cost is manifested in the context of the community . therefore , fluctuating selection will be driven by enhanced infection costs in both resource deplete and replete conditions . as such , enhanced infection is likely to be an important driver of fluctuating population dynamics facilitating long - term host - parasite coexistence in nature and should be taken into consideration in future ecological models of fluctuating selection . let us now consider the matter from the parasite 's perspective . as discussed above , bacterial microdiversity in genomic regions responsible for viral susceptibility this raises the question of what enables persistence of viruses if only a small fraction of the potential host population is actually available for infection ? this is especially acute both because viruses constantly lose some hosts due to mutations that provide resistance to it , and because of the low rate of counter - mutation for the reinfection of these hosts . one possible explanation is mutations in viruses that enable them to switch hosts after the original host gained resistance . tail fiber diversification due to point mutations and domain swapping , are well known phenomena that can lead to a change in host - range for the phage . another possibility is that enhanced infection supplies a virus with new infection opportunities without any change occurring in the virus . indeed , the same resistance mutation in a host that caused a narrowing of host range for one virus can sometimes provide other viruses with new infection capabilities . taken from the perspective of a single virus , a virus that constantly loses some hosts due to resistance , profits from interactions between other host - virus pairs : resistance mutations and selection for resistance in these other interactions , can provide the first virus with greater infectivity for some hosts and even afford it with additional hosts ( fig . in this light we should consider the host range of viruses as a dynamic set of bacterial strains , as its host range includes bacteria which are just one mutation away from serving as hosts ( fig . initially ( a ) two virus - host pairs ( v1-h1 and v2-h2 ) are present in a common environment , but do not cross infect each other . following a mutation , h1 gains resistance to v1 giving rise to h1 ( b ) . in this scenario thus the population size of h1 will increase while that of h1 will decline due to viral infection ( depicted by a smaller circle ) . the survival of v1 now depends on its ability to gain a new host . to keep up in an arms race ( c ) a counter - mutation in v1 gives rise to a host range mutant ( v1 ) which can reinfect h1 , thereby enabling the recoupling of the virus - host pair . alternatively , passive host switching occurs through enhanced infection ( d ) , whereby v1 is provided with a new host ( h2 ) due to a mutation in h2 that conferred resistance to , and was selected by , another virus , v2 ( d ) . thus an independent interaction between h2 and v2 provided the v1 population with another host without any mutation in v1 . in summation , the arms race and enhanced infection lead to a dynamic host range for v1 . despite the fact that this virus lost its immediate host ( h1 ) through a resistance mutation these bacterial types are only one mutation away from serving as its immediate hosts , as a single mutation in these bacteria or in v1 can lead to productive infection . we propose that due to this constant fresh serving of potential hosts , the virus can sustain its population at times when the abundance of its initial host is extremely low and the chance of contact is minimal . this continuous , passive supply of new hosts is thus likely to compensate at least partially for the asymmetry in counter - mutation in phages . this would enable viral persistence in the face of changes in host availability and abundance that result from mutation induced resistance and fluctuating host dynamics , respectively . in summary , natural communities are complex systems whose members are constantly interacting with each other . the diversity of the players in this system are the outcome of millions of years of large - scale coevolution between viruses and their various hosts , during which host mutations have led to hypervariability in gene sequence and gene content in viral susceptibility regions of microbial genomes . enhanced infection can serve as a driver of fluctuating selection among these diverse subpopulations which would ultimately prevent any single subpopulation from taking over the entire population . enhanced infection also leads to passive host - switching in viruses that provides a constant supply of alternative hosts to the virus . this is especially important for viral persistence during periods of low abundances , and thus low contact rates , with preexisting susceptible hosts and likely compensates for the inherent asymmetry in the degree of phage counter - mutations . moreover , this passive switching is also expected to prevent arms race - like super - resistant hosts from developing . these features of the system illustrate the importance of the biotic context in which an organism lives . simple experimental set - ups that are detached from this biotic complexity neglect this aspect of antagonistic coexistence . therefore , efforts are needed to develop meaningful experimental systems and models that take this complexity into account .

bacteria and their viruses ( phages ) are antagonists , yet have coexisted in nature for billions of years . models proposed to explain the paradox of antagonistic coexistence generally reach two types of solutions : arms race - like dynamics that lead to hosts and viruses with increasing resistance and infection ranges ; and population fluctuations between diverse host and viral types due to a metabolic cost of resistance . recently , we found that populations of the marine cyanobacterium , prochlorococcus , consist of cells with extreme hypervariability in gene sequence and gene content in a viral susceptibility region of the genome . furthermore , we found a novel cost of resistance where resistance to one set of viruses is accompanied by changes in infection dynamics by other viruses . in this combined mini - review and commentary paper we discuss these findings in the context of existing ecological , evolutionary and genetic models of host - virus coexistence . we suggest that this coexistence is governed mainly by fluctuations between microbial subpopulations with differing viral susceptibility regions and that these fluctuations are driven by both metabolic and enhanced infection costs of resistance . furthermore , we suggest that enhanced infection leads to passive host - switching by viruses , preventing the development of hosts with universal resistance . these findings highlight the vital importance of community complexity for host - virus coexistence .

The Arms Race Fluctuating Selection Kill the Winner Genetic Models of Host-Parasite Coevolution Hyperdiversity in Viral Susceptibility Regions Enhanced Infection, a Novel Cost of Resistance Enhanced Infection Leads to Passive Host-Switching in Viruses Summary

one of the current evolutionary models put forward to explain antagonistic coexistence is the continual arms race between bacterial hosts and their viral parasites . in this model , mutations occur in the host that confer resistance to viruses and thus prevent infection and decimation of the host population . subsequently , mutations occur in the virus that enables it to re - infect the recently emergent resistant population . hence the coexistence of host and parasite is maintained through repeated cycles of host mutation and viral counter - mutation . under this model selection is directional with a continuous increase in the breadth of resistance and infectivity of host and parasite , respectively , resulting in generalist hosts and viruses . despite this directionality , these antagonists often remain in the same evolutionary position relative to each other in a red queen - like manner . however , it is important to point out that red queen dynamics do not necessarily lead to directional selection and therefore the terms arms race and red queen dynamics are not interchangeable . despite the wide recognition of the arms race model in the scientific community , most coevolutionary experimental studies indicate that the arms race between bacteria and viruses does not continue indefinitely , often being limited to no more than a few cycles . the first is due to genetic constraints on the phage for counter - mutation , whereby a resistant host emerges yet no subsequent phage acquires a mutation enabling it to reinfect this resistant strain . in such situations resistant bacteria would come to dominate the population with a concomitant decline in virus numbers that could even lead to viral extinction . the second factor likely to limit the arms race is related to metabolic constraints on the host that are associated with resistance . this can result , for example , if host nutrient acquisition proteins at the surface of the host also serve as viral receptors . this metabolic limitation is a cost of resistance that is manifested as a reduction in growth rate and thus lowers fitness of the mutants , making the resistant host less competitive than previous hosts . in light of the limitations described above , an alternative evolutionary model has been suggested to explain antagonistic coexistence between bacterial hosts and viral parasites , that of density dependent fluctuating selection . in this model , mutants emerge that lead to diverse bacterial and viral populations . different to the arms race model , however , mutations do not need to be continuously produced and the emergent bacteria and viruses have different rather than greater resistance and infectivity ranges , respectively . furthermore , while a metabolic cost of resistance is considered to limit the extent of the arms race , such a cost is a basic assumption of fluctuating selection . this cost of resistance prevents competitive exclusion of susceptible hosts within which viral populations propagate . to illustrate the fluctuating nature of population dynamics in this model we will describe a simplified situation of a susceptible and a resistant host and a single parasite . as the abundance of the fast - growing susceptible host rises to dominate the population , so does the contact rate with its abundant parasite , resulting in increased host mortality and a subsequent decline in the susceptible host population . concomitantly , the abundance of the parasite decreases due to the obligatory requirement of susceptible hosts for parasite propagation . in this manner neither resistant nor susceptible strains are driven to extinction , and the latter allow viral populations to also persist . thus , abundances of resistant cells , susceptible hosts and parasites oscillate in the community over time due to the negative density dependent selection driven by alternating selection pressures : viral selection for resistant mutants and competitive selection for faster growing hosts . similar to fluctuating selection , kill the winner assumes a metabolic cost for resistance , and is linked to resource utilization . the outcome of both models is fluctuations in population composition resulting in a diverse community . for example , kill the winner assumes that a particular host can be infected by only one phage and that one phage can infect only a single host . these ecological and evolutionary models of fluctuating selection can not be easily reconciled with existing experimental data . first , they assume a tradeoff between viral resistance and fitness and generally relate this tradeoff to competition for resources that negatively impact the growth rate of the resistant bacterium . while resistance is often accompanied by a decrease in growth rate relative to the susceptible cells from which they were derived , it is also quite common that a growth rate cost of resistance is not detected . furthermore , while mutations conferring resistance to infection are sometimes found in nutrient uptake associated genes , some are associated with lipopolysaccharide and cell - wall biosynthesis genes . additionally , even when a growth rate cost of resistance does appear , compensatory mutations can evolve and thus decrease , if not eliminate the cost . model predicts that under viral predation the bacterial population will be highly diverse and that the dominant species in the marine pelagic system will not exceed a density threshold of ~10,000 cells per ml . this is in direct contrast to the high densities of dominant bacterial groups present in the marine environment . for example , single ecotypes of the dominant photosynthetic cyanobacterium , prochlorococcus , reach 200,000 cells per ml and that of the heterotrophic bacterium , pelagibacter , can reach 125,000 cells per ml . therefore other factors must be taken into consideration if we are to adequately explain long - term antagonistic coexistence . at the genetic level , two models of infection - resistance specificity between hosts and parasites are commonly considered . the gene - for - gene model relates to the recognition between a specific host receptor and a matching pathogen elicitor molecule that leads to host resistance . as in the arms race however , at the population level , this would lead to the fixation of the generalist , and seeing as this does nt occur in nature , these models invoke a metabolic cost associated with generalism that would lead to fluctuating selection . the matching - alleles model assumes that an exact match between a host and parasite is needed for infection and therefore each pathogen is specific to , and can only infect , a single host . furthermore , in this model if mutations occur they cause a change in the range of resistance and infectivity , but a single parasite still only infects a single host ( similar to the assumption used in the kill the winner hypothesis ) . therefore this model does not permit universal infectivity or resistance by assuming a genetic constraint on directional selection of an arms race . such populations are controlled by fluctuating selection between different genotypes and population diversity is maintained without a metabolic cost associated with resistance . experimental data suggest that the gene - for - gene model is found in plant - pathogen systems and that matching - alleles - like interactions occur at some level in vertebrate immunity systems . these genetic models may actually be at either ends of a specificity continuum with specificity more realistically lying somewhere in the middle . while this is a purely theoretical combination of these two models , it predicts that a single parasite can both efficiently infect a specific host ( as in the matching - alleles model ) as well as infect other hosts with lower efficiency ( as in the gene - for - gene model ) in an imperfect lock and key manner . the above suggestion that genetic diversity within populations can be maintained without a metabolic cost of resistance is seldom incorporated into models of fluctuating selection ( but see refs . below we describe our recent findings of hyperdiversity in viral susceptibility regions of cyanobacterial genomes and argue that such genetic microdiversity , together with mutations that lead to changes in infection dynamics , facilitates fluctuations at the subpopulation level , providing experimental evidence in line with the ideas outlined in agrawal and lively . we investigated bacterial resistance to viral infection using the cyanobacterium prochlorococcus and its viruses as a model system , as it exemplifies a microbe that coexists with antagonistic viruses at high abundances in the marine environment . using four ancestral laboratory strains belonging to two high - light adapted prochlorococcus ecotypes , we isolated 77 substrains , each experimentally selected for resistance to one of 10 different viruses . the genomic characterization of these resistant substrains revealed mutations in a variety of predicted cell - surface genes . the resistance conferring mutations were in strain specific genes that localized primarily to a single hypervariable region of the genome , known as genomic island 4 . this region is highly variable in terms of gene content and gene sequence diversity when comparing among strains from the same ecotype . this is in stark contrast to the high degree of gene synteny and gene sequence identity in most other regions of the genome . the strain specific nature of the genes in this genomic region , suggests that viral selection has promoted the loss of susceptibility genes accompanied by the lateral gain of new genes with similar cell - surface functions , but with different viral recognition determinants . the high diversity of this genome region in natural populations of prochlorococcus suggests that it is constantly losing and gaining genes and accumulating other mutations . therefore this genome region can be considered a viral susceptibility region that is prone to enhanced diversification , that has developed over long evolutionary scales , and that is continuing to develop today in response to viral selection pressure . the hypervariability resulting from this viral selection pressure has given rise to prochlorococcus populations in the oceans that are composed of various subpopulations with diverse viral susceptibility regions . since each subpopulation is susceptible to a different set of viruses no single viral type can cause the collapse of the entire prochlorococcus population . we suggest that fluctuating selection acts at this subpopulation level facilitating long - term host - virus coexistence despite high densities of the overall host population . thus this genome mechanism of diversification of viral susceptibility regions , both through nucleotide polymorphisms as well as loss and gain of cell - surface genes , is potentially widespread among a variety of host - virus systems . therefore , when addressing antagonistic host - virus coexistence in abundant populations in the environment , the microdiversity in viral susceptibility regions of these populations needs to betaken into consideration . the challenge now will be to determine what constitutes a discrete subpopulation and whether these can be discerned from the standpoint of host - virus interactions . many of the mutants in our study exhibited a reduced growth rate , including those with a mutation in the phosphorus uptake gene and others with mutations in genes involved in lipopolysaccharide and cell wall biosynthesis and modification . we found that a number of these strains exhibited a novel type of cost of resistance , that of enhanced susceptibility to other viruses . in these strains , the exact same mutations that conferred resistance to one set of viruses brought about enhanced infection dynamics by other viruses . in at least two cases this was due to more rapid adsorption by these other viruses to the resistant mutant relative to the ancestral strain . additional situations were also observed , whereby resistant strains were infected more slowly by other viruses . moreover , studies by others have shown that mutations that conferred resistance to one virus enabled infection by another virus that was incapable of infecting the ancestral bacterium from which the resistant strain was derived . therefore , a bacterial mutation can be under varying levels of selection pressures due to different degrees of infectivity by distinct viruses , including those belonging to different viral families . these findings indicate that infection is not a binary off or on trait , but rather that infectivity can be considered a continuum that ranges from complete resistance , through varying infection efficiencies , to highly infective . who showed drastic differences in infection efficiency by the same virus for different strains of flavobacteria isolated from the marine environment . based on these findings , we propose that the enhanced infection cost of resistance can serve as the tradeoff between viral resistance and fitness and drive fluctuating selection . this novel cost of resistance can , therefore , explain antagonistic coexistence , even when there is no metabolic cost associated with it . the continuum of infection efficiencies resulting from enhanced infection , that would lead to the generation of fluctuating selection between diverse genotypes without the requirement for a metabolic cost of resistance , provides empirical evidence for the combined theoretical model proposed by agrawal and lively ( described above ) . it should be noted that fluctuations between host subpopulations can be driven by viruses even when new mutations conferring resistance have neither type of cost , as long as a particular bacterium can be infected by more than one virus and that each resistance mutation affects the ability of only one of them to infect the host . however , these fluctuations will be much more common and considerably more pronounced when resistance is accompanied with the enhanced infection cost ( fig . this is because mutation - induced enhanced infection and susceptibility to a changing set of viruses dramatically increases the probability for infection relative to mutations that confer resistance alone . this is due both to a greater chance for the mutant to meet infectious viruses ( fig . 2 ) and the rapidity of the ensuing infection once these viruses are encountered . furthermore , enhanced infection would prevent the development of generalist hosts with a wide range of resistance to co - occurring viruses , and would thus preclude the directional selection expected from a continuous arms race ( fig . enhanced infection can , however , support red queen dynamics , in which mutations serve to enhance diversity within host and virus populations without any directionality of selection . schematic representation of bacterial population dynamics with and without enhanced infection . in this simplified depiction , the ancestral host is susceptible to viruses a , b and d each of which infect the host with equal efficiency . this ancestral host is not initially susceptible to virus c. in the absence of enhanced infection , declines in the abundance of the host ( red line ) result from contact with viruses a , b and d but not with virus c and reduce the population to the same abundance . with the existence of enhanced infection , declines in the abundance of the host ( blue line ) result from interactions with all 4 viruses and are to different lower bounds . in this latter scenario , the mutation conferring resistance to virus a increased the infection efficiency of virus b for the emergent resistant strain , and the mutation conferring resistance to virus a or b made it susceptible to virus c. this depiction assumes that there is no growth rate cost to mutations and no virus counter - mutations occur . enhanced infection leads to shifts in the phages exerting population control . in a given environment a host population ( large oval shape ) encounters diverse viral types ( small geometric symbols ) with different capacities for infecting the host . non - infective viruses are positioned at the top corner , viruses with high infectivity ( rapid ) are at the bottom left corner and viruses with low infectivity ( slow ) are at the bottom right corner of each infectivity triangle . over time the host acquires mutations that change the ability of viruses to infect it , thus changing their position in the infectivity triangle . to aid traceability the color of the viruses represents their initial capacity for infection in the t0 triangle while their position at subsequent time points ( t1 and t2 ) represents their current capacity for infection . in the left panel host mutations conferring resistance to viruses are not associated with enhanced infection by other viruses and therefore viral shifts in infection capacity are unidirectional . in this case , the accumulation of resistance mutations leads to a generalist bacterium that , in the absence of a metabolic cost of resistance or a counter - mutation in the virus , would come to dominate the population . in the right panel host mutations conferring resistance to viruses are associated with enhanced infection leading to bidirectional changes in infection capabilities by other viruses . in this case , the accumulation of resistance mutations results in shifts in the viruses capable of infecting the host rather than a reduction in the number of viruses that can infect it . therefore enhanced infection prevents a single bacterial strain from taking over the population by continuous shifts in the virus exerting control , and thus helps maintain bacterial diversity . therefore resistance costs , whether caused by a metabolic or enhanced infection cost , can drive fluctuating selection . while metabolic costs are linked to environmental conditions through competition for resource availability , the enhanced infection cost is manifested in the context of the community . therefore , fluctuating selection will be driven by enhanced infection costs in both resource deplete and replete conditions . as such , enhanced infection is likely to be an important driver of fluctuating population dynamics facilitating long - term host - parasite coexistence in nature and should be taken into consideration in future ecological models of fluctuating selection . as discussed above , bacterial microdiversity in genomic regions responsible for viral susceptibility this raises the question of what enables persistence of viruses if only a small fraction of the potential host population is actually available for infection ? this is especially acute both because viruses constantly lose some hosts due to mutations that provide resistance to it , and because of the low rate of counter - mutation for the reinfection of these hosts . tail fiber diversification due to point mutations and domain swapping , are well known phenomena that can lead to a change in host - range for the phage . another possibility is that enhanced infection supplies a virus with new infection opportunities without any change occurring in the virus . indeed , the same resistance mutation in a host that caused a narrowing of host range for one virus can sometimes provide other viruses with new infection capabilities . taken from the perspective of a single virus , a virus that constantly loses some hosts due to resistance , profits from interactions between other host - virus pairs : resistance mutations and selection for resistance in these other interactions , can provide the first virus with greater infectivity for some hosts and even afford it with additional hosts ( fig . in this light we should consider the host range of viruses as a dynamic set of bacterial strains , as its host range includes bacteria which are just one mutation away from serving as hosts ( fig . initially ( a ) two virus - host pairs ( v1-h1 and v2-h2 ) are present in a common environment , but do not cross infect each other . following a mutation , h1 gains resistance to v1 giving rise to h1 ( b ) . in this scenario thus the population size of h1 will increase while that of h1 will decline due to viral infection ( depicted by a smaller circle ) . to keep up in an arms race ( c ) a counter - mutation in v1 gives rise to a host range mutant ( v1 ) which can reinfect h1 , thereby enabling the recoupling of the virus - host pair . alternatively , passive host switching occurs through enhanced infection ( d ) , whereby v1 is provided with a new host ( h2 ) due to a mutation in h2 that conferred resistance to , and was selected by , another virus , v2 ( d ) . in summation , the arms race and enhanced infection lead to a dynamic host range for v1 . despite the fact that this virus lost its immediate host ( h1 ) through a resistance mutation these bacterial types are only one mutation away from serving as its immediate hosts , as a single mutation in these bacteria or in v1 can lead to productive infection . we propose that due to this constant fresh serving of potential hosts , the virus can sustain its population at times when the abundance of its initial host is extremely low and the chance of contact is minimal . this would enable viral persistence in the face of changes in host availability and abundance that result from mutation induced resistance and fluctuating host dynamics , respectively . the diversity of the players in this system are the outcome of millions of years of large - scale coevolution between viruses and their various hosts , during which host mutations have led to hypervariability in gene sequence and gene content in viral susceptibility regions of microbial genomes . enhanced infection can serve as a driver of fluctuating selection among these diverse subpopulations which would ultimately prevent any single subpopulation from taking over the entire population . enhanced infection also leads to passive host - switching in viruses that provides a constant supply of alternative hosts to the virus . this is especially important for viral persistence during periods of low abundances , and thus low contact rates , with preexisting susceptible hosts and likely compensates for the inherent asymmetry in the degree of phage counter - mutations . moreover , this passive switching is also expected to prevent arms race - like super - resistant hosts from developing . these features of the system illustrate the importance of the biotic context in which an organism lives . simple experimental set - ups that are detached from this biotic complexity neglect this aspect of antagonistic coexistence .

human immunodeficiency virus ( hiv ) and tuberculosis ( tb ) co - infection constitutes a high - burden disease in thailand , where 12,890 individuals died from tb in 2009 and where hiv accounts for 17% of tb cases , the highest figure in the south - east asian region.1 early case identification followed by adequate treatment is the cornerstone of tb control programs . thailand s rank of 18th among 22 high - burden countries for tb is partly attributed to inadequate case evaluation and treatment default , which also result in increased deaths and contribute to underperformance on the world health organization ( who ) target for treatment success ( 81% versus 85%).1 efforts to improve tb diagnosis by new technology have commonly faced the challenges of high equipment cost , lack of skilled technicians , and excessive turnaround time.2 rapid immunochromatographic test ( ict ) technology may be the exception to this rule : when used as a serodiagnostic test for tb antibodies ( ict - tb ) , this technology is affordable , simple and safe to use , and produces results within 25 minutes.3 however , in an hiv - infected population , several antigens may be recognized by the antibodies , thereby limiting the diagnostic validity of such tests.4 previous studies have reported a wide range of sensitivity of ict - tb tests in hiv patients ( from 0% to 80% ) ; specificity was also very variable , ranging from 39% to 97.4%.48 a landmark study , conducted by the who special programme for research and training in tropical diseases in 2008 , showed poor performance of 19 commercial rapid diagnostic tests in hiv patients , with sensitivity reduced by an average of 22%43% compared with that in hiv - negative patients.9 in thai settings , only two diagnostic validity studies , on the use of lipoarabinomannan and 38-kd recombinant antigens using pleural effusion and serum , have been reported.4,5 in an intensified effort to find cases of active tb in thailand , a general screening tool , which disregards hiv status , is currently being used ; this tool consists of simple questions relating to signs and symptoms of tb . from a recent review , the best predictors for tb diagnosis in hiv patients living in resource - constrained settings are presence of cough ( for any duration ) , fever , night sweats , or weight loss.10 other signs , symptoms , and simple laboratory tests proposed by other studies are fatigue,11,12 shaking chills,12 loss of appetite,12 difficulty in breathing,12 chest pain,12 abdominal pain,12 nausea / vomiting,12 body mass index ( bmi),11,12 lymphadenopathy,11,12 hemoglobin level of < 10 g / dl,12 tuberculin skin test positivity,12 hospitalization upon registration,12 lack of antiretroviral ( arv ) therapy,11,12 chest radiograph abnormalities,11,13,14 and cd4 cell count.11,14 however , the proposed combinations of predictors , as well as the performance of such predictor combinations , varies between studies . the validity of such existing screening tools is unknown in the setting of thailand ; furthermore , the value of an ict - tb test used in combination with clinical predictors has yet to be evaluated in thailand . therefore , the objective of this study was to identify clinical characteristics , including demographics , signs , and symptoms that may be used to predict the presence of active tb , as well as to evaluate the diagnostic value of ict - tb testing over such clinical predictors in subgroups of hiv patients stratified by clinical risk scores . the study protocol was approved by two institutional review boards : the faculty of medicine , chiang mai university and lampang regional hospital . all hiv patients aged 18 years or older seeking medical care or reporting for annual checkup at either a tb clinic or an arv clinic between april 2009 and may 2010 , regardless of tb signs and symptoms , were eligible for the study . outpatient data , including clinical signs and symptoms , and blood samples were collected on the first day . study settings included maesai district hospital , a community hospital in chiang rai , and lampang hospital , a general hospital in lampang , thailand . patients who were receiving tb treatment or isoniazid prophylaxis treatment ( ipt ) at the time of or within 1 year prior to recruitment were excluded . the attending physician or trained nursing staff interviewed participants and recorded data in a case record form . data collected included patient demographics and clinical data , comprising medical history , history of tb diagnosis , history of tb contact , and arv prescription . data regarding duration of signs and symptoms were also collected ; these included cough , cough with sputum , hemoptysis , fever , diarrhea , weight loss , night sweats , dyspnea , chest pain , nausea or vomiting , abdominal pain , loss of appetite , fatigue , shaking chills , and size of lymphadenopathy . laboratory results at recruitment , such as abnormal chest radiograph , complete blood count , and cd4 cell count , were also collected . as the index test , a locally produced ict - tb test containing a membrane strip pre - coated with colloid gold - conjugated recombinant microbacterium tuberculosis antigens ( 38 , 16 , and 6 kd ) was used . in this test , the patient s sample migrates chromatographically along the membrane to the test region , where a visible line forms if an antigen - antibody - antigen - gold particle complex forms ( positive result ) . the absence of a visible color band within the test window indicates a negative result . the test was interpreted as invalid if the reference and the control bands did not appear.15 blood serum specimens , approximately 100 l , were obtained on the day of recruitment by research staff who had received training in the implementation of the ict - tb test . all patients were tested with the ict - tb on the day of enrolment or not later than 3 days after enrolment . results were interpreted within 20 minutes by three interpreters blinded to each other s findings and to the patient s clinical information . when at least two readers indicated a positive result , the result was recorded as positive . criteria for a definitive tb diagnosis were defined as smear positivity , positive culture of sputum or other specimens , positive clinical and radiological evaluation , response to tb treatment , or histological characteristics indicative of tb in biopsy specimens , as is the standard clinical practice . only for registered tb cases , sputum or other specimens first , predictors with a p - value of < 0.05 were selected by univariate logistic regression . second , using multivariate logistic regression with a forward method of model selection,16 predictors of tb in the final model were selected not only from statistical significance but also from clinical significance . models with / without the ict - tb test were compared by adding the ict - tb test results into the clinical predictor model . to assess whether the model prediction was improved by the ict - tb , two methods were used : the area under receiver operating characteristic curve ( auroc ) method , and the net reclassification improvement ( nri ) method.17 models with and without ict - tb were validated and assigned classical diagnostic values . for the scoring scheme , each variable from the final model was weighted by its coefficient and transformed to an item score . the cutoff points of total scores , determined by statistical significance and the yield of prediction , were used to classify hiv patients into the low- or high - risk groups . the positive predictive value ( ppv ) of the ict - tb test for each category was compared with the prior probability ( prevalence ) by the fisher s exact probability test . the number of patients required in the study was estimated based on previous information of an odds ratio of 9.6 and 16.9% prevalence in the non - tb group.15 thus , to achieve 90% power and a significant finding at alpha = 0.05 , 18 active tb patients were required . given the prevalence of active tb ( 17% ) in the study population,18 106 hiv patients were needed ; in order to compensate for 20% missing data , the final minimum number of hiv patients needed was 128 . the study protocol was approved by two institutional review boards : the faculty of medicine , chiang mai university and lampang regional hospital . all hiv patients aged 18 years or older seeking medical care or reporting for annual checkup at either a tb clinic or an arv clinic between april 2009 and may 2010 , regardless of tb signs and symptoms , were eligible for the study . outpatient data , including clinical signs and symptoms , and blood samples were collected on the first day . study settings included maesai district hospital , a community hospital in chiang rai , and lampang hospital , a general hospital in lampang , thailand . patients who were receiving tb treatment or isoniazid prophylaxis treatment ( ipt ) at the time of or within 1 year prior to recruitment were excluded . the attending physician or trained nursing staff interviewed participants and recorded data in a case record form . data collected included patient demographics and clinical data , comprising medical history , history of tb diagnosis , history of tb contact , and arv prescription . data regarding duration of signs and symptoms were also collected ; these included cough , cough with sputum , hemoptysis , fever , diarrhea , weight loss , night sweats , dyspnea , chest pain , nausea or vomiting , abdominal pain , loss of appetite , fatigue , shaking chills , and size of lymphadenopathy . laboratory results at recruitment , such as abnormal chest radiograph , complete blood count , and cd4 cell count , were also collected . as the index test , a locally produced ict - tb test containing a membrane strip pre - coated with colloid gold - conjugated recombinant microbacterium tuberculosis antigens ( 38 , 16 , and 6 kd ) was used . in this test , the patient s sample migrates chromatographically along the membrane to the test region , where a visible line forms if an antigen - antibody - antigen - gold particle complex forms ( positive result ) . the absence of a visible color band within the test window indicates a negative result . the test was interpreted as invalid if the reference and the control bands did not appear.15 blood serum specimens , approximately 100 l , were obtained on the day of recruitment by research staff who had received training in the implementation of the ict - tb test . all patients were tested with the ict - tb on the day of enrolment or not later than 3 days after enrolment . results were interpreted within 20 minutes by three interpreters blinded to each other s findings and to the patient s clinical information . when at least two readers indicated a positive result , the result was recorded as positive . criteria for a definitive tb diagnosis were defined as smear positivity , positive culture of sputum or other specimens , positive clinical and radiological evaluation , response to tb treatment , or histological characteristics indicative of tb in biopsy specimens , as is the standard clinical practice . only for registered tb cases , sputum or other specimens as the index test , a locally produced ict - tb test containing a membrane strip pre - coated with colloid gold - conjugated recombinant microbacterium tuberculosis antigens ( 38 , 16 , and 6 kd ) was used . in this test , the patient s sample migrates chromatographically along the membrane to the test region , where a visible line forms if an antigen - antibody - antigen - gold particle complex forms ( positive result ) . the absence of a visible color band within the test window indicates a negative result . the test was interpreted as invalid if the reference and the control bands did not appear.15 blood serum specimens , approximately 100 l , were obtained on the day of recruitment by research staff who had received training in the implementation of the ict - tb test . all patients were tested with the ict - tb on the day of enrolment or not later than 3 days after enrolment . results were interpreted within 20 minutes by three interpreters blinded to each other s findings and to the patient s clinical information . when at least two readers indicated a positive result , the result was recorded as positive . criteria for a definitive tb diagnosis were defined as smear positivity , positive culture of sputum or other specimens , positive clinical and radiological evaluation , response to tb treatment , or histological characteristics indicative of tb in biopsy specimens , as is the standard clinical practice . only for registered tb cases , sputum or other specimens first , predictors with a p - value of < 0.05 were selected by univariate logistic regression . second , using multivariate logistic regression with a forward method of model selection,16 predictors of tb in the final model were selected not only from statistical significance but also from clinical significance . models with / without the ict - tb test were compared by adding the ict - tb test results into the clinical predictor model . to assess whether the model prediction was improved by the ict - tb , two methods were used : the area under receiver operating characteristic curve ( auroc ) method , and the net reclassification improvement ( nri ) method.17 models with and without ict - tb were validated and assigned classical diagnostic values . for the scoring scheme , each variable from the final model was weighted by its coefficient and transformed to an item score . the cutoff points of total scores , determined by statistical significance and the yield of prediction , were used to classify hiv patients into the low- or high - risk groups . the positive predictive value ( ppv ) of the ict - tb test for each category was compared with the prior probability ( prevalence ) by the fisher s exact probability test . the number of patients required in the study was estimated based on previous information of an odds ratio of 9.6 and 16.9% prevalence in the non - tb group.15 thus , to achieve 90% power and a significant finding at alpha = 0.05 , 18 active tb patients were required . given the prevalence of active tb ( 17% ) in the study population,18 106 hiv patients were needed ; in order to compensate for 20% missing data , the final minimum number of hiv patients needed was 128 . of 213 hiv patients eligible for the study , seven ( 3.3% ) were excluded because of ongoing tb and ipt treatment at the time of enrolment . the final analysis included 206 hiv patients , all of whom underwent the ict - tb tests . of these , 37 ( 18% ) were diagnosed with active tb , including 33 ( 94.3% ) with pulmonary tb and four ( 5.7% ) with extrapulmonary tb cases ( figure 1 ) . using univariate logistic regression , the following patient characteristics , signs , and symptoms were significant : bmi , history of contact , cd4 count level , arv prescribed , cough , fever , weight loss , dyspnea , chest pain , loss of appetite , fatigue , and shaking chills ( tables 1 and 2 ) . the predictors that remained in the final model were cough , shaking chills , bmi , and arv prescribed , with cd4 clustered in the model ( table 3 ) . although those with a positive ict - tb test result were 5.05 times more likely to have active tb ( 95% confidence interval , 0.6638.48 ) compared with those who tested negative , the ict - tb test itself did not add any statistical significance to the final model ( table 3 ) . these predictors explained 90.2% of the probability ( auroc ) of active tb . when the ict - tb test result was added in the model , it increased the probability of active tb to 91.6% ; this increase was not statistically significant ( p = 0.40 ) ( figure 2 ) . this nonimprovement of the model by the ict - tb test result was also confirmed by the nri analysis ( p = 0.51 ) . adding the ict - tb test result did not enhance any of the diagnostic values in the validation of the model ( without ict - tb versus with ict - tb ) : sensitivity , 48.0% versus 52.0% ; specificity , 95.3% versus 94.7% ; ppv , 63.2% versus 61.9% ; and npv , 91.7% versus 92.2% . this study further investigated whether the ict - tb would enhance the diagnosis in subgroups of individuals with low versus high risk of having tb . the subgroups were categorized by a risk score system developed from each selected predictor , weighted by the coefficient from the final model , and transformed by dividing the value by the smallest coefficient ( 1.5 for cough and bmi ) . the value derived was rounded up to the nearest whole number to assign a final score ; scores ranged from 0 to 11 ( table 4 ) . when hiv patients were classified into low tb risk ( scores 7 ) and high tb risk ( scores > 7 ) groups , the ppv of ict - tb test ( post - test probability ) increased insignificantly from prevalence ( pre - test probability ) in both the low risk ( from 12.5% to 27.3% , p = 0.17 ) and high risk groups ( from 78.6% to 80.8% , p = 0.73 ) ( table 5 ) . in january 2011 , who reported the lack of accuracy of serological tests for tb in hiv populations;19 however , the present study is the first to assess whether addition of the ict - tb test result extends the diagnostic value of a screening tool based on clinical predictors in thailand . no statistically significant additional value over the four clinical factors selected was found , although the prediction probability , demonstrated from roc area curves , increased from 90.2% to 91.6% . furthermore , we externally validated the best performing rule for tb diagnosis in hiv patients recommended by who , ie , presence of cough for any duration , fever , night sweats , or weight loss , in our dataset.10 the result was in accordance with the present authors findings . auroc without the ict - tb was 85.5% , compared with 86.3% with inclusion of the ict - tb test result ; this increase was not statistically significant ( p = 0.19 ) . subgroup analysis was performed to assess any added diagnostic value in low and high tb risk groups , stratified by a clinical risk score ; post - test probability of the ict - tb did not increase significantly from pre - test probability in either subgroup , confirming a nonbenefit of the ict - tb test over the screening tool in hiv patients . the ict - tb test used for the study is one among several commercial kits available in this country . because antibody responses to m. tuberculosis antigens may be heterogeneous for a number of reasons , multiple antigens were used in the ict - tb test to increase the performance of the test.15 the added diagnostic value may vary with the use of ict - tb tests containing different antigens however , the authors of this paper considered that the ict - tb test selected for use in this study should perform best since it is a locally produced test using antigens derived from an endogenous m. tuberculosis strain and had shown higher diagnostic accuracy than an imported test.20 the combination of factors best predicting active tb in hiv patients was as follows : bmi , < 19 kg / m ; cough for > 2 weeks ; shaking chills for 1 week ; and lack of arv prescription . this is also partly consistent with predictors from other studies , ie , bmi of 18 kg / m,12 and cough for > 2 weeks.12 other predictors found associated with active tb in other studies , but not in the present study , were weight loss,10 night sweats,10 fever,10 lymphadenopathy,11,12 and general weakness;11,12 these discrepant results are likely due to population differences . in some previous studies , an abnormal chest radiograph was used as one of the predictors and was proven to increase sensitivity in detection of active tb.10,13,14 however , in this study , abnormal chest radiograph was excluded as a predictor because it was used as one of the criteria for tb definition , which was likely to introduce incorporation bias . from an economic perspective , using the ict - tb may not seem to be particularly burdensome due to its low cost ( less than us$5 ) . however , given the nonbenefit of the test , the thai ministry of public health budget may save more than us$8,00010,000 per year by solely following the screening tool developed in this study ; these funds could be reallocated to subsidize other hiv - related care programs , such as tb prevention in the population at large . first , m. tuberculosis culture was not used as a gold standard for diagnosis due to limited accessibility in many areas . in the present study , 14 ( 37.8% ) of 37 tb cases sent for m. tuberculosis culture yielded a result ; of these , 13 ( 92.9% ) were culture - positive , reflecting a high consistence with the reference standard used in the study . given the small number of cases with culture results , m. tuberculosis culture could not be used as a reference in regression analysis . a second limitation may be the generalizability of the study , which may be limited due to the variability of the predicting factors , prevalence of tb severity , and spectrum of the target population . therefore , validation of the model is required before it can be adopted as routine in other health care settings . third , some variables had missing values , such as cd4 cell counts ( 12.1% ) and bmi ( 2.9% ) . results of multiple imputation analysis showed the same tendency , with slightly different effect sizes for each predictor . in both the original analysis and that using multiple imputation data , the ict - tb test did not significantly add to the clinical predictors ( p = 0.07 ) . during model fitting , cd4 levels were found statistically significant only in univariate analysis ; however , given its clinical significance , this variable was taken into account by clustering in the final model . in future research , most hiv / tb patients with cd4 cell counts less than 200 cells/l have low immunity and can be diagnosed with tb by routine clinical predictors ( without the ict - tb ) ; therefore , this group may be prone to selection bias . additionally , in a previous study , the ict - tb test performed better when the cd4 cell count was more than 200 cells/l.5 lastly , the relatively small number of active tb patients in this study may limit the power of analysis for some variables ; future research with a larger sample is warranted to confirm the results of the present study . however , given that the present study is prospective in nature and patients were followed up for 2 months , misclassification of tb diagnosis should be minimal ; this is a strength of the study . in this study setting , the ict - tb test did not improve tb diagnosis compared with the 4 clinical predictors in the overall group and any subgroups of hiv patients , as classified by clinical risk scores . further research will be necessary to apply and compare the diagnostic performance of the clinical algorithm suggested by who with the clinical predictors used in this study .

purpose : the value of an immunochromatographic test for tuberculosis ( ict - tb ) combined with clinical predictors has yet to be evaluated in thailand . this study aimed to assess any additional diagnostic value of an ict - tb test over that of clinical predictors in a group of human immunodeficiency virus ( hiv ) patients as well as in subgroups of hiv patients classified by clinical risk scores.patients and methods : an extended cross - sectional study was conducted at a community hospital in chiang rai and a general hospital in lampang . hiv patients registered between april 2009 and may 2010 were screened by a locally made ict - tb test , including 38 , 16 , and 6 kd microbacterium tuberculosis antigens , as well as by routine evaluations for tb diagnosis . demographic data , medical history , signs , and symptoms were recorded . participants were followed up for 2 months for final ascertainment of tb diagnosis.results:of 206 patients , 37 ( 18% ) had tb . four clinical predictors were identified : low body mass index ( < 19 kg / m2 ) , prolonged cough ( duration > 2 weeks ) , shaking chills ( 1 week ) , and no use of antiretrovirals . the area under the receiver operating curve was 90.2% ; adding the ict - tb test result increased the area nonsignificantly to 91.6% ( p = 0.40 ) . when patients were categorized by risk scores derived from selected clinical predictors into low ( scores 7 ) and high ( scores > 7 ) tb risk groups , a positive ict - tb test increased the positive predictive value nonsignificantly in the low risk group ( from 12.5% to 27.3% , p = 0.17 ) and the high risk group ( from 78.6% to 80.8% , p = 0.73).conclusion : in this study setting , the ict - tb test did not enhance tb diagnosis over the four clinical predictors in the overall group or any subgroups of hiv patients classified by clinical risk scores .

Introduction Materials and methods Study settings and population Data collection Index and reference tests ICT-TB Reference tests Statistical analysis Results Discussion Conclusion

human immunodeficiency virus ( hiv ) and tuberculosis ( tb ) co - infection constitutes a high - burden disease in thailand , where 12,890 individuals died from tb in 2009 and where hiv accounts for 17% of tb cases , the highest figure in the south - east asian region.1 early case identification followed by adequate treatment is the cornerstone of tb control programs . thailand s rank of 18th among 22 high - burden countries for tb is partly attributed to inadequate case evaluation and treatment default , which also result in increased deaths and contribute to underperformance on the world health organization ( who ) target for treatment success ( 81% versus 85%).1 efforts to improve tb diagnosis by new technology have commonly faced the challenges of high equipment cost , lack of skilled technicians , and excessive turnaround time.2 rapid immunochromatographic test ( ict ) technology may be the exception to this rule : when used as a serodiagnostic test for tb antibodies ( ict - tb ) , this technology is affordable , simple and safe to use , and produces results within 25 minutes.3 however , in an hiv - infected population , several antigens may be recognized by the antibodies , thereby limiting the diagnostic validity of such tests.4 previous studies have reported a wide range of sensitivity of ict - tb tests in hiv patients ( from 0% to 80% ) ; specificity was also very variable , ranging from 39% to 97.4%.48 a landmark study , conducted by the who special programme for research and training in tropical diseases in 2008 , showed poor performance of 19 commercial rapid diagnostic tests in hiv patients , with sensitivity reduced by an average of 22%43% compared with that in hiv - negative patients.9 in thai settings , only two diagnostic validity studies , on the use of lipoarabinomannan and 38-kd recombinant antigens using pleural effusion and serum , have been reported.4,5 in an intensified effort to find cases of active tb in thailand , a general screening tool , which disregards hiv status , is currently being used ; this tool consists of simple questions relating to signs and symptoms of tb . from a recent review , the best predictors for tb diagnosis in hiv patients living in resource - constrained settings are presence of cough ( for any duration ) , fever , night sweats , or weight loss.10 other signs , symptoms , and simple laboratory tests proposed by other studies are fatigue,11,12 shaking chills,12 loss of appetite,12 difficulty in breathing,12 chest pain,12 abdominal pain,12 nausea / vomiting,12 body mass index ( bmi),11,12 lymphadenopathy,11,12 hemoglobin level of < 10 g / dl,12 tuberculin skin test positivity,12 hospitalization upon registration,12 lack of antiretroviral ( arv ) therapy,11,12 chest radiograph abnormalities,11,13,14 and cd4 cell count.11,14 however , the proposed combinations of predictors , as well as the performance of such predictor combinations , varies between studies . the validity of such existing screening tools is unknown in the setting of thailand ; furthermore , the value of an ict - tb test used in combination with clinical predictors has yet to be evaluated in thailand . therefore , the objective of this study was to identify clinical characteristics , including demographics , signs , and symptoms that may be used to predict the presence of active tb , as well as to evaluate the diagnostic value of ict - tb testing over such clinical predictors in subgroups of hiv patients stratified by clinical risk scores . all hiv patients aged 18 years or older seeking medical care or reporting for annual checkup at either a tb clinic or an arv clinic between april 2009 and may 2010 , regardless of tb signs and symptoms , were eligible for the study . outpatient data , including clinical signs and symptoms , and blood samples were collected on the first day . study settings included maesai district hospital , a community hospital in chiang rai , and lampang hospital , a general hospital in lampang , thailand . data collected included patient demographics and clinical data , comprising medical history , history of tb diagnosis , history of tb contact , and arv prescription . data regarding duration of signs and symptoms were also collected ; these included cough , cough with sputum , hemoptysis , fever , diarrhea , weight loss , night sweats , dyspnea , chest pain , nausea or vomiting , abdominal pain , loss of appetite , fatigue , shaking chills , and size of lymphadenopathy . as the index test , a locally produced ict - tb test containing a membrane strip pre - coated with colloid gold - conjugated recombinant microbacterium tuberculosis antigens ( 38 , 16 , and 6 kd ) was used . in this test , the patient s sample migrates chromatographically along the membrane to the test region , where a visible line forms if an antigen - antibody - antigen - gold particle complex forms ( positive result ) . the test was interpreted as invalid if the reference and the control bands did not appear.15 blood serum specimens , approximately 100 l , were obtained on the day of recruitment by research staff who had received training in the implementation of the ict - tb test . all patients were tested with the ict - tb on the day of enrolment or not later than 3 days after enrolment . criteria for a definitive tb diagnosis were defined as smear positivity , positive culture of sputum or other specimens , positive clinical and radiological evaluation , response to tb treatment , or histological characteristics indicative of tb in biopsy specimens , as is the standard clinical practice . second , using multivariate logistic regression with a forward method of model selection,16 predictors of tb in the final model were selected not only from statistical significance but also from clinical significance . models with / without the ict - tb test were compared by adding the ict - tb test results into the clinical predictor model . to assess whether the model prediction was improved by the ict - tb , two methods were used : the area under receiver operating characteristic curve ( auroc ) method , and the net reclassification improvement ( nri ) method.17 models with and without ict - tb were validated and assigned classical diagnostic values . the cutoff points of total scores , determined by statistical significance and the yield of prediction , were used to classify hiv patients into the low- or high - risk groups . the positive predictive value ( ppv ) of the ict - tb test for each category was compared with the prior probability ( prevalence ) by the fisher s exact probability test . the number of patients required in the study was estimated based on previous information of an odds ratio of 9.6 and 16.9% prevalence in the non - tb group.15 thus , to achieve 90% power and a significant finding at alpha = 0.05 , 18 active tb patients were required . given the prevalence of active tb ( 17% ) in the study population,18 106 hiv patients were needed ; in order to compensate for 20% missing data , the final minimum number of hiv patients needed was 128 . all hiv patients aged 18 years or older seeking medical care or reporting for annual checkup at either a tb clinic or an arv clinic between april 2009 and may 2010 , regardless of tb signs and symptoms , were eligible for the study . outpatient data , including clinical signs and symptoms , and blood samples were collected on the first day . study settings included maesai district hospital , a community hospital in chiang rai , and lampang hospital , a general hospital in lampang , thailand . data collected included patient demographics and clinical data , comprising medical history , history of tb diagnosis , history of tb contact , and arv prescription . data regarding duration of signs and symptoms were also collected ; these included cough , cough with sputum , hemoptysis , fever , diarrhea , weight loss , night sweats , dyspnea , chest pain , nausea or vomiting , abdominal pain , loss of appetite , fatigue , shaking chills , and size of lymphadenopathy . as the index test , a locally produced ict - tb test containing a membrane strip pre - coated with colloid gold - conjugated recombinant microbacterium tuberculosis antigens ( 38 , 16 , and 6 kd ) was used . in this test , the patient s sample migrates chromatographically along the membrane to the test region , where a visible line forms if an antigen - antibody - antigen - gold particle complex forms ( positive result ) . the test was interpreted as invalid if the reference and the control bands did not appear.15 blood serum specimens , approximately 100 l , were obtained on the day of recruitment by research staff who had received training in the implementation of the ict - tb test . all patients were tested with the ict - tb on the day of enrolment or not later than 3 days after enrolment . criteria for a definitive tb diagnosis were defined as smear positivity , positive culture of sputum or other specimens , positive clinical and radiological evaluation , response to tb treatment , or histological characteristics indicative of tb in biopsy specimens , as is the standard clinical practice . only for registered tb cases , sputum or other specimens as the index test , a locally produced ict - tb test containing a membrane strip pre - coated with colloid gold - conjugated recombinant microbacterium tuberculosis antigens ( 38 , 16 , and 6 kd ) was used . in this test , the patient s sample migrates chromatographically along the membrane to the test region , where a visible line forms if an antigen - antibody - antigen - gold particle complex forms ( positive result ) . the test was interpreted as invalid if the reference and the control bands did not appear.15 blood serum specimens , approximately 100 l , were obtained on the day of recruitment by research staff who had received training in the implementation of the ict - tb test . all patients were tested with the ict - tb on the day of enrolment or not later than 3 days after enrolment . criteria for a definitive tb diagnosis were defined as smear positivity , positive culture of sputum or other specimens , positive clinical and radiological evaluation , response to tb treatment , or histological characteristics indicative of tb in biopsy specimens , as is the standard clinical practice . models with / without the ict - tb test were compared by adding the ict - tb test results into the clinical predictor model . to assess whether the model prediction was improved by the ict - tb , two methods were used : the area under receiver operating characteristic curve ( auroc ) method , and the net reclassification improvement ( nri ) method.17 models with and without ict - tb were validated and assigned classical diagnostic values . the cutoff points of total scores , determined by statistical significance and the yield of prediction , were used to classify hiv patients into the low- or high - risk groups . the positive predictive value ( ppv ) of the ict - tb test for each category was compared with the prior probability ( prevalence ) by the fisher s exact probability test . the number of patients required in the study was estimated based on previous information of an odds ratio of 9.6 and 16.9% prevalence in the non - tb group.15 thus , to achieve 90% power and a significant finding at alpha = 0.05 , 18 active tb patients were required . given the prevalence of active tb ( 17% ) in the study population,18 106 hiv patients were needed ; in order to compensate for 20% missing data , the final minimum number of hiv patients needed was 128 . the final analysis included 206 hiv patients , all of whom underwent the ict - tb tests . of these , 37 ( 18% ) were diagnosed with active tb , including 33 ( 94.3% ) with pulmonary tb and four ( 5.7% ) with extrapulmonary tb cases ( figure 1 ) . using univariate logistic regression , the following patient characteristics , signs , and symptoms were significant : bmi , history of contact , cd4 count level , arv prescribed , cough , fever , weight loss , dyspnea , chest pain , loss of appetite , fatigue , and shaking chills ( tables 1 and 2 ) . the predictors that remained in the final model were cough , shaking chills , bmi , and arv prescribed , with cd4 clustered in the model ( table 3 ) . although those with a positive ict - tb test result were 5.05 times more likely to have active tb ( 95% confidence interval , 0.6638.48 ) compared with those who tested negative , the ict - tb test itself did not add any statistical significance to the final model ( table 3 ) . when the ict - tb test result was added in the model , it increased the probability of active tb to 91.6% ; this increase was not statistically significant ( p = 0.40 ) ( figure 2 ) . this nonimprovement of the model by the ict - tb test result was also confirmed by the nri analysis ( p = 0.51 ) . adding the ict - tb test result did not enhance any of the diagnostic values in the validation of the model ( without ict - tb versus with ict - tb ) : sensitivity , 48.0% versus 52.0% ; specificity , 95.3% versus 94.7% ; ppv , 63.2% versus 61.9% ; and npv , 91.7% versus 92.2% . this study further investigated whether the ict - tb would enhance the diagnosis in subgroups of individuals with low versus high risk of having tb . the subgroups were categorized by a risk score system developed from each selected predictor , weighted by the coefficient from the final model , and transformed by dividing the value by the smallest coefficient ( 1.5 for cough and bmi ) . when hiv patients were classified into low tb risk ( scores 7 ) and high tb risk ( scores > 7 ) groups , the ppv of ict - tb test ( post - test probability ) increased insignificantly from prevalence ( pre - test probability ) in both the low risk ( from 12.5% to 27.3% , p = 0.17 ) and high risk groups ( from 78.6% to 80.8% , p = 0.73 ) ( table 5 ) . in january 2011 , who reported the lack of accuracy of serological tests for tb in hiv populations;19 however , the present study is the first to assess whether addition of the ict - tb test result extends the diagnostic value of a screening tool based on clinical predictors in thailand . no statistically significant additional value over the four clinical factors selected was found , although the prediction probability , demonstrated from roc area curves , increased from 90.2% to 91.6% . furthermore , we externally validated the best performing rule for tb diagnosis in hiv patients recommended by who , ie , presence of cough for any duration , fever , night sweats , or weight loss , in our dataset.10 the result was in accordance with the present authors findings . auroc without the ict - tb was 85.5% , compared with 86.3% with inclusion of the ict - tb test result ; this increase was not statistically significant ( p = 0.19 ) . subgroup analysis was performed to assess any added diagnostic value in low and high tb risk groups , stratified by a clinical risk score ; post - test probability of the ict - tb did not increase significantly from pre - test probability in either subgroup , confirming a nonbenefit of the ict - tb test over the screening tool in hiv patients . the ict - tb test used for the study is one among several commercial kits available in this country . because antibody responses to m. tuberculosis antigens may be heterogeneous for a number of reasons , multiple antigens were used in the ict - tb test to increase the performance of the test.15 the added diagnostic value may vary with the use of ict - tb tests containing different antigens however , the authors of this paper considered that the ict - tb test selected for use in this study should perform best since it is a locally produced test using antigens derived from an endogenous m. tuberculosis strain and had shown higher diagnostic accuracy than an imported test.20 the combination of factors best predicting active tb in hiv patients was as follows : bmi , < 19 kg / m ; cough for > 2 weeks ; shaking chills for 1 week ; and lack of arv prescription . this is also partly consistent with predictors from other studies , ie , bmi of 18 kg / m,12 and cough for > 2 weeks.12 other predictors found associated with active tb in other studies , but not in the present study , were weight loss,10 night sweats,10 fever,10 lymphadenopathy,11,12 and general weakness;11,12 these discrepant results are likely due to population differences . in some previous studies , an abnormal chest radiograph was used as one of the predictors and was proven to increase sensitivity in detection of active tb.10,13,14 however , in this study , abnormal chest radiograph was excluded as a predictor because it was used as one of the criteria for tb definition , which was likely to introduce incorporation bias . from an economic perspective , using the ict - tb may not seem to be particularly burdensome due to its low cost ( less than us$5 ) . however , given the nonbenefit of the test , the thai ministry of public health budget may save more than us$8,00010,000 per year by solely following the screening tool developed in this study ; these funds could be reallocated to subsidize other hiv - related care programs , such as tb prevention in the population at large . a second limitation may be the generalizability of the study , which may be limited due to the variability of the predicting factors , prevalence of tb severity , and spectrum of the target population . in both the original analysis and that using multiple imputation data , the ict - tb test did not significantly add to the clinical predictors ( p = 0.07 ) . in future research , most hiv / tb patients with cd4 cell counts less than 200 cells/l have low immunity and can be diagnosed with tb by routine clinical predictors ( without the ict - tb ) ; therefore , this group may be prone to selection bias . additionally , in a previous study , the ict - tb test performed better when the cd4 cell count was more than 200 cells/l.5 lastly , the relatively small number of active tb patients in this study may limit the power of analysis for some variables ; future research with a larger sample is warranted to confirm the results of the present study . however , given that the present study is prospective in nature and patients were followed up for 2 months , misclassification of tb diagnosis should be minimal ; this is a strength of the study . in this study setting , the ict - tb test did not improve tb diagnosis compared with the 4 clinical predictors in the overall group and any subgroups of hiv patients , as classified by clinical risk scores . further research will be necessary to apply and compare the diagnostic performance of the clinical algorithm suggested by who with the clinical predictors used in this study .

dendritic cells ( dcs ) , originally described by steinman and cohn , serve as a crucial link between innate and adaptive immunity . although they represent only a small population of leukocytes , they are the most powerful antigen presenting cells ( apc ) with the unique ability to activate naive t cells . as sentinel members of the innate immune system , dcs respond to antigens and molecules containing pathogen - associated molecular patterns ( pamps ) or damage - associated molecular patterns ( damps)so - called danger signals by the generation of protective cytokines . as members of the acquired immune system , dcs respond to these harmful molecules by efficient antigen uptake , processing , and presentation , and hence dcs are crucial in the initiation of adaptive immune responses . besides their potent capacity to stimulate naive t cells , effector t cells and memory t cells , as well as b cells , they are also involved in the maintenance of tolerance against harmless ( auto)antigens [ 4 , 5 ] . because of their immunoregulatory capacities and because very small numbers of activated dcs are highly efficient at generating immune responses against antigens , dcs have been vigorously used in clinical trials in order to elicit or amplify immune responses against cancer and chronic infectious diseases . although an impressive amount of data has been obtained from these clinical trials completed thus far , the outcomes were not in line with initial expectations [ 810 ] . a critical issue in the development of dc - based vaccines is that their ability to stimulate immune responses depends largely on the activation state of dcs . in this paper , we discuss the continuous quest for the best in vitro conditions in order to generate clinical - grade dcs with a potent immunogenic potential . for this , we explore the molecular and cellular mechanisms underlying adequate immune responses and focus on optimal dc culture regimens and activation stimuli in humans . dcs originate from cd34 haematopoietic stem cells in the bone marrow and circulate as precursors through the blood stream to target tissues . additionally , it is well established that during physiological stress , monocytes are also a source of dc precursors and differentiate into immature dcs in the presence of gm - csf and a variety of other cytokines . immature dcs take residence at sites of potential antigen entry and are specialized in antigen capturing and processing . they recognize the so - called pathogen - associated molecular patterns ( pamps ) which are evolutionary conserved structures , including microbial lipids , carbohydrates , nucleic acids and intermediates of viral replication ( double - stranded ( ds)rna ) , via pattern recognition receptors ( prrs ) . there are several types of prrs that are involved in innate recognition of pathogens , including toll - like receptors ( tlrs ) , nucleotide - binding - oligomerization - domain-(nod - like ) receptors , interferon ( ifn - induced ) dsrna - activated protein kinase ( pkr ) , and rig - i - like helicases . once dcs have captured a foreign nonself - antigen , they undergo a highly regulated maturation process and remodel into fully activated antigen - presenting dcs capable to elicit effective immune responses . indeed , mature dcs express high levels of several costimulatory molecules as well as major histocompatibility complex ( mhc ) molecules on their surface . maturation of dcs also induces the production of chemokines that attract naive and memory t cells . during the maturation process , dcs exit the nonlymphoid tissues to migrate via afferent lymph to lymphoid tissues subsequently , mature dcs will activate ( naive ) t- and b - lymphocytes that recognize the presented antigen as peptide - mhc complexes on the surface of the dc . yet , additionally , positive amplification of antigen presentation via costimulation and secretion of various cytokines is also crucial to induce proper immune responses [ 3 , 15 ] ( cfr . besides the above - delineated classical view of the dc life cycle , it has gradually become clear that dcs do not represent a homogeneous population . briefly , the first division is the distinction between plasmacytoid and myeloid or conventional dcs ( cdcs ) . plasmacytoid dcs ( pdcs ) , also referred to as type i ifn - producing cells ( ipcs ) , are the key effectors in the innate immune system because of their extra - ordinary capacity to produce type i ifn upon viral infection [ 16 , 17 ] . the conventional dcs can be further subdivided according to their localization : ( i ) lymphoid organ - resident dcs , ( ii ) peripheral tissue - resident dcs ( e.g. , langerhans cells and interstitial dcs ) , and ( iii ) circulating dcs . in human blood , differences in dc subsets can be identified based on differential expression of specific markers : pdcs express cd303 ( bdca-2 ) , cd304 ( bdca-4 ) , and cd123 ( il-3r ) , whereas cdcs are characterized by their expression of cd1c ( bdca-1 ) and cd11c [ 18 , 19 ] . in addition , pdcs and cdcs also express a different set of toll - like receptors tlrs . in brief , pdcs express mainly tlr7 and tlr9 , whereas cdcs exhibit strong expression of tlr1 , tlr2 , tlr3 , tlr4 , and tlr8 . accordingly , pdcs mainly recognize viral components and produce a large amount of ifn-. in contrast , cdcs recognize bacterial components and produce proinflammatory cytokines such as tnf- , il-6 , and il-12p70 to activate proinflammatory t - cell subsets [ t helper type 1 ( th1)/th17 ] and consequently recruit cytotoxic t - lymphocytes ( ctl ) . because of the unique biological function of each dc subset , it was proposed that a specific dc lineage determines the outcome of t - cell contact , that is , tolerance or immunity . indeed , it was initially thought that cdcs were inducers of immunity , while pdcs induced tolerance . however , nowadays pdcs are believed to be the key effector cells in the early antiviral innate immune response by producing large amounts of type i interferons upon viral infection . furthermore , it has been shown that pdcs augment immune responses by cross - talking with cdcs by the production of ifn- , thereby playing a key role in effective stimulation of adaptive immunity as well . in addition to ifn- production , it has been demonstrated that mouse pdcs also express cd40l , which activates cdcs to produce il-12p70 ( figure 1 ) . recently , several groups identified a unique human dc subset ( cd11cbdca-3 ) as the homologue of mouse cd8 dc [ 2326 ] . of particular importance is their superior antigen cross - presentation capacity , expression of the xc chemokine receptor 1 ( xcr1 ) , and their capacity to produce high levels of bioactive il-12p70 . initially , it was suggested that bdca-3 dcs and bdca-1 dcs may represent maturational stages of the same cell type . the fact that bdca-3 expression is induced on a reasonable proportion of bdca-1 dcs after culture - induced maturation may be considered an argument in favour of the former concept . however , since the same observation was also made for il-3-stimulated pdc , such data could also be taken as an argument in favour of a similar relationship between bdca-3 dcs and pdcs . nowadays , it is well accepted that bdca-3 dcs represent a unique myeloid dc subset that effectively activates cd8 ctl , in analogy with mouse cd8 dcs . this supports a potential key role for the myeloid bdca-3 dc subset in immunity to viruses , as well as other intracellular pathogens [ 2830 ] and may have important implications in the design of human dc vaccines . therapeutic vaccines to treat chronic infectious diseases ( such as human immunodeficiency virus ( hiv ) , cytomegalovirus ( cmv ) , hepatitis b virus ( hbv ) , and hepatitis c virus ( hcv ) ) , or numerous tumor types ( including melanoma , leukaemia , breast , and prostate cancer ) mainly aim to induce antigen - specific cell - mediated immunity to clear infected cells and eliminate tumor cells . recent studies have shown that dcs play a critical role in directing effector t - cell responses towards a th1 , th2 , th17 , or regulatory t cell ( treg ) response [ 31 , 32 ] . briefly , upon maturation , dcs upregulate the expression of certain products necessary to supply t lymphocytes with the 3 signals that will determine their activation status and general fate : antigen - specific signalling via the t cell receptor ( tcr ) , mediated by the binding of mhc - peptide complexes to the tcr drives the initial interaction between dcs and t cells ( i.e. , signal 1 ) . costimulation by surface molecules on apc , such as dcs , can either amplify or regulate the interaction with t cells ( i.e. , signal 2 ) . costimulatory molecules can be divided in two classes : ig superfamily members , including cd28 , that interact with several members of the b7 family ( cd80/cd86 ) [ 3537 ] on the one hand , and tnf receptor superfamily members , including cd27 and cd40 , that bind to membrane - bound proteins of the tnf superfamily [ 35 , 38 , 39 ] on the other hand . cd28 is expressed on t cells , and is the receptor for cd80 and cd86 expressed on activated apc . ligation of cd28 provides costimulatory signals required for t cell activation : ( i ) altering the threshold level of tcr ligation required for activation , ( ii ) reducing the time needed to stimulate naive t cells and ( iii ) enhancing the magnitude of the t cell response . without cd28 signalling , the t cell would either become apoptotic or anergic . the b7 family of costimulatory molecules has been extensively reviewed elsewhere [ 41 , 42 ] . cd27 , a member of the tnf receptor superfamily , is constitutively expressed on the surface of naive t cells , in contrast to other members of the tnf receptor family and can thus play a role during the initiation of t cell responses . the contribution of cd27 to the immune response is dependent upon cd70 expression . while during primary t cell activation there seems to be a certain redundancy in cd80/cd86 and cd70 costimulation , it is triggering of cd27 on t - lymphocytes by its ligand cd70 that enhances the magnitude of antigen - specific cytotoxic t cell reponses [ 38 , 45 ] , which is required for effective immunotherapy . cd27/cd70 interaction increases the initial expansion and survival of antigen - specific t cells and improves their cytotoxic capacity . furthermore , a recent study has shown that cd70 expressed on mouse dec205 cdcs represents an il-12p70-independent th1-inducing factor ( vide infra ) . taken together , enhancing cd70 expression on dcs would lead to the development of a vaccine strategy capable of facilitating the cd27/cd70 interaction , and hence the induction of an adequate anti - tumor or antiviral immune response . finally , mature dcs can secrete a variety of pro- and anti - inflammatory cytokines for differentiation from naive t cells to effector t cells ( i.e. , signal 3 ) . one well - studied third signal agent is interleukin ( il)-12p70 for the induction of th1 and ctl , which are essential for efficient tumor / pathogen rejection . il-12p70 is a multifunctional proinflammatory cytokine with pleiotropic effects and comprises two subunits : p35 and p40 . highly - coordinated p35 and p40 gene expression results in the formation of the biologically active form il-12p70 and is essential for initiation of an effective immune response . indeed , il-12p70 activates natural killer ( nk ) and t cells to produce mainly ifn- , it favours the generation of ctl and it enhances the cytotoxic activation of activated nk cells . besides the activation of innate and antigen - specific adaptive immunity against the tumor cells , the antitumor effects of il-12p70 are based on the ability to inhibit tumor angiogenesis through ifn- [ 52 , 53 ] . in addition , il-12p70 is crucial in the early phase of host defence against microbial infections [ 52 , 54 , 55 ] , where it is produced within a few hours after bacterial , fungal or parasitic infection . thus , to develop an efficient vaccine against tumors or chronic infectious diseases , dcs producing the biologically active form il-12p70 are desired . rather than simply recruiting th1 cells and ctl , vaccines should be designed to recruit other cellular arms of the immune system as well , for example , nk cells and antibody - producing b cells . in this perspective , it has been shown that dcs also play a key role in the activation of nk cells that can have powerful effects against tumor cells , particularly those with attenuated mhc expression . indeed , in response to dc - derived cytokines , such as il-12p70 and il-18 , nk cells are able to produce ifn- . in turn , exposure to signals provided by activated nk cells subsequently induces dcs to mature into a highly stimulatory phenotype that produces sustained il-12p70 , thereby promoting adaptive immunity [ 59 , 60 ] . overall , these findings support the concept to include dc - nk interactions in order to improve dc - based immunotherapy . furthermore , recent studies that have resulted in reappraisal of the potential of antibodies in the control of tumors and viruses support the strategy that dc - based vaccines should also be designed with antibody production in mind [ 6163 ] . in addition to priming of t cells and nk cells , the group of banchereau have recently demonstrated that dcs may also directly signal naive b - cell differentiation through the production il-12p70 and indirectly by promoting the differentiation of il-21-producing t follicular helper cells ( tfh ) in an il-12p70-dependent manner [ 65 , 66 ] . these observations suggest that il-12p70 could constitute a potent vaccine adjuvant in situations when both the cellular and humoral arms of the immune system are required , such as cancer [ 62 , 63 ] and hiv . indeed , studies with rhesus macaques have concluded that il-12p70 enhances the induction of specific antibody responses in vivo when used as vaccine adjuvant [ 6769 ] . noteworthy , il-12p70 also possesses a number of powerful nonimmunologically related anticancer activities . for example , il-12p70 plays a role as an antiangiogenic agent that can strongly inhibit the formation of neovasculature . taken together , the goal of many dc - based vaccination protocols is to cultivate dcs that are capable of expressing immunostimulatory cytokines ( il-12p70 ) and costimulatory molecules ( cd70 ) in parallel with antigen presentation . since expression of costimulatory molecules and cytokine secretion can be influenced by environmental signals during dc maturation , it is necessary to find an optimal cytokine environment for dc maturation in order to create a powerful vaccine against several cancer types or chronic infectious diseases . various attempts have subsequently been made in order to harness dc to achieve most powerful immunity , including strategies to enhance or stabilise antigen - specific stimulation , as well as essential costimulatory modulation of dcs . to maximize the efficiency and stability of antigen presentation by dcs , several strategies have been developed . these include direct in vivo delivery of antigen to circulating dcs in patients , as well as a variety of ways for in vitro loading of dcs with antigen . indeed , antigens coupled to antibodies specific for dc markers , such as 33d1 or dec-205 , have already been used in preclinical models to deliver antigens to dcs in vivo . additionally , dcs transduced ex vivo with tumor- or viral - derived mrna or dna [ 7274 ] , fused with tumor cells [ 75 , 76 ] , or directly loaded with tumor- or viral - derived peptides [ 77 , 78 ] have been tested for the induction of antigen - specific immune responses in vitro and in vivo . while the use of peptides as a source of antigen has several limitations when implementing clinical trials with antigen - loaded dcs , including human leukocyte antigen ( hla ) restriction as well as a limited number of identified immunodominant tumor- and virus - associated antigens , we and others [ 8084 ] have previously shown that dcs transfected with mrna - encoding antigens are superior to other loading strategies to induce immune responses . in general , there are several advantages regarding the use of mrna for antigen loading of dcs [ 72 , 85 ] as compared to tumor - associated peptides . mrna transfection will generate multiple antigenic epitopes , possibly more immunogenic than those already characterized , independent of the patient 's hla haplotype . in addition , mrna can be isolated and amplified from autologous tumor or virally infected cells in order to obtain mrna encoding patient - specific antigens [ 8688 ] . moreover , because mrna only has a short half - life and does not integrate in the host genome , genetic modification of dcs by mrna electroporation is considered to be highly safe and an easily applicable clinical tool . the earliest studies on dc vaccination were initiated in 1993 and utilized whole blood leukapheresis products with subsequent gradient centrifugation procedures to enrich for rare immature dc precursors of the peripheral blood before antigen loading and maturation . however , because of low yield of circulating dcs and difficulty to obtain them , the clinical utility of dc vaccines was initially limited . in a second attempt to directly isolate dcs from peripheral blood , moreover , although blood dcs from patients with a malignant or chronic infectious disease may seem to have normal distributions , they might have some functional defects , such as a lower expression of costimulatory molecules or an impaired capacity to stimulate autologous antigen - specific t cells [ 92 , 93 ] . currently , dcs for vaccination studies are generally obtained in large numbers after in vitro generation . at first , human dcs were cultured from cd34 haematopoietic progenitors in the presence of granulocyte - macrophage colony stimulating factor ( gm - csf ) and tumor necrosis factor alpha ( tnf- ) [ 94 , 95 ] . however , only few studies that used cd34-derived dc preparations for vaccination protocols in clinical phase i studies have been reported [ 96 , 97 ] . nowadays , generating dcs from peripheral blood cd14 monocytes is a generally - accepted method and is extensively used in experimental and clinical vaccination studies . in doing so , large numbers of monocyte - derived ( mo-)dcs are obtained without necessity for pretreatment of donors with any cytokines to mobilize dc progenitor cells . yet , the design of dc - based clinical trials varies greatly , including dc preparation , and therefore , standardization and further improvement for clinical use are needed . while a combination of granulocyte - macrophage colony - stimulating factor ( gm - csf ) with il-4 is most commonly used to induce immature dcs from monocytes [ 100 , 101 ] , a variety of other cytokines , such as ifn- [ 102104 ] , tnf- [ 105 , 106 ] , and il-15 have been used in combination with gm - csf for this purpose . in this perspective , santini et al . as well as arimoto - miyamoto et al . reported independently that ifn- induces rapid differentiation of freshly isolated gm - csf - treated human monocytes into mo - dcs endowed with potent functional activities , both in vitro and in vivo [ 102 , 103 ] , possibly mediated by ifn--dependent induction of cd70 expression . it must be noted however that ifn- also induces activity of rnases , and can not therefore be used in in vitro culture regimens for dcs when mrna - based in vitro modification of dcs is wanted . in addition , others have demonstrated that cd14 monocytes respond to il-15 by undergoing morphological transformation and acquiring characteristic dc features that facilitate antigen - specific responses of t cells . in contrast to ifn--modulated dcs , mrna electroporation appeared to serve as an efficient antigen - loading strategy for il-15-treated dcs . furthermore , chomarat et al . described that tnf- facilitates the induction of adaptive immunity also by promoting dc differentiation from cd14 blood precursors in vitro . however , it has been reported in contrast that tnf--treated semi mature dcs induce tolerance in experimental acute encephalitis ( eae ) , a mouse model for multiple sclerosis . moreover , due to strong plastic adherence before and to a lesser extent also after maturation , il-15 and tnf- treatment for dc generation results in a lower dc yield . consequently , the well - established and generally used combination of gm - csf and il-4 [ 100 , 101 ] is the most efficient method to obtain mo - dcs that express acceptable levels of cd70 with minimal loss of cells by adherence and with good compatibility with a mrna approach . regardless of how they are generated , it is important that dcs are activated to a mature phenotype , since immature dcs are no longer considered as competent candidates for vaccination trials because of their low t - cell activation potential [ 113115 ] . most dc culture regimens that have been commonly employed in clinical trials have activated dcs through the use of individual cytokines associated with inflammation or inflammatory cytokine cocktails . indeed , in an attempt to resemble a physiological environment for dc maturation , balanced cocktails of maturation agents that may be the most representative of various inflammatory states have often been used . in 1996 , romani was the first to describe a method to mature dcs from human blood by using a conditioned medium containing an unidentified cytokine mixture produced by adherent peripheral blood mononuclear cells ( pbmc ) stimulated by human immunoglobulins or fixed staphylococcus aureus cowan i strain . only one year later , morse and colleagues described a way to mature mo - dcs by adding tnf- to the culture medium . tnf- appeared to enhance the number of cells expressing the maturation marker cd83 , which seemed to be the most potent allostimulatory cells in mixed lymphocyte reactions . also , jonuleit et al . reported for the first time a well - defined cytokine cocktail to induce dc maturation , consisting of il-1 , il-6 , tnf- , and pge2 . this combination of proinflammatory mediators represents current golden standard for activation of dcs , although the concentration of the diverse components varies among studies . fully - mature dcs induced by this combination of inflammatory cytokines have been consistently observed as superior to immature dcs in promoting a higher degree of specific t - cell priming in vitro and in vivo . while pge2 increases the expression of ccr7 and hence the capacity of dcs to migrate to the regional lymph nodes through chemotaxis by ccl-19 and/or -21 , pge2 also inhibits il-12p70 secretion by dcs . although some details remain incompletely clarified , expression of il-12p70 appears to be under unusually tight regulation and requires at least 2 signals activating both myd88 ( myeloid differentiation factor 88)- and trif ( tir domain - containing adapter - inducing ifn-)-dependent pathways simultaneously for maximal expression [ 119 , 120 ] . of note , tlrs , commonly used for activation of dcs , are divided in those that are myd88-dependent and those that are trif - dependent , hence explaining observed requirements of multiple tlr engagement for maximized il-12p70 production . in this perspective , mature dcs with the potential to produce high amounts of biologically active il-12p70 ( 1015 ng / ml ) were obtained by mailliard et al . in 2004 , who used a combination of il-1 , tnf- , ifn- , ifn- , and poly i : c . although these mature mo - dcs displayed a slightly decreased migratory capacity , they induced significantly more antigen - specific cytotoxic t cells than did the golden standard counterparts , dependent on the high il-12p70 secretion . in 2007 , zobywalski et al . proposed a cytokine cocktail consisting of tnf- , il-1 , ifn- , r848 and pge2 as the best cocktail to allow large - scale processing of clinical - grade mo - dcs with the capacity to secrete il-12p70 . addition of poly i : c to this cocktail significantly increased il-12p70 production even more , yet it disabled the mature dcs to express the transgene after exogenous rna electroporation and it led to a decline in cell viability . dohnal et al . used a mixture of lps and ifn- to mature dcs . although high il-12p70 secretion by mature mo - dcs was previously attributed to the addition of ifn- , ifn- also appeared to be responsible for the low migratory ability of dcs cultivated in the presence of lps and ifn- [ 122 , 123 ] . nevertheless , this migratory problem could be fixed by including pge2 in the maturation - inducing cytokine cocktail [ 123 , 124 ] . in addition , whereas dc maturation by tlr ligand alone ( including lps , cpg , and poly i : c ) has been reported to increase expression of classical activation markers as well as many inflammatory cytokines , a tlr agonist alone does not result in a substantial cd8 t - cell response , which is probably due to no or very low levels of il-12p70 secretion as well as insufficient induction of cd70 by tlr ligand stimulation alone . according to sanchez et al . , expression of cd70 on mo - dcs requires combined tlr / cd40 stimulation . in preliminary experiments , we experienced that addition of ifn- alone to a cocktail of proinflammatory cytokines is neither enough for optimal cd70 induction on mo - dc ( unpublished data ) . in contrast , addition of ifn- in combination with the tlr7/8 agonist r848 to the standard maturation cocktail from which il-6 was omitted resulted in a significant increase in cd70 expression ( unpublished data ) . from the above - mentioned observations , it may be evident that each compound added to a cytokine cocktail can influence dc phenotype and function in its own way and the ideal maturation mixture still needs to be well considered . maturation cocktail to prime th1-polarizing mo - dcs must contain pge2 [ 123 , 124 ] , for its migration - inducing potential , a tlr ligand ( e.g. , lps or r848 ( own unpublished data ) , but not poly i : c ) in combination with cd40l or ifn- , and some proinflammatory cytokines that have a positive impact on dc maturation ( e.g. , tnf- and il-1 ) . in addition , the cocktail must be free of il-6 which has been described to inhibit il-12p70 secretion [ 34 , 56 ] , while il-4 and il-10 need to be eliminated from the cocktail as well , since these cytokines prevent cd70 expression . alternatively , one can optimise dc immunogenicity through molecular modification of the cells , for example , by selective overexpression of genes encoding immune - stimulatory signals ( e.g. , il-12p70 [ 127 , 128 ] , cd40 or cd40 ligands [ 129 , 130 ] , and cd80/cd86 ) or by selective downmodulation of negative regulatory molecules , such as il-10 [ 131 , 132 ] , ido , socs1 [ 134 , 135 ] , and tgf - beta . mo - dc generation as well as maturation does not solely depend on the cytokine environment , but can also be influenced by oxygen levels , culture media and medium supplements . mo - dcs are generally differentiated ex vivo in incubators that maintain atmospheric oxygen levels of 21% o2 in combination with 5% co2 . indeed , the oxygen levels in tissues are usually 35% , whereas approximately 12% in arterial blood . in many inflamed and tumor tissues , even extremely low oxygen levels ( < 1% ) have been found . therefore it is evident that dcs experience rapid changes of oxygen supply during their migration in different tissues . although it is well recognized that tissue microenvironments are involved in regulating the development and function of immune cells , including b- and t cells , only few studies have investigated the effect of hypoxia ( < 1% oxygen saturation ) or physiological oxygen levels ( 3% oxygen saturation ) on the differentiation of human dcs from progenitors and their maturation . yang et al . reported that monocytes remain able to differentiate into dcs under hypoxia . however , these hypoxia - conditioned dcs displayed poor t cell - stimulatory activity and shifted towards a th2-stimulatory phenotype , presumably as a consequence of the marked reduction of mhc class ii and costimulatory molecule expression , as well as of reduced th1-polarizing cytokine secretion [ 140 , 141 ] . the observed inhibition of dc function by hypoxia could possibly explain why most tumors can efficiently escape from host immune surveillance . . showed only one year later that reoxygenation of hypoxia - differentiated dcs results in complete recovery of their mature phenotype and function , including a strong ability of the reoxygenated dcs to drive immune responses towards a proinflammatory th1/th17 direction . besides hypoxic conditions , one study investigated the influence of physiological oxygen levels on dc physiology and antigen - presenting capacity . surprisingly , no difference in expression of surface molecules ( cd54 , cd40 , cd83 , cd86 , hla - dr , cxcr4 , ccr7 ) nor secretion of tnf- , il-6 , and il-10 was observed between dc cultures under physiological ( 3% ) or atmospherical ( 21% ) oxygen levels . albeit that dcs stimulated with lps or cd40l under physiological o2 conditions secreted higher amounts of il-12p70 , these dcs did not elicit increased cd8 t - cell responses in vitro , as measured by ifn- secretion . taken together , there is still some controversy on whether physiologically or atmospherically oxygen levels must be used for dc culture and not enough data exist to robustly support a conclusion . for optimal production of clinical - grade dcs from peripheral blood monocytes , it is also important to choose the appropriate culture medium as well as potential serum supplements . initially , most mo - dcs used for clinical trials were generated in medium supplemented with plasma or serum , such as fetal bovine serum ( fbs ) containing xenologous proteins . for this , fbs can be immunogenic and possibly transfer bovine - related infections , including bovine spongiform encephalopathy [ 98 , 142 ] . however also the use of autologous or allogeneic ( pooled ) serum derived from patients or healthy controls , respectively , might lead to undesired immunomodulatory ingredients that can affect dc phenotype and function . therefore , it is clear that by eliminating the need for serum , an undesirable variable is removed making the medium more defined and consistent [ 142 , 143 ] . for this reason , several clinical - grade serum - free media are now commercially available and have been tested , including xvivo15 , xvivo20 , and aimv [ 98 , 144 , 145 ] . although so far only a small amount of studies have compared mo - dcs differentiated in serum - free medium with cells cultured in medium containing serum , they all agree that serum - containing media were more able to generate mature mo - dcs as compared with serum - free media [ 143 , 145 , 146 ] . the latter resulted mainly in the generation of semimature mo - dcs that express cd83 ( a mature dc marker ) as well as cd1a ( an immature dc marker [ 98 , 101 ] ) , and were slightly but consistently less able to produce il12p70 in response to maturation - inducing stimuli [ 142 , 143 ] . other characteristics , including yield , surface expression of maturation markers , in vitro survival , migratory capacities and induction of lymphocyte proliferation , were comparable between dcs differentiated in serum - free or serum - containing media [ 99 , 143 , 145 , 146 ] . in vivo assays following transfer of such mo - dcs generated in serum - free medium into humans are needed to decide whether the limited difference in cd1a expression and cytokine production is of true biological relevance . despite the use of mature dcs in vaccination trials , results from multiple clinical trials with dc - based vaccines have been contradictory and only fractions of enrolled patients show potent antitumor or antiviral immune responses with moderate clinical response rates ( approximately 1015% ) ( reviewed in [ 10 , 148151 ] ) or partial control of viremia and immune reconstitution [ 77 , 152154 ] , respectively . several studies suggested that this is because of inefficient activation of th1-polarized responses due to incomplete dc maturation [ 155157 ] . for this , different strategies are currently being pursued in order to improve the efficacy and outcome of dc - based cancer vaccines . considering the above - mentioned powerful immune - stimulatory properties possessed by il-12p70 , dc - based vaccination strategies may consistently benefit from incorporation or endogenous induction of this cytokine . in a first phase i clinical trial by the group of czerniecki , 13 breast cancer subjects were injected intranodally with short - term dcs activated with a cytokine - cocktail consisting of ifn- and lps in order to induce il-12p70-secreting dcs . the authors reported induction of robust detectable immunity as evidenced by in vitro monitoring of circulating vaccine - induced antigen - specific cd4 and cd8 t cells , as well as both t - and b - cell infiltrates into tumor region as well as dramatic reductions in tumor volume . additionally , dohnal et al . also showed the safety and feasibility of ifn-/lps - activated dcs for the treatment of paediatric cancer patients . besides that no adverse events were reported , they also demonstrated the potential of il-12p70-secreting dcs to induce cellular immune responses . it should , however , be noted that traxlmayr et al . reported il-12p70-dependent proliferation of immunosuppressive t cells in cancer patients vaccinated with il-12p70-secreting dcs , pointing to a negative regulatory feedback mechanism for dc - controlled immune responses . furthermore , it has been demonstrated by others [ 160162 ] that dcs electroporated with mrna encoding cd40 ligand , cd70 , and constitutively active toll - like receptor 4 , so - called trimix dcs , display increased potential for the induction and amplification of tumor - specific responses in patients with advanced melanoma . noteworthy , a positive delayed - type hypersensitivity assay ( dth ) postvaccination correlated with il-12p70 secreting capacity of vaccinated dcs . one of the major obstacles against successful dc vaccination , is certain immunosuppressive mechanisms triggered by the tumor cells or viruses . indeed , under the influence of the tumorigenic microenvironment , the host dcs may acquire a tolerogenic phenotype . these tumor - conditioned dcs could , in return , produce a variety of immunosuppressive molecules and thus further supporting tumor immune escape . we ( unpublished data ) and others have previously shown that dc differentiation and functional activities are tightly regulated by this cytokine [ 165 , 166 ] . in return , dcs can secrete il-10 and effectively inhibit t - cell activation . additionally , numerous viruses , including human cmv , hiv , herpes simplex virus type 1 , and measles virus , target dcs [ 167 , 168 ] , and have evolved strategies to specifically modulate dc phenotype and/or function , thereby promoting virus - mediated immune escape . for example , dcs infected by human cmv are characterized by reduced expression of mhc class i and ii molecules , costimulatory molecules , and proinflammatory cytokines , which consequently results in reduced t - cell activation . nowadays , emerging evidence indicates that one of the most effective ways to enhance the efficacy of dc - based immunotherapy is by targeting the negative arm of immune regulation . for future clinical trials , this may be achieved by the use of small interfering rna ( sirna ) for knocking down il-10 expression by dcs [ 131 , 132 ] , or other negative regulatory molecules , such as indoleamine 2,3-dioxygenase ( ido ) , suppressors of cytokine signalling 1 ( socs1 ) [ 134 , 135 ] , and transforming growth factor ( tgf)-beta . indeed , inhibition of expression of these regulatory molecules has been demonstrated to significantly enhance the abilities of dcs to present tumor antigens , to produce il-12p70 , and to induce effectively antitumor responses . with respect to tackle different arms of the immune system , many different approaches are currently being pursued . in particular , considering the distinct ability of different dc subsets in inducing both innate and adaptive immunity , the exploitation of specific subsets of dcs to elicit the desired immune response is foreseen . although pdcs primarily contribute to innate antiviral immune responses by producing ifn-/ , this ability also has been reported to activate other dcs , including those involved in cross - priming , and consequently greater activation of adaptive immune responses . in doing so hence , combination therapies aiming at interaction of pdcs and cdcs to stimulate t - cell priming , and hence effective anti - tumor or antiviral immunity are needed in cancer patients and chronically infected patients ( figure 1 ) . additionally , differentiation of monocytes into dcs with cocktails including gm - csf and il-15 will generate cells with the phenotype and characteristics of langerhans cells ( lc ) , which are far more efficient in vitro in priming antigen - specific cd8 t cells than dcs derived with gm - csf and il-4 [ 111 , 171 ] . as described by others , lcs are very efficient in cross - presenting peptides to cd8 t cells , which acquire potent cytotoxicity and are able to efficiently kill target cells , including tumor cell lines that express peptide - hla complex , only at low amounts in an il-15-dependent manner . the pivotal role of lc to allow maximal stimulation of both humoral and cellular immune responses , supports the important concept for targeting lc in the design of vaccines that aim at eliciting strong cellular immune responses [ 66 , 173 , 174 ] . the recent identification of human cd141 dcs that can effectively cross - present antigens has clear implications for the design of new therapies to treat cancers and infectious diseases with improved efficacy . it has been reported that the limiting cytokine for the development of the murine counterpart is the fms - related tyrosine kinase 3 ligand ( flt3l ) , rather than gm - csf or m - csf , which has major influence on the development of inflammatory and migratory dcs [ 175177 ] . however , although poulin et al . made a first attempt to delineate in vitro culturing conditions for the generation of cd141 dcs from human progenitor cells , further optimization of such protocols is necessary to allow for their use in adoptive transfer immunotherapy approaches . current efforts for dc - based modalities have been compromised by a failure to utilize the full potential of dcs . however , even though only limited success rates have been achieved to date , dc vaccination remains a promising immunological approach against tumors and/or viruses and deserves further exploration . alternative strategies to enhance dc immunogenicity by functional conditioning and molecular modifications have been investigated in vitro . the different findings discussed here , indicate that dcs can indeed be functionally conditioned and genetically modified to acquire an enhanced immunogenic phenotype . for this , time has come to bring dc - based immunotherapy to the next level and implement above - mentioned observations in a standardized regimen for alternatively conditioned dcs . results from first clinical trials will subsequently reveal their potential in order to improve treatment of cancer and chronic infections .

earlier investigations have revealed a surprising complexity and variety in the range of interaction between cells of the innate and adaptive immune system . our understanding of the specialized roles of dendritic cell ( dc ) subsets in innate and adaptive immune responses has been significantly advanced over the years . because of their immunoregulatory capacities and because very small numbers of activated dc are highly efficient at generating immune responses against antigens , dcs have been vigorously used in clinical trials in order to elicit or amplify immune responses against cancer and chronic infectious diseases . a better insight in dc immunobiology and function has stimulated many new ideas regarding the potential ways forward to improve dc therapy in a more fundamental way . here , we discuss the continuous search for optimal in vitro conditions in order to generate clinical - grade dc with a potent immunogenic potential . for this , we explore the molecular and cellular mechanisms underlying adequate immune responses and focus on most favourable dc culture regimens and activation stimuli in humans . we envisage that by combining each of the features outlined in the current paper into a unified strategy , dc - based vaccines may advance to a higher level of effectiveness .

1. Introduction 2. Origin and Subsets of DCs 3. The Immune System against Cancer and 4. Harnessing DCs for Clinical Use 5. Taking DC into the Clinic 6. Conclusion

dendritic cells ( dcs ) , originally described by steinman and cohn , serve as a crucial link between innate and adaptive immunity . as sentinel members of the innate immune system , dcs respond to antigens and molecules containing pathogen - associated molecular patterns ( pamps ) or damage - associated molecular patterns ( damps)so - called danger signals by the generation of protective cytokines . as members of the acquired immune system , dcs respond to these harmful molecules by efficient antigen uptake , processing , and presentation , and hence dcs are crucial in the initiation of adaptive immune responses . because of their immunoregulatory capacities and because very small numbers of activated dcs are highly efficient at generating immune responses against antigens , dcs have been vigorously used in clinical trials in order to elicit or amplify immune responses against cancer and chronic infectious diseases . although an impressive amount of data has been obtained from these clinical trials completed thus far , the outcomes were not in line with initial expectations [ 810 ] . a critical issue in the development of dc - based vaccines is that their ability to stimulate immune responses depends largely on the activation state of dcs . in this paper , we discuss the continuous quest for the best in vitro conditions in order to generate clinical - grade dcs with a potent immunogenic potential . for this , we explore the molecular and cellular mechanisms underlying adequate immune responses and focus on optimal dc culture regimens and activation stimuli in humans . once dcs have captured a foreign nonself - antigen , they undergo a highly regulated maturation process and remodel into fully activated antigen - presenting dcs capable to elicit effective immune responses . during the maturation process , dcs exit the nonlymphoid tissues to migrate via afferent lymph to lymphoid tissues subsequently , mature dcs will activate ( naive ) t- and b - lymphocytes that recognize the presented antigen as peptide - mhc complexes on the surface of the dc . yet , additionally , positive amplification of antigen presentation via costimulation and secretion of various cytokines is also crucial to induce proper immune responses [ 3 , 15 ] ( cfr . plasmacytoid dcs ( pdcs ) , also referred to as type i ifn - producing cells ( ipcs ) , are the key effectors in the innate immune system because of their extra - ordinary capacity to produce type i ifn upon viral infection [ 16 , 17 ] . in human blood , differences in dc subsets can be identified based on differential expression of specific markers : pdcs express cd303 ( bdca-2 ) , cd304 ( bdca-4 ) , and cd123 ( il-3r ) , whereas cdcs are characterized by their expression of cd1c ( bdca-1 ) and cd11c [ 18 , 19 ] . because of the unique biological function of each dc subset , it was proposed that a specific dc lineage determines the outcome of t - cell contact , that is , tolerance or immunity . furthermore , it has been shown that pdcs augment immune responses by cross - talking with cdcs by the production of ifn- , thereby playing a key role in effective stimulation of adaptive immunity as well . of particular importance is their superior antigen cross - presentation capacity , expression of the xc chemokine receptor 1 ( xcr1 ) , and their capacity to produce high levels of bioactive il-12p70 . therapeutic vaccines to treat chronic infectious diseases ( such as human immunodeficiency virus ( hiv ) , cytomegalovirus ( cmv ) , hepatitis b virus ( hbv ) , and hepatitis c virus ( hcv ) ) , or numerous tumor types ( including melanoma , leukaemia , breast , and prostate cancer ) mainly aim to induce antigen - specific cell - mediated immunity to clear infected cells and eliminate tumor cells . recent studies have shown that dcs play a critical role in directing effector t - cell responses towards a th1 , th2 , th17 , or regulatory t cell ( treg ) response [ 31 , 32 ] . briefly , upon maturation , dcs upregulate the expression of certain products necessary to supply t lymphocytes with the 3 signals that will determine their activation status and general fate : antigen - specific signalling via the t cell receptor ( tcr ) , mediated by the binding of mhc - peptide complexes to the tcr drives the initial interaction between dcs and t cells ( i.e. ligation of cd28 provides costimulatory signals required for t cell activation : ( i ) altering the threshold level of tcr ligation required for activation , ( ii ) reducing the time needed to stimulate naive t cells and ( iii ) enhancing the magnitude of the t cell response . the b7 family of costimulatory molecules has been extensively reviewed elsewhere [ 41 , 42 ] . highly - coordinated p35 and p40 gene expression results in the formation of the biologically active form il-12p70 and is essential for initiation of an effective immune response . besides the activation of innate and antigen - specific adaptive immunity against the tumor cells , the antitumor effects of il-12p70 are based on the ability to inhibit tumor angiogenesis through ifn- [ 52 , 53 ] . thus , to develop an efficient vaccine against tumors or chronic infectious diseases , dcs producing the biologically active form il-12p70 are desired . rather than simply recruiting th1 cells and ctl , vaccines should be designed to recruit other cellular arms of the immune system as well , for example , nk cells and antibody - producing b cells . in this perspective , it has been shown that dcs also play a key role in the activation of nk cells that can have powerful effects against tumor cells , particularly those with attenuated mhc expression . indeed , in response to dc - derived cytokines , such as il-12p70 and il-18 , nk cells are able to produce ifn- . overall , these findings support the concept to include dc - nk interactions in order to improve dc - based immunotherapy . furthermore , recent studies that have resulted in reappraisal of the potential of antibodies in the control of tumors and viruses support the strategy that dc - based vaccines should also be designed with antibody production in mind [ 6163 ] . these observations suggest that il-12p70 could constitute a potent vaccine adjuvant in situations when both the cellular and humoral arms of the immune system are required , such as cancer [ 62 , 63 ] and hiv . taken together , the goal of many dc - based vaccination protocols is to cultivate dcs that are capable of expressing immunostimulatory cytokines ( il-12p70 ) and costimulatory molecules ( cd70 ) in parallel with antigen presentation . since expression of costimulatory molecules and cytokine secretion can be influenced by environmental signals during dc maturation , it is necessary to find an optimal cytokine environment for dc maturation in order to create a powerful vaccine against several cancer types or chronic infectious diseases . various attempts have subsequently been made in order to harness dc to achieve most powerful immunity , including strategies to enhance or stabilise antigen - specific stimulation , as well as essential costimulatory modulation of dcs . additionally , dcs transduced ex vivo with tumor- or viral - derived mrna or dna [ 7274 ] , fused with tumor cells [ 75 , 76 ] , or directly loaded with tumor- or viral - derived peptides [ 77 , 78 ] have been tested for the induction of antigen - specific immune responses in vitro and in vivo . while the use of peptides as a source of antigen has several limitations when implementing clinical trials with antigen - loaded dcs , including human leukocyte antigen ( hla ) restriction as well as a limited number of identified immunodominant tumor- and virus - associated antigens , we and others [ 8084 ] have previously shown that dcs transfected with mrna - encoding antigens are superior to other loading strategies to induce immune responses . mrna transfection will generate multiple antigenic epitopes , possibly more immunogenic than those already characterized , independent of the patient 's hla haplotype . in addition , mrna can be isolated and amplified from autologous tumor or virally infected cells in order to obtain mrna encoding patient - specific antigens [ 8688 ] . moreover , because mrna only has a short half - life and does not integrate in the host genome , genetic modification of dcs by mrna electroporation is considered to be highly safe and an easily applicable clinical tool . the earliest studies on dc vaccination were initiated in 1993 and utilized whole blood leukapheresis products with subsequent gradient centrifugation procedures to enrich for rare immature dc precursors of the peripheral blood before antigen loading and maturation . in a second attempt to directly isolate dcs from peripheral blood , moreover , although blood dcs from patients with a malignant or chronic infectious disease may seem to have normal distributions , they might have some functional defects , such as a lower expression of costimulatory molecules or an impaired capacity to stimulate autologous antigen - specific t cells [ 92 , 93 ] . currently , dcs for vaccination studies are generally obtained in large numbers after in vitro generation . at first , human dcs were cultured from cd34 haematopoietic progenitors in the presence of granulocyte - macrophage colony stimulating factor ( gm - csf ) and tumor necrosis factor alpha ( tnf- ) [ 94 , 95 ] . however , only few studies that used cd34-derived dc preparations for vaccination protocols in clinical phase i studies have been reported [ 96 , 97 ] . in doing so , large numbers of monocyte - derived ( mo-)dcs are obtained without necessity for pretreatment of donors with any cytokines to mobilize dc progenitor cells . yet , the design of dc - based clinical trials varies greatly , including dc preparation , and therefore , standardization and further improvement for clinical use are needed . while a combination of granulocyte - macrophage colony - stimulating factor ( gm - csf ) with il-4 is most commonly used to induce immature dcs from monocytes [ 100 , 101 ] , a variety of other cytokines , such as ifn- [ 102104 ] , tnf- [ 105 , 106 ] , and il-15 have been used in combination with gm - csf for this purpose . reported independently that ifn- induces rapid differentiation of freshly isolated gm - csf - treated human monocytes into mo - dcs endowed with potent functional activities , both in vitro and in vivo [ 102 , 103 ] , possibly mediated by ifn--dependent induction of cd70 expression . it must be noted however that ifn- also induces activity of rnases , and can not therefore be used in in vitro culture regimens for dcs when mrna - based in vitro modification of dcs is wanted . described that tnf- facilitates the induction of adaptive immunity also by promoting dc differentiation from cd14 blood precursors in vitro . however , it has been reported in contrast that tnf--treated semi mature dcs induce tolerance in experimental acute encephalitis ( eae ) , a mouse model for multiple sclerosis . moreover , due to strong plastic adherence before and to a lesser extent also after maturation , il-15 and tnf- treatment for dc generation results in a lower dc yield . regardless of how they are generated , it is important that dcs are activated to a mature phenotype , since immature dcs are no longer considered as competent candidates for vaccination trials because of their low t - cell activation potential [ 113115 ] . most dc culture regimens that have been commonly employed in clinical trials have activated dcs through the use of individual cytokines associated with inflammation or inflammatory cytokine cocktails . fully - mature dcs induced by this combination of inflammatory cytokines have been consistently observed as superior to immature dcs in promoting a higher degree of specific t - cell priming in vitro and in vivo . in this perspective , mature dcs with the potential to produce high amounts of biologically active il-12p70 ( 1015 ng / ml ) were obtained by mailliard et al . proposed a cytokine cocktail consisting of tnf- , il-1 , ifn- , r848 and pge2 as the best cocktail to allow large - scale processing of clinical - grade mo - dcs with the capacity to secrete il-12p70 . in addition , whereas dc maturation by tlr ligand alone ( including lps , cpg , and poly i : c ) has been reported to increase expression of classical activation markers as well as many inflammatory cytokines , a tlr agonist alone does not result in a substantial cd8 t - cell response , which is probably due to no or very low levels of il-12p70 secretion as well as insufficient induction of cd70 by tlr ligand stimulation alone . in preliminary experiments , we experienced that addition of ifn- alone to a cocktail of proinflammatory cytokines is neither enough for optimal cd70 induction on mo - dc ( unpublished data ) . from the above - mentioned observations , it may be evident that each compound added to a cytokine cocktail can influence dc phenotype and function in its own way and the ideal maturation mixture still needs to be well considered . although it is well recognized that tissue microenvironments are involved in regulating the development and function of immune cells , including b- and t cells , only few studies have investigated the effect of hypoxia ( < 1% oxygen saturation ) or physiological oxygen levels ( 3% oxygen saturation ) on the differentiation of human dcs from progenitors and their maturation . showed only one year later that reoxygenation of hypoxia - differentiated dcs results in complete recovery of their mature phenotype and function , including a strong ability of the reoxygenated dcs to drive immune responses towards a proinflammatory th1/th17 direction . for optimal production of clinical - grade dcs from peripheral blood monocytes , it is also important to choose the appropriate culture medium as well as potential serum supplements . for this , fbs can be immunogenic and possibly transfer bovine - related infections , including bovine spongiform encephalopathy [ 98 , 142 ] . for this reason , several clinical - grade serum - free media are now commercially available and have been tested , including xvivo15 , xvivo20 , and aimv [ 98 , 144 , 145 ] . the latter resulted mainly in the generation of semimature mo - dcs that express cd83 ( a mature dc marker ) as well as cd1a ( an immature dc marker [ 98 , 101 ] ) , and were slightly but consistently less able to produce il12p70 in response to maturation - inducing stimuli [ 142 , 143 ] . other characteristics , including yield , surface expression of maturation markers , in vitro survival , migratory capacities and induction of lymphocyte proliferation , were comparable between dcs differentiated in serum - free or serum - containing media [ 99 , 143 , 145 , 146 ] . despite the use of mature dcs in vaccination trials , results from multiple clinical trials with dc - based vaccines have been contradictory and only fractions of enrolled patients show potent antitumor or antiviral immune responses with moderate clinical response rates ( approximately 1015% ) ( reviewed in [ 10 , 148151 ] ) or partial control of viremia and immune reconstitution [ 77 , 152154 ] , respectively . for this , different strategies are currently being pursued in order to improve the efficacy and outcome of dc - based cancer vaccines . considering the above - mentioned powerful immune - stimulatory properties possessed by il-12p70 , dc - based vaccination strategies may consistently benefit from incorporation or endogenous induction of this cytokine . in a first phase i clinical trial by the group of czerniecki , 13 breast cancer subjects were injected intranodally with short - term dcs activated with a cytokine - cocktail consisting of ifn- and lps in order to induce il-12p70-secreting dcs . besides that no adverse events were reported , they also demonstrated the potential of il-12p70-secreting dcs to induce cellular immune responses . reported il-12p70-dependent proliferation of immunosuppressive t cells in cancer patients vaccinated with il-12p70-secreting dcs , pointing to a negative regulatory feedback mechanism for dc - controlled immune responses . furthermore , it has been demonstrated by others [ 160162 ] that dcs electroporated with mrna encoding cd40 ligand , cd70 , and constitutively active toll - like receptor 4 , so - called trimix dcs , display increased potential for the induction and amplification of tumor - specific responses in patients with advanced melanoma . indeed , under the influence of the tumorigenic microenvironment , the host dcs may acquire a tolerogenic phenotype . in return , dcs can secrete il-10 and effectively inhibit t - cell activation . nowadays , emerging evidence indicates that one of the most effective ways to enhance the efficacy of dc - based immunotherapy is by targeting the negative arm of immune regulation . for future clinical trials , this may be achieved by the use of small interfering rna ( sirna ) for knocking down il-10 expression by dcs [ 131 , 132 ] , or other negative regulatory molecules , such as indoleamine 2,3-dioxygenase ( ido ) , suppressors of cytokine signalling 1 ( socs1 ) [ 134 , 135 ] , and transforming growth factor ( tgf)-beta . indeed , inhibition of expression of these regulatory molecules has been demonstrated to significantly enhance the abilities of dcs to present tumor antigens , to produce il-12p70 , and to induce effectively antitumor responses . with respect to tackle different arms of the immune system , many different approaches are currently being pursued . in particular , considering the distinct ability of different dc subsets in inducing both innate and adaptive immunity , the exploitation of specific subsets of dcs to elicit the desired immune response is foreseen . although pdcs primarily contribute to innate antiviral immune responses by producing ifn-/ , this ability also has been reported to activate other dcs , including those involved in cross - priming , and consequently greater activation of adaptive immune responses . the pivotal role of lc to allow maximal stimulation of both humoral and cellular immune responses , supports the important concept for targeting lc in the design of vaccines that aim at eliciting strong cellular immune responses [ 66 , 173 , 174 ] . it has been reported that the limiting cytokine for the development of the murine counterpart is the fms - related tyrosine kinase 3 ligand ( flt3l ) , rather than gm - csf or m - csf , which has major influence on the development of inflammatory and migratory dcs [ 175177 ] . made a first attempt to delineate in vitro culturing conditions for the generation of cd141 dcs from human progenitor cells , further optimization of such protocols is necessary to allow for their use in adoptive transfer immunotherapy approaches . current efforts for dc - based modalities have been compromised by a failure to utilize the full potential of dcs . however , even though only limited success rates have been achieved to date , dc vaccination remains a promising immunological approach against tumors and/or viruses and deserves further exploration . alternative strategies to enhance dc immunogenicity by functional conditioning and molecular modifications have been investigated in vitro . for this , time has come to bring dc - based immunotherapy to the next level and implement above - mentioned observations in a standardized regimen for alternatively conditioned dcs . results from first clinical trials will subsequently reveal their potential in order to improve treatment of cancer and chronic infections .

the conformational behavior of dna in solution is driven by a subtle interplay among various intramolecular degrees of freedom and interactions with the environment . for example , when in a b form , correlated motions of the backbone , sugar and glycosidic dna torsions lead to the dynamical transitions between bi and bii states , as well as the extent of local sampling of geometries characteristic of the a form dna as manifested in sugar puckering transitions between the south and north conformations . on a global scale , the balance between bonded and nonbonded intramolecular parameters along with interactions with the environment influence the conformational equilibrium between the a , b , and z forms of dna . moreover , interactions with mobile ions and hydration effects are known to contribute to the overall dna stability and regulate a number of critical polymeric properties , such as persistence length and stiffness . over the past decade , computational modeling of dna had some successes in predicting selected properties of dna and helped to rationalize many dna related experiments . in particular , modeling allowed for atomistic investigations of dna energetics , mechanisms of structural transitions and the impact of environmental effects on dna information that is not readily accessible to experimental techniques , such as x - ray crystallography and nmr . these studies were based on additive force fields ( ff ) for dna , including charmm , amber , bristol - myers squibb , and gromos . while successful in many respects , these models show a number of limitations , potentially associated with the lack of explicit electronic polarization in the model , including limited treatment in the equilibrium between the different conformations of dna as well as relative energies of different conformations . while continued developments in the additive ffs have partially overcome these limitations , it is anticipated that extending the form of the potential energy function to include the explicit treatment of electronic polarization may lead to improvements in dna ffs , including yielding a more accurate description of the underlying physical forces dictating the structure and dynamics of dna . recently , we have presented the first - generation charmm polarizable ff for dna based on the classical drude oscillator formalism . briefly , electronic induction in the model is represented by introducing a massless charged particle ( i.e. , the drude oscillator ) attached to each polarizable atom by a harmonic spring . in practice , charge redistribution in response to changes in the local electric field is approximated by self - consistently updating the positions of the auxiliary particles to their local energy minima for any given configuration of the atoms in the system . this self - consistent field ( scf ) approach takes into account the permanent electric field due to the fixed charges and the contribution of the induced dipoles to the electric field . in the context of molecular dynamics ( md ) simulations , the scf calculation is substituted with an extended lagrangian integrator that treats the drude oscillators as dynamic variables , allowing for the computational efficiency required for long - time md simulations . to date , the drude model has been implemented in charmm , namd and chemshell quantum mechanics / molecular mechanics ( qm / mm ) . as discussed elsewhere , due to the need of use of a 1 fs integration time step ( versus the 2 fs time step customarily used in additive md simulations ) , use of the drude polarizable ff in simulations yields an approximately 4-fold overhead compared to additive simulations . in addition , a module , the drude prepper has been added to the charmm - gui , facilitating the conversion of charmm psf and coordinate files into the corresponding files required for drude - based simulations including example inputs for charmm and namd . in our published study on development of the drude force field for dna we focused mainly on details of parametrization of the highly correlated backbone , sugar , and glycosidic torsion angles , and on extensive validation of the final parameters by comparing md simulation results with a wide range of experimental data on dna in solution . a notable observation from those simulations was the significant enhancement and variation of base dipole moments as compared to the c36 additive ff , indicating that the underlying physical forces dictating the properties of dna are significantly different in the polarizable model . however , the discussion in that paper covered only a small part of the optimization process , not addressing such critical aspects of the modeling as fine - tuning of the interactions between dna and mobile ions and the balance between dna electrostatic and hydration effects , subtle corrections in the internal base parameters , and the impact of these on the overall dna stability and subtler conformational phenomena , including bi / bii transitions and a - to - b equilibrium . presently , we focus on the above aspects of the drude ff development and additional testing of the optimized dna parameters against a range of experimental data and theoretical estimations . our optimization procedure involves approaches ranging from qm calculations on various model compounds and their complexes to investigating the hydration and osmotic properties of the chemically relevant ionic systems to studying the impact of the mobile ions on dna structural stability . we note that the final results presented here are based on the identical set of parameters developed in our earlier study on the drude force field for dna . some results are also presented for a default parameter set , which is that based on direct transfer of parameters optimized targeting small model compounds representative of dna with only minimal adjustment of selected dihedral parameters . in the present study we used a number of small model systems to probe how electrostatic , hydration and other effects in the dna duplex impact overall dna stability , as well as subtler aspects of dna structural behavior in solution . those systems are shown in figure 1 . dihedral angles shown for 1 are defined as follows : = o3p o5c5 , = c4c3o3p , = c3o3p o5 and 4 = c3c4o4c1. in 4 and 5 , r = ch3 or h. model compound 1 , composed of the sugar and a 3 methylated phosphate group ( figure 1 ) was previously used to study correlations between and torsions in the context of bi / bii transitions in dna . in our recent parametrization study we discussed in detail the optimization of these and other dihedral angles . in the present study , by analyzing individual energetic contributions of 1 , we demonstrate that , in addition to the torsion parameters , adjustments of electrostatic parameters ( partial charges ) was necessary to achieve satisfactory agreement with both qm data on this model compound and experimental data on bi / bii transitions and a - to - b equilibrium in dna duplexes in solution . qm data for 1 , as previously published , are represented by a 2d versus potential energy surface based on structure optimizations at the mp2/6 - 31(+)g(d ) level with and being sampled in increments of 15 , while restraining the overall conformation to that of the canonical b dna form . specifically , one of the sugar moiety dihedral angles , 4 , and the backbone torsion , , were restrained . analogous mm calculations were carried out using the charmm program and the drude polarizable force field . the model was minimized first using the adopted - basis newton raphson ( abnr ) algorithm before performing the 2d dihedral potential energy scan . dihedral harmonic restraints of 10 kcal / mol / rad were used with minimizations performed to a gradient of 10 kcal / mol / . alteration of partial charges in 1 prompted further investigation of hydration properties of a smaller model compound , 2 , dimethylphosphate ( dmp ) , which is representative of the phosphate group in the dna backbone . the alteration of the electrostatic parameters to correct the behavior of 1 leads to changes in the hydration of 2 . the optimization was performed by targeting , simultaneously , reproduction of the hydration free energy of dmp and the gas - phase heterodimeric interactions with water . water monohydrates , using gaussian 09 , were performed with dmp fixed in the geometry obtained from gas phase optimization at the mp2/6 - 31+g * level of theory and the water molecule fixed in the geometry of the swm4-ndp model . three minimum energy conformations of dmp were considered : gg , tt , and gt , where g and t stand for gauche and trans , respectively . the relative orientations of the dmp and water molecules were also fixed , as shown in figure 2 , with only the interaction distances optimized at the mp2/6 - 31+g * level of theory . interaction energies were then determined via single point calculations at the ri - mp2/cc - pvqz level of theory , performed on the mp2/6 - 31+g * optimized geometries . the single point calculations were performed using the program q - chem , with counterpoise correction included to account for basis set superposition error ( bsse ) . mm polarizable calculations were performed with charmm using the swm4-ndp water model and restraints and orientations identical to those used in the qm calculations . schematic representation of the interaction orientation between dmp anion and water molecules . while analysis was performed on the basis of water the numbering of the water orientations corresponds to that used in table 2 and tables s1 and s2 of the supporting information . another important aspect of dna modeling is the ability of the ff to properly treat interactions between dna and mobile ions . the first approach deals with the detailed analysis of the na and cl distributions around a dna duplex , ignoring sequence specific effects . the second approach deals with gas - phase calculations on model systems involving interactions of na with individual nucleic acid bases , as well as with dmp . in particular , na was positioned at distances ranging from 1 to10 away from the chemically relevant atoms at various orientations relative to the base . n1 of adenine , n3 of cytosine , n1 of guanine and n3 of thymine were chosen for probing interactions with na . in the case of guanine and thymine , whose n atoms are protonated , out - of - plane interactions with na were studied . additionally , calculations on the ade - ade / na trimer model system were performed , with na placed at distances [ 110 ] from the bases in the in - plane orientation and bases fixed in the relative orientation corresponding to b form dna . analogous calculations were carried out for dmp / na dimers , probing interactions among the phosphate group and na , with the latter positioned at various orientations and distances from the anionic o1p / o2p and ester o3/o4 oxygen atoms . visual representations of the model systems are provided in the results and discussion section along with the corresponding qm and mm energy profiles . the distance increment in positioning na from the atom of interest was 0.1 in the range of [ 14 ] , and 1 at larger separations . qm potential energy profiles for all complexes were based on gas phase structures optimized at the mp2/6 - 31++g(2df,2pd ) level of theory using gaussian 09 with counterpoise corrections applied to account for bsse . equivalent single point calculations were then performed using the drude polarizable force field for nucleic acids . additional analysis of the nucleic acid bases was performed on their geometries and vibrational spectra , motivated by preliminary simulations of the drew dickerson ( dd or ecori ) dodecamer , indicating that the conformational behavior of the individual nucleic acid bases may contribute to dna instability . this prompted a partial reoptimization of the internal parameters for the nucleic acid bases . as discussed below , this mainly involved manual adjustment of the base valence angles leading to a better representation of the vibrational frequencies and improvements in the geometries . target data for bonds and angles involving hydrogen atoms were obtained from the previously published qm geometries optimized at the mp2/6 - 31+g * level of theory , while all other bond length and angle parameters were optimized to reproduce target crystallographic geometries obtained from the cambridge structural database survey . charmm vibrational spectra were calculated using the molvib utility of the charmm program , with the target infrared spectra calculated ab initio at the mp2/6 - 31+g * level of theory . internal coordinate assignment was performed according to the method of pulay et al . , and a scale factor of 0.9434 was applied to qm calculated normal modes to account for limitations in the level of theory used . hydration free energies , ghyd , were calculated via the free energy perturbation ( fep ) method using the protocol of deng and roux . in this approach , the lj potential is separated into purely repulsive and attractive components using the weeks chandler simulation systems involved a periodic box containing a single dmp molecule solvated by 250 swm4-ndp water molecules . simulations were performed using charmm and the velocity verlet integrator , which includes treatment of drude particles via an extended lagrangian formalism . the shake algorithm was employed to constrain covalent bonds to hydrogen , and an integration time step of 1 fs was used . simulations were performed in the npt ensemble at 298.15 k and 1 atm , using a nos electrostatic interactions were treated using particle mesh ewald ( pme ) summation with a coupling parameter of 0.34 and a sixth - order spline for mesh interpolation . the system was equilibrated for 100 ps at each value of the thermodynamic coupling parameter , , after which the system was simulated for 200 ps of production md . for the calculation of dispersive and electrostatic contributions to ghyd , was assigned values ranging from 0.0 to 1.0 in equally spaced increments of 0.1 . for the repulsive term , extra windows were required in the low region , such that took additional values of 0.05 , 0.15 , 0.25 , and 0.35 . the dispersive and electrostatic contributions to ghyd were calculated using thermodynamic integration ( ti ) , with the repulsive contribution calculated using a soft - core scheme , and unbiased using the weighted histogram analysis method ( wham ) . because these simulations were performed under periodic boundary conditions using the particle sum ( p - sum ) convention , the interface potential between the bulk liquid and the external dielectric medium is zero . in reality , solvation of a charged species across a liquid / vacuum interface also depends on a nonzero and water - model - dependent interface potential ( lv ) . therefore , an additional contribution to the free energy , equal to qionlv , must be accounted for when performing calculations using the p - sum convention under periodic boundary conditions . when considering a system consisting of a monovalent anion solvated with swm4-ndp water the gas phase contribution to the hydration free energy was calculated using an identical procedure , but considered only an isolated dmp molecule in the gas phase , rather than a single molecule solvated in a water box . system setup , md simulation protocol , and the calculation of osmotic pressures for the bulk electrolyte solutions closely follow the method developed by roux and co - workers . three aqueous electrolyte solutions of ( 1 ) na and cl ions , ( 2 ) na and dmp ions , and ( 3 ) na and acetate ions were simulated at two molal concentrations each , approximately corresponding to solutions having molarities of 1 m and 3 m , respectively ( see below ) . each simulation system contains three compartments separated by two virtual semipermeable membranes aligned with the xy - plane . the electrolyte compartment centered on the absolute origin of coordinates is enveloped by compartments containing pure water . water can freely pass through the membranes separating electrolyte and water solutions , while the ions are subject to the force of the half - harmonic planar potential on each side of the ionic compartment whose action mimics the ideal behavior of a semipermeable membrane inducing osmosis . a representative snapshot from md simulation of the na / dmp electrolyte solution at 3 m concentration is shown in figure 3 . the instantaneous osmotic pressure , = fmemb / a , equals the instantaneous total force on the membranes,1divided by the total area of the membranes , a. in this expression , k is the force constant of the membrane potential , 10 kcal/(mol ) , d = 48 is the linear size of the cubic ionic compartment , and zi is the z - coordinate of the i ion . values of the instantaneous force were recorded every 0.5 ps , and the total md simulation run was for 70 ns for each simulated electrolyte system . the convergence of md simulations was verified by calculating osmotic pressures from the two halves of the md trajectory . standard deviations around the averaged values were obtained based on seven 10 ns blocks of the total md trajectories . snapshot from an md simulation of the 3 m aqueous solution of the dmp and na ions . sodium ions are yellow , dmp ions are cyan , and water is red / white . given the values of experimentally measured osmotic pressure coefficient , =/id , the experimental value of the osmotic pressure at molal concentration , m , is determined as follows:23where r is the gas constant , t is temperature in kelvin , vm is the molar volume of pure water at 1 bar and room temperature , and is the total number of cations and anions per formula unit ( in our case of 1/1 electrolyte solutions = 2 ) . it is important to note that , following the convention used in experimental measurements , the osmotic pressure of an ideal solution in the above expression is proportional to the molal concentration of the electrolyte , m , which differs from the traditionally used molar concentration m. therefore , to enable direct comparison with the experiment , the actual molal concentrations need to be computed from md simulations based on the values of mean densities of the cations , anions , and pure water in the ionic compartment as follows:4where overbars indicate ensemble averages , and msalt is the molar concentration of the salt . the resulting molal concentration is then used to obtain the osmotic pressure of an ideal solution via eqs 2 and 3 . an identical approach for computing molal concentrations and osmotic pressures is used in ref ( 74 ) . md simulation protocol for studying electrolyte solutions is analogous to that described below for the dna duplexes . the first option allows for the three dimensions of the periodic cell to fluctuate independently , while the second one keeps the ratio of the unit cell constant in the xy - plane while allowing fluctuations along the z axis in the course of md simulation . in addition , the tclforce script of namd was utilized to impose semiharmonic restraints on ionic species during md simulations , as elaborated above . simulations were carried out with the charmm drude polarizable and the additive c36 force fields . each simulated system included 45 000 particles , including atoms , lone pairs , and drude particles , in the case of the polarizable model and 26 000 atoms in the additive model . simulated systems included the ecori dodecamer and junfos 16-mer whose structural behavior was extensively investigated in our original study . additionally , we studied a shorter 10-base - pair dna oligomer ( 1dcv ) . all dna molecules were solvated with the polarizable swm4-ndp water model in cubic boxes with water molecules extending at least 15 from the dna surface , with addition of neutralizing na ions and an extra 120 mm of nacl salt . initial configurations were generated by simulating the analogous additive charmm36 ( c36 ) systems for several nanoseconds according to the protocol described elsewhere , taking the last snapshot from these runs as inputs for subsequent drude simulations . charmm was used for generating the polarizable drude system , self - consistent relaxation of the drude particle positions , and short equilibration of the whole system , as elaborated elsewhere . for the production md simulation namd ( version 2.9 ) was used whose self - consistent treatment of the drude polarizable model is based on a dual thermostatting scheme and langevin dynamics . in particular , real atoms and polarizable degrees of freedom ( drude particles ) were coupled to the thermostats at 300 and 1 k , respectively . electrostatic interactions were treated using the pme summation with a coupling parameter of 0.34 and a sixth - order spline for mesh interpolation . nonbonded pair lists were maintained out to 16 , and a real space cutoff of 12 was used for the electrostatic and lennard - jones ( lj ) terms . all covalent bonds involving hydrogen as well as the intramolecular geometries of water molecules were constrained using the settle algorithm . the hardwall feature enabled the use of a 1 fs time step in md simulations . as previously described , this feature is associated with a hard wall reflective term in the potential energy function that has been added to resolve the polarization catastrophe problem in drude md simulations . this term was invoked only when drude particles moved > 0.2 away from their parent nuclei during md simulations . to analyze in detail the na and cl distributions around a dna duplex , we calculated their radial distribution functions ( rdfs ) from md simulations following the approach developed by savelyev et al . each rdf was based on first defining the ion dna separation as the closest distance between the dna molecule and the particular ion . ion distance histograms from each snapshot of the md simulation . to obtain the rdfs , the histograms were normalized by the volume jacobian , playing the role of the configurational entropy of ions structured around dna . the number of neighbors within a distance r from a given object is5where is the bulk particle concentration , g(r ) is the rdf ( pair correlation function ) , and j(r ) is the volume jacobian . the latter is defined as the volume of a shell , equidistant from the dna surface ( see figure 4a , b ) , which was numerically calculated as a function of a distance from the dna surface ( figure 4b ) . to minimize end effects , only the inner region of the dna duplex ( eight internal base pairs in the case of the 12-bais - pair ecori dodecamer ) was used for computing the rdfs . this was achieved by clipping the periodic boundary cell with two parallel planes that pass through the centers of the upper four and the lower four dna base pairs and perpendicular to the line connecting these centers . side ( a ) and top ( b ) views of the dna and equidistant shell ( in red ) from its surface . j(r ) is the volume jacobian whose numerical value as a function of the distance from dna surface is shown in c. it is seen that the function increases monotonically only after 4 from the dna , from which it can be approximated by the cylindrical jacobian often used for calculation of the ionic distributions around dna . to avoid end effects , ionic distributions were analyzed in the central region of the dna , as shown in a. it is worth mentioning that the present method of computing j(r ) and rdf differs from the standard procedures relying on either spherical or cylindrical symmetries . for example , although dna is on average cylindrically symmetric and the use of the cylindrical jacobian is reasonable , such an approach leads to an overestimation of the counterion condensation at small distances from the dna surface . this is important , in particular , when calculating the absolute number of ions contributing to the various rdf peaks and subsequently determining the fraction of the dna charge neutralized by condensed counterions a critical test for evaluating the quality of the force field used to model ion / dna interactions ( see below ) . the numerical volume jacobian , taking into account the complex and irregular shape of the dna segment , is characterized by an unusual nonmonotonic behavior in the vicinity of the dna oligomer ( figure 4c ) . it is seen that only at distances more than 5 from the dna surface can the monotonically increasing jacobian be approximated by the cylindrical one . dna distance histograms , computed over snapshots saved every 4 ps from the 100 ns simulations , were normalized by the corresponding numerical jacobian . three - dimensional grids with lattice spacings of 0.5 were used to calculate both the ion - dna distance histograms and the volume jacobian . the biochemical algorithm library ( ball ) was used to implement the computational analysis subroutines . additionally , ball was used for writing code for reimaging / recentering of md trajectories in cases when dna strands ( treated as independent segments ) appeared separated in the course of simulation because of the periodic boundary conditions . this postprocessing was necessary for subsequent calculations of the volume jacobian and rdfs , and also other overall structural dna properties , such as root - mean - square ( rms ) deviations and watson crick base - pairing . earlier stages of the drude force field optimization for dna were aimed at resolving overall duplex instability , while later published efforts addressed finer details of the dna structural behavior after the duplex stability was achieved . the resulting drude dna parameters were subjected to an extensive validation focusing on the details of the key backbone , sugar and glycosidic dihedral angles , whose correlated motions influence two critical aspects of the dna structural behavior in solution the proper treatment of the bi / bii transitions and the equilibrium between a and b forms dna . in the present work we present a description of earlier parametrization of selected bonded parameters in the bases and nonbonded parameters regulating electrostatic and van der waals interactions between mobile ions , dna atoms and water molecules . these interactions are strongly interdependent and must be properly balanced , with the balance extending to the rest of the ff . for example , mitigating electrostatic interactions between selected atoms along the dna backbone by varying their default partial charges proved necessary for achieving experimentally and computationally predicted bi - to - bii transition frequencies and the dynamical puckering transitions of the sugars , as well as better consistency with gas - phase qm data . such alterations , however , disrupted the existing balance of various energetic contributions having an effect on , e.g. , dna hydration properties , requiring adjustments of lj parameters that impact specific interactions with water molecules . those adjustments , in turn , prompted calculations of the hydration free energies and osmotic pressure coefficients for a number of biologically relevant ions in aqueous solution and comparison of md simulation results with available experimental data . another complication that was encountered involved the interactions between the dna and mobile ions . while the individual ions and base nonbond parameters were carefully optimized , that optimization did not involve direct interactions of the ions with the bases . in the ff , these off - diagonal interactions are treated with the electrostatic parameters for the individual moieties along with lj parameters obtained from combining rules , the lorentz berthelot combining rules in the present case . with the additive force field , this approximation generally worked well , although limitations in the approach have been discussed . however , with the drude force field , the direct use of the electrostatic and combining rule lj parameters has been shown to be more problematic , leading to optimization of both terms in specific cases via the use of through - space thole interactions ( nbthole ) and pair - specific lj parameters ( nbfix ) , respectively . for example , nbthole terms have been used to fine - tune the interactions of counterion pairs based on osmotic pressures and the pair - specific lj parameters have been used to improve free energies of hydration . with dna , an imbalance between the base and counterions leads to the interactions between them being significantly overestimated , leading to destabilization of the dna . to correct this , gas phase qm calculations of interactions of the nucleic acid bases and na ion were used to initially optimize the off - diagonal parameters , followed by md simulations of the fully solvated dna duplex in a salt buffer with analysis focused on reproduction of predictions from counterion condensation theory . following several iterations , pair - specific lj parameters for the ions and nucleic acid base hydrogen bond acceptor atoms were obtained that achieve satisfactory agreement with both the qm and counterion condensation theory target data . initial nonbond parameters for the dna ff involved the published base and atomic ion parameters ; however , their application lead to instabilities in simulations of duplex dna . as shown in figure s1 of the supporting information , simulations of the ecori dodecamer resulted in rms deviations from canonical b conformation which increased irreversibly after 10 ns and 15 ns of md simulations , respectively , when only neutralizing na ions and na ions together with an extra 120 mm nacl salt were included in the simulation system . the direct impact of the salt concentration on the longevity of the dna s stability domain suggested the potential cause for such behavior , which turned out to be associated with the anomalously favorable interactions among na ions and the dna oligomer . we first computed the dna / na rdfs and compared the results from polarizable drude and analogous additive c36 md simulations ( see figure s2 , supporting information ) . such comparison revealed that na condensed on dna to a much greater extent in the polarizable model , with that increase largely involving direct contact of na with dna . subsequent rdf analysis of specific sites on the dna ( figure s3 , supporting information ) identified stronger binding of na in the central aatt tract of the ecori oligomer . analysis was then performed on the frequency of na binding to these four internal residues ( figure s4 ) revealing that while , on average , na ions interact more frequently with the phosphate groups in the additive model ( figure s4a ) , there is an enhanced binding of the sodium to the interior of the dna in the drude model , namely , to the n1/n6 atoms of the adenine and n3/o4 atoms of the thymine ( figure s4b , supporting information ) . because these atoms participate in the thy - ade watson crick base - pairing ( n3hn1 and o4h n6 hydrogen bonds ) , it became clear that the cause for the dna structural disruption was na competing for those interactions . the above analysis prompted the calculation of qm and mm interactions between the individual nucleic acid bases and na . additionally , as the aa stacked pair was indicated to be problematic ( figure s4c , supporting information ) , calculations on the ade - ade / natrimer complex were performed , with the adenine bases fixed in the b dna conformation . as is evident , the default drude parameters largely overestimate the attraction between na and selected atoms of the bases at short distances . the mm and qm energies differ by 1215 kcal / mol for ade and cyt bases ( figure 5a , d ) , and by 8 kcal / mol for thy and gua bases ( figure 5b , c ) . ade / na trimer for which mm and qm differ by nearly 40 kcal / mol ( figure 5e ) . clearly , these results point to the default drude parameters leading to overestimation of the base - na interactions ultimately destabilizing duplex dna . qm and empirical drude potential energy surfaces as functions of the distance between na ion and selected atoms of the nucleic acid bases ( a d ) , or of a a stacked bases ( e ) . to overcome the overestimation of the base - na interactions , we considered the use of both the nbthole and pair - specific lj ( nbfix option in charmm ) approaches . while both approaches could alleviate the problem , as shown in figure 5e , use of pair - specific lj terms was considered to be more practical as a number of other pair - specific lj terms are implemented in the drude ff . the nbfix approach is based on assigning small repulsive cores ( i.e. , lj radii ) to the particles of interest to mitigate coulomb interaction among them . to resolve the problem , we modified only the values of rmin and did not alter the lj depth ( ) from that obtained from the combining rules . such modifications have been made for lj interactions between na and the following atoms : ( 1 ) n1 , n3 , n6 of adenine ; ( 2 ) n1 , n2 , n3 of guanine ; ( 3 ) n1 , n3 , n6 of thymine ; and ( 4 ) n4 , n3 of cytosine . in practice , because of the repeating atom types in all four bases , only three unique pair - specific lj terms ( e.g. , nbfixes ) were introduced . as seen from figure 5 , as anticipated , these adjustments led to improved stability of duplex dna in solution , as judged from rms differences versus b - form dna for the ecori dodecamer ( see figure s5 ) . during preliminary duplex dna md simulations , significant out - of - plane distortions of individual bases lead to dna instability on times exceeding 30 ns ( figure s5 , supporting information ) . visual examination of the base geometries during the course of the simulations revealed that excessive out - of - plane distortions were evident for non - hydrogen atoms as well as for the amino groups . the combined effect of these distortions was the formation of hydrogen bond - like interactions between the stacked bases in the duplex dna , in particular , the amino groups with the carbonyl oxygen atoms . interestingly , similar deformations were recently observed from gas - phase qm calculations on model systems composed of the two stacked bases , whose overall geometries were fixed in either b or a dna conformations based on selected sets of internal coordinates , but allowed to relax otherwise . while the effect of these out - of - plane distortions in duplex dna in solution is not clear , such base deformations clearly need to be limited to achieve a stable dna structure . to understand the cause of the distortions , analysis of the amino groups in the adenine and cytosine bases demonstrated that the out - of - plane displacement of the nh2 group hydrogen atoms was associated with the sum of the valence angles around nitrogen atoms being smaller than 360 ( figure s6 , supporting information ) . a similar analysis was also performed for the base s 5- and 6-membered rings whose planarity is characterized by the sum of all inner valence angles being 540 and 720 , respectively ( not shown ) . adjustments of the internal base parameters were performed targeting crystal survey geometries and qm geometries and vibrational spectra of individual bases with their geometries restrained to be planar . based on the above observations , optimization focused on the equilibrium values for the valence angles , subjected to the following restraints : ( 1 ) the sum of the valence angles around the planar centers , such as amino groups , should be 360 ; and ( 2 ) the sum of the inner valence angles in the 5- and 6-membered rings should be 540 and 720 , respectively . although these were just a few restraints , performing such adjustments posed a challenge because of the repeating atom types in chemically different bases , making it difficult for these conditions to be satisfied for all in - plane centers and/or rings . however , the final parameters significantly improved both the agreement of the drude model with both the experimental geometries and qm frequencies of the bases , as shown in tables s1s8 of the supporting information . following these corrections , the dna duplex was observed to be stable on time scales of 100 ns based on md simulations of the ecor1 dodecamer ( figure s5 ) , as well as simulations of other duplexes presented in our previous study . once extended simulations of dna in solution were possible , more subtle conformational effects were addressed . these included transitions between the bi and bii substates of the canonical b form dna and pucker transitions of the sugar groups from the dominant south conformation to the occasionally visited north conformation in b form simulations . model compound 1 was designed to study energetics of the correlated motions among the and backbone dihedral angles in the context of the bi / bii transitions in duplex dna . qm energetic data on 1 for 2d energy scans of versus torsions along with experimentally determined populations of the bi and bii states were used as target data for optimization of the and torsions in the context of the initial drude parameters . as the optimization proceeded , it was necessary to significantly sacrifice the quality of agreement with the qm data to obtain satisfactory sampling of the bi and bii states . the resulting drude 2d versus energy surface showed the model to significantly overstabilize the bii state of 1 as compared to the qm surface , as shown in figure 6a , b , respectively . in addition to the overall shapes of the qm and mm landscapes differing dramatically , the low energy pathways connecting bi and bii states were in poor agreement . moreover , while bi - to - bii transitions were occurring , no experimentally observed transitions in sugar pucker from the south to north conformation , an important prerequisite for stabilizing a - form dna under appropriate conditions , occurred . this is important as the a / b and bi / bii conformational equilibrium are strongly coupled , with the bii substate known to be strongly anticorrelated with sampling of the north sugar conformation characteristic of the a form dna . qm and empirical drude 2d potential energy surfaces of versus for model compound 1 ( t3ps ) . also depicted are structures of the lowest ( bii ) and highest ( 240/240 ) energy conformations ( c ) of landscape a generated by nonoptimized drude parameters . some of the 1d representative and energy profiles are provided in the supporting information , figure s7 . this involved energetic analysis of conformations of 1 having the smallest and the largest relative energies ; those states are indicated by blue circles in figure 6a with the approximate values of the / angles being 240/240 for the highest and 245/175 ( bii ) for the lowest energetic states . comparison of individual energetic contributions ( bonded , lj and electrostatic ) in these conformations revealed that their electrostatic contributions differ by 8 kcal / mol in favor of the bii state , a contribution almost entirely responsible for the total energetic difference of 7 kcal / mol . examination of the geometries of these conformations suggested this difference to be attributed to the attraction between the anionic o1p / o2p atoms of the phosphate and the positively charged c3 carbon of the sugar ring . this distance is shorter by 1 in the bii conformation , as shown in figure 6c . due to this favorable interaction , the c3o3p valence angle is significantly deformed , being 109 instead of 120 as observed in a survey of dna crystal structures . despite being a local effect , the polymeric nature of dna would lead to this difference potentially causing an elongated dna duplex . to overcome this issue , we altered the default electrostatic parameters by making the partial charges for the o1p / o2p atoms less negative by 0.1e , and the c3 atom less positive by 0.2e . following this change and additional optimization , it was observed that the agreement between the qm and mm data for 1 was significantly improved ( figure 6d ) as was the agreement with experiment for the bi / bii equilibrium and puckering of the sugar moieties in duplex dna . in addition , the mm model better reproduced experimental data for selected valence and dihedral angles in duplex dna . in the empirical versus potential energy surface , the flattening of the landscape was achieved by mitigating the electrostatic interactions . to facilitate the comparison between qm and mm outcomes , we also included in the supporting information several representative 1d potential energy profiles as a function of or obtained for model compound 1 being in either bi or bii conformation ( figure s7 ) . figure 7 shows time series of the and torsions sampled in the ecori dodecamer using the final parameters , demonstrating frequent transitions in a bimodal fashion corresponding to bi / bii transitions . additionally , time series for the sugar pseudorotation angles indicate pucker transitions from the north to south conformation occurring on the experimentally observed time scale of 200 ps . figure s8 of supporting information also demonstrates bi / bii transitions and sugar pucker transitions to occur on the level of a single nucleotide . thus , adjustments of the partial atomic charges lead to significant improvements in the conformational properties in the context of the gas phase model compound qm data and the condensed phase duplex dna data . time series and probability distribution functions from the md simulation of the ecori dodecamer for and backbone torsions , as well as for the sugar puckering obtained for the drude parameters optimized based on model compound 2 . transitions between bi ( 190 , 270 ) and bii ( 270 , 180 ) states , and north ( p 15 ) and south ( p 175 ) sugar conformations are evident . based on these time series , both bi / bii transitions and sugar pucker transitions occur on time scales of 200 ps , consistent with the qm data for model compound 2 and nmr experimental data , respectively . due to the changes in dna backbone electrostatics it was necessary to reevaluate the hydration of the phosphodiester backbone . this was performed based on the ability of the force field to reproduce the hydration free energy as well as the qm minimum interactions energies with individual water molecules using dmp as the model compound ( figure 1 ) . the experimental hydration free energy of dmp is not available in the literature , but it can be estimated using the thermodynamic cycle shown in figure 8 . g1 is determined to be = 325 4 kcal / mol from gas phase acidity data , and g3 = 1.75 kcal / mol is obtained from the pka of protonated dmp ( hdmp ) . to estimate g2 , the hydration free energy of hdmp , we start with the known hydration free energy of a closely related compound , trimethylphosphate ( tmp ) , which has a hydration free energy of 8.7 kcal / mol . we then use qm to compute the relative hydration free energy between hdmp and tmp : a qm amsol calculation reveals it to be 3.4 kcal / mol , giving an estimate of 12.1 kcal / mol for g2 . alternatively , the relative hydration free energy between hdmp and tmp may be determined from the free energies of hydration of acetic acid , 6.7 kcal / mol , and methyl acetate , 3.32 kcal / mol , resulting in the same value for g2 = 12.1 kcal / mol . for the hydration free energy of the proton , g(h ) , a value of 258.8 kcal / mol is used . putting all the contributions together gives an estimate for the experimental hydration free energy of dmp of 78.4 5 kcal / mol . table 1 shows the hydration free energy for dmp computed at different stages of the parameter optimization . it is seen that the above - described change in charges for the o1p / o2p oxygen and c3 carbon atoms resulted in the hydration free energies being less favorable by 14 kcal / mol , significantly lower than the experimental estimate . to correct this , we systematically modified lj parameters of selected atoms in dmp , varying both the well depth ( ) and rmin terms . the final value of the hydration free energy obtained using the optimized drude model parameters was 74 kcal / mol , in good agreement with the experimental estimate . to further test the quality of the model , the gas phase qm and mm minimum interaction energies for dmp water monohydrates in different orientation were determined . this analysis is important as it indicates whether the relative energies of hydrogen bonds with different atoms on dmp are correctly reproduced , information that the hydration free energy can not provide . a total of nine different water positions were considered , as shown in figure 2 . the minimum interaction energies and distances with dmp in the gg conformation , corresponding to the geometry of the phosphate group encountered in dna duplexes , are shown in table 2 . minimum interaction energies for the other two minimum energy conformations of dmp , gt and tt , are provided in tables s9 and s10 , respectively , of the supporting information . it is clear from the data that the gas phase interactions with water are uniformly well reproduced . in general , the results indicate that an appropriate balance between the electrostatic and lj parameters has been achieved . the structure of the dmp was first optimized at mp2/6 - 31+g * level of theory . next , the distances between water molecule and dmp were optimized at mp2/6 - 31+g * level of theory with the rest of the dimer fixed . qm interaction energies were calculated at the ri - mp2/cc - pvqz level of theory using counterpoise correction . distances for the qm optimized geometries 26 are between the water hydrogen and o11/o13 oxygen atom of dmp . for geometry 9 , the distance is between water oxygen and the dmp phosphorus . as evidenced above for the base - na interactions , the proper balance of the nonbond portion of the force field is essential . to investigate this for the phosphodiester backbone and the nacl salt , osmotic pressure coefficient is a measure of the solution nonideality , describing how much the behavior of the real electrolyte solution deviates from that of an ideal solution at a particular concentration . thermodynamically , osmotic pressure is related to the second osmotic coefficient in the osmotic virial series for a nonideal solution , with that coefficient , in turn , related to the effective interaction potential ( potential of mean force ) between a pair of ions . thus , reproduction of osmotic pressures is an important requirement directly related to the ff s ability to properly model interionic interactions and ensure those interactions are properly balanced with the ion systems studied included aqueous solutions of na and cl ions , na and dmp ions , and na and acetate ions , each at low and high molal concentrations ( 1 m and 3 m ) . experimental values for the osmotic pressures of na / cl electrolytes are available for a wide range of concentrations , [ 06 m ] . for na / dmp solution , experimental osmotic pressures are known only at low salt concentrations ( 1 m ) . therefore , an additional electrolyte system composed of na and acetate , due to the acetate oxygen atoms being chemically similar to the anionic oxygens of dmp , was simulated at 1 m and 3 m , concentrations for which experimental osmotic pressure coefficients are available . based on the osmotic pressure calculations , optimization of selected table 3 shows the osmotic pressures produced by the final drude parameters along with experimental values and results from the additive c36 ff . table 4 contains the actual molal concentrations in the ionic slab from the md simulations , required for direct determination with the experimental data . comparison of the results in table 3 shows the level of agreement to be quite good . the drude model is typically in improved agreement with experiment over c36 , although the additive model is in better agreement for the na+/acetate solution . given the overall good agreement between the drude and experimental data , the current parametrization provides a plausible description of osmotic properties of biologically relevant ionic solutions . utilizing amber force field . while experimental data for osmotic pressure of the na / dmp system is not available at high concentrations , the value obtained from a md simulation study of this system at 3 m is presented in table 3 . the value was determined in a study aimed at improved lj parametrization of specific ion pairs in the amber force field . as can be seen , the result from that study is in a good agreement with the additive c36 result , while the drude simulation produced a noticeably higher value . given the underestimation of the osmotic pressure for the na / acetate system , this result indicates that the experimental value may be significantly higher than that obtained from the additive ffs . our final test of the drude model is focused on details of the ionic structuring around the dna duplex . as demonstrated above , excessive condensation of counterions to the dna may lead to disruption of the dna structure . numerous prior experimental and theoretical studies focused on sequence specific ionic binding , such as determining whether and how long counterions reside in dna grooves , or competitive ionic binding to specific dna sites . while these aspects will be addressed in future studies , for validation we will focus on the overall ionic condensation . ionic distribution around dna was characterized by computing rdfs , with averaging over the central region of the dna duplexes . such an approach allows for a direct comparison of the md simulation results with the counterion condensation ( cc ) theory . in their seminal work , onsager and manning demonstrated that cc must occur on linear rods that are uniformly charged above some critical charge density . the dna molecule , being linear and carrying high charge per unit length , is well above this threshold . one can estimate from these simple arguments that 76% of the total dna charge is expected to be neutralized by condensed counterions within a 9 manning radius . rdfs for na and cl ions shown in figure 9 were computed from md simulations of the ecori dodecamer in the 120 mm nacl aqueous solution using both the polarizable drude and additive c36 models . analogous data for another two dna systems , a longer junfos ( 16-base - pair ) and a shorter 1dcv ( 10-base - pair ) duplex are provided in the supporting information ( figure s9 ) . in all graphs , na rdfs are characterized by three prominent peaks , indicating the formation of ionic shells around polyanionic dna . the onset of such an ionic structuring when approaching dna from the bulk is commonly associated with the manning s radius of cc theory . as seen from the plots , counterions are structured within 9 from dna surface in both ffs , coinciding with the cc predictions . this result is consistent with the definition of beveridge and co - workers for the condensed counterions as lying within a 9 region from the dna surface , where ions are structured with respect to dna . na / dna and cl / dna rdfs from the drude ( black ) and additive ( red ) md simulations of the ecori dodecamer computed based on the closest approach , as elaborated in the methods section . the computed ionic rdfs were used to calculate the fraction of the dna residual charge neutralized by the ions . to this end , rdfs for na and cl were integrated from 0 to 9 , according to eq 5 , and the resulting number of co - ions and counterions were summed and divided by dna residual charge . drude and additive result for dd , junfos , and 1dcv dna systems are shown in table 5 . the agreement with the cc estimates is clearly better for the drude ff simulations , which predict 77% , 79% , and 75% of the dna residual charge to be neutralized by ions within the manning radius for ecori , 1dcv and junfos , respectively . these results indicate that the drude model faithfully captures basic features of the interplay between dna duplexes and the surrounding ionic atmosphere , offering an improvement over the additive c36 ff , and further indicating the quality of the nonbonded parameters for dna and mobile ions . in the present study we addressed a number of critically important aspects of the development of the drude polarizable force field for dna . these issues included treatment of base - na interactions , proper treatment of the base intramolecular parameters , balancing dna electrostatic and hydration effects , and how they impact the overall dna stability and regulate subtler aspects of dna conformational behavior . for example , when drude parameters developed for the nucleic acid bases were applied to a dna duplex in solution , the dna structure became unstable after 25 ns of md simulation time because of significant out - of - plane deformations of individual bases . the problem was overcome by a partial reoptimization of base valence angle parameter based on reproduction of experimental geometries and qm geometries and vibrational spectra . next , based on the qm model compound calculations , altering the default electrostatic charges of selected backbone dna atoms was shown to be necessary to improve bi / bii conformational equilibrium and to better reproduce the dynamical sugar pucker transitions observed experimentally . such changes , however , required readjustment of selected lj parameters dictating interactions between dna and water , as well as dna and mobile ions . these modifications were based on simultaneous targeting a range of experimental and qm data , such as the hydration free energy for dmp and the qm gas - phase interactions of dmp with water molecules , qm data on small model compounds representative of the bases and dmp- interacting with na ion , and experimental data on osmotic pressure measurements . a critical test of the ffs ability to capture basic features of the distribution of mobile ions around dna involved comparing predictions from counterion condensation theory with those based on computing ion / dna rdfs obtained from md simulations . our results indicate that drude simulations generate a physically plausible picture of ionic distribution around dna , reproducing the theoretical estimates of the fraction of neutralized residual dna charge by the condensed ions . notably , the drude results show an improvement over the c36 ff , indicating the improved physical description of the forces dictating the ionic solvation of dna due to the explicit treatment of electronic polarizability . in summary , we focused on balancing the electrostatics and hydration of the dna backbone by optimizing selected drude parameters for interactions between dna , mobile ions and water molecules . our initial efforts were aimed at stabilizing an overall dna structure in solution , which included improvements in the base valence angle parameters , while later parameter modifications addressed finer details of the dna conformational behavior , such as bi / bii transitions and the equilibrium between north and south states of the sugar moiety . force field validation presented in this study , together with the earlier demonstrated ability of the force field to adequately model all aspects of the b form dna , as well as to stabilize the a form in low water activity environment , indicate that the current drude polarizable model for dna is suitable for computational studies of various dna molecules under varying conditions on the 100 ns time scale . in addition , the availability of drude polarizable parameters for proteins , lipids , and carbohydrates will allow for simulation studies of heterogeneous biological systems .

recently we presented a first - generation all - atom drude polarizable force field for dna based on the classical drude oscillator model , focusing on optimization of key dihedral angles followed by extensive validation of the force field parameters . presently , we describe the procedure for balancing the electrostatic interactions between ions , water , and dna as required for development of the drude force field for dna . the proper balance of these interactions is shown to impact dna stability and subtler conformational properties , including the conformational equilibrium between the bi and bii states , and the a and b forms of dna . the parametrization efforts were simultaneously guided by gas - phase quantum mechanics ( qm ) data on small model compounds and condensed - phase experimental data on the hydration and osmotic properties of biologically relevant ions and their solutions , as well as theoretical predictions for ionic distribution around dna oligomer . in addition , fine - tuning of the internal base parameters was performed to obtain the final dna model . notably , the drude model is shown to more accurately reproduce counterion condensation theory predictions of dna charge neutralization by the condensed ions as compared to the charmm36 additive dna force field , indicating an improved physical description of the forces dictating the ionic solvation of dna due to the explicit treatment of electronic polarizability . in combination with the polarizable dna force field , the availability of drude polarizable parameters for proteins , lipids , and carbohydrates will allow for simulation studies of heterogeneous biological systems .

Introduction Computational Methods Results and Discussion Conclusions

for example , when in a b form , correlated motions of the backbone , sugar and glycosidic dna torsions lead to the dynamical transitions between bi and bii states , as well as the extent of local sampling of geometries characteristic of the a form dna as manifested in sugar puckering transitions between the south and north conformations . on a global scale , the balance between bonded and nonbonded intramolecular parameters along with interactions with the environment influence the conformational equilibrium between the a , b , and z forms of dna . moreover , interactions with mobile ions and hydration effects are known to contribute to the overall dna stability and regulate a number of critical polymeric properties , such as persistence length and stiffness . while successful in many respects , these models show a number of limitations , potentially associated with the lack of explicit electronic polarization in the model , including limited treatment in the equilibrium between the different conformations of dna as well as relative energies of different conformations . while continued developments in the additive ffs have partially overcome these limitations , it is anticipated that extending the form of the potential energy function to include the explicit treatment of electronic polarization may lead to improvements in dna ffs , including yielding a more accurate description of the underlying physical forces dictating the structure and dynamics of dna . recently , we have presented the first - generation charmm polarizable ff for dna based on the classical drude oscillator formalism . to date , the drude model has been implemented in charmm , namd and chemshell quantum mechanics / molecular mechanics ( qm / mm ) . as discussed elsewhere , due to the need of use of a 1 fs integration time step ( versus the 2 fs time step customarily used in additive md simulations ) , use of the drude polarizable ff in simulations yields an approximately 4-fold overhead compared to additive simulations . in addition , a module , the drude prepper has been added to the charmm - gui , facilitating the conversion of charmm psf and coordinate files into the corresponding files required for drude - based simulations including example inputs for charmm and namd . in our published study on development of the drude force field for dna we focused mainly on details of parametrization of the highly correlated backbone , sugar , and glycosidic torsion angles , and on extensive validation of the final parameters by comparing md simulation results with a wide range of experimental data on dna in solution . a notable observation from those simulations was the significant enhancement and variation of base dipole moments as compared to the c36 additive ff , indicating that the underlying physical forces dictating the properties of dna are significantly different in the polarizable model . however , the discussion in that paper covered only a small part of the optimization process , not addressing such critical aspects of the modeling as fine - tuning of the interactions between dna and mobile ions and the balance between dna electrostatic and hydration effects , subtle corrections in the internal base parameters , and the impact of these on the overall dna stability and subtler conformational phenomena , including bi / bii transitions and a - to - b equilibrium . presently , we focus on the above aspects of the drude ff development and additional testing of the optimized dna parameters against a range of experimental data and theoretical estimations . our optimization procedure involves approaches ranging from qm calculations on various model compounds and their complexes to investigating the hydration and osmotic properties of the chemically relevant ionic systems to studying the impact of the mobile ions on dna structural stability . we note that the final results presented here are based on the identical set of parameters developed in our earlier study on the drude force field for dna . in the present study we used a number of small model systems to probe how electrostatic , hydration and other effects in the dna duplex impact overall dna stability , as well as subtler aspects of dna structural behavior in solution . in the present study , by analyzing individual energetic contributions of 1 , we demonstrate that , in addition to the torsion parameters , adjustments of electrostatic parameters ( partial charges ) was necessary to achieve satisfactory agreement with both qm data on this model compound and experimental data on bi / bii transitions and a - to - b equilibrium in dna duplexes in solution . specifically , one of the sugar moiety dihedral angles , 4 , and the backbone torsion , , were restrained . analogous mm calculations were carried out using the charmm program and the drude polarizable force field . the optimization was performed by targeting , simultaneously , reproduction of the hydration free energy of dmp and the gas - phase heterodimeric interactions with water . the second approach deals with gas - phase calculations on model systems involving interactions of na with individual nucleic acid bases , as well as with dmp . equivalent single point calculations were then performed using the drude polarizable force field for nucleic acids . water can freely pass through the membranes separating electrolyte and water solutions , while the ions are subject to the force of the half - harmonic planar potential on each side of the ionic compartment whose action mimics the ideal behavior of a semipermeable membrane inducing osmosis . the instantaneous osmotic pressure , = fmemb / a , equals the instantaneous total force on the membranes,1divided by the total area of the membranes , a. in this expression , k is the force constant of the membrane potential , 10 kcal/(mol ) , d = 48 is the linear size of the cubic ionic compartment , and zi is the z - coordinate of the i ion . it is important to note that , following the convention used in experimental measurements , the osmotic pressure of an ideal solution in the above expression is proportional to the molal concentration of the electrolyte , m , which differs from the traditionally used molar concentration m. therefore , to enable direct comparison with the experiment , the actual molal concentrations need to be computed from md simulations based on the values of mean densities of the cations , anions , and pure water in the ionic compartment as follows:4where overbars indicate ensemble averages , and msalt is the molar concentration of the salt . each simulated system included 45 000 particles , including atoms , lone pairs , and drude particles , in the case of the polarizable model and 26 000 atoms in the additive model . charmm was used for generating the polarizable drude system , self - consistent relaxation of the drude particle positions , and short equilibration of the whole system , as elaborated elsewhere . for the production md simulation namd ( version 2.9 ) was used whose self - consistent treatment of the drude polarizable model is based on a dual thermostatting scheme and langevin dynamics . to obtain the rdfs , the histograms were normalized by the volume jacobian , playing the role of the configurational entropy of ions structured around dna . j(r ) is the volume jacobian whose numerical value as a function of the distance from dna surface is shown in c. it is seen that the function increases monotonically only after 4 from the dna , from which it can be approximated by the cylindrical jacobian often used for calculation of the ionic distributions around dna . this is important , in particular , when calculating the absolute number of ions contributing to the various rdf peaks and subsequently determining the fraction of the dna charge neutralized by condensed counterions a critical test for evaluating the quality of the force field used to model ion / dna interactions ( see below ) . earlier stages of the drude force field optimization for dna were aimed at resolving overall duplex instability , while later published efforts addressed finer details of the dna structural behavior after the duplex stability was achieved . the resulting drude dna parameters were subjected to an extensive validation focusing on the details of the key backbone , sugar and glycosidic dihedral angles , whose correlated motions influence two critical aspects of the dna structural behavior in solution the proper treatment of the bi / bii transitions and the equilibrium between a and b forms dna . these interactions are strongly interdependent and must be properly balanced , with the balance extending to the rest of the ff . for example , mitigating electrostatic interactions between selected atoms along the dna backbone by varying their default partial charges proved necessary for achieving experimentally and computationally predicted bi - to - bii transition frequencies and the dynamical puckering transitions of the sugars , as well as better consistency with gas - phase qm data . those adjustments , in turn , prompted calculations of the hydration free energies and osmotic pressure coefficients for a number of biologically relevant ions in aqueous solution and comparison of md simulation results with available experimental data . however , with the drude force field , the direct use of the electrostatic and combining rule lj parameters has been shown to be more problematic , leading to optimization of both terms in specific cases via the use of through - space thole interactions ( nbthole ) and pair - specific lj parameters ( nbfix ) , respectively . following several iterations , pair - specific lj parameters for the ions and nucleic acid base hydrogen bond acceptor atoms were obtained that achieve satisfactory agreement with both the qm and counterion condensation theory target data . the direct impact of the salt concentration on the longevity of the dna s stability domain suggested the potential cause for such behavior , which turned out to be associated with the anomalously favorable interactions among na ions and the dna oligomer . analysis was then performed on the frequency of na binding to these four internal residues ( figure s4 ) revealing that while , on average , na ions interact more frequently with the phosphate groups in the additive model ( figure s4a ) , there is an enhanced binding of the sodium to the interior of the dna in the drude model , namely , to the n1/n6 atoms of the adenine and n3/o4 atoms of the thymine ( figure s4b , supporting information ) . the combined effect of these distortions was the formation of hydrogen bond - like interactions between the stacked bases in the duplex dna , in particular , the amino groups with the carbonyl oxygen atoms . interestingly , similar deformations were recently observed from gas - phase qm calculations on model systems composed of the two stacked bases , whose overall geometries were fixed in either b or a dna conformations based on selected sets of internal coordinates , but allowed to relax otherwise . adjustments of the internal base parameters were performed targeting crystal survey geometries and qm geometries and vibrational spectra of individual bases with their geometries restrained to be planar . however , the final parameters significantly improved both the agreement of the drude model with both the experimental geometries and qm frequencies of the bases , as shown in tables s1s8 of the supporting information . following these corrections , the dna duplex was observed to be stable on time scales of 100 ns based on md simulations of the ecor1 dodecamer ( figure s5 ) , as well as simulations of other duplexes presented in our previous study . these included transitions between the bi and bii substates of the canonical b form dna and pucker transitions of the sugar groups from the dominant south conformation to the occasionally visited north conformation in b form simulations . qm energetic data on 1 for 2d energy scans of versus torsions along with experimentally determined populations of the bi and bii states were used as target data for optimization of the and torsions in the context of the initial drude parameters . as the optimization proceeded , it was necessary to significantly sacrifice the quality of agreement with the qm data to obtain satisfactory sampling of the bi and bii states . in addition to the overall shapes of the qm and mm landscapes differing dramatically , the low energy pathways connecting bi and bii states were in poor agreement . examination of the geometries of these conformations suggested this difference to be attributed to the attraction between the anionic o1p / o2p atoms of the phosphate and the positively charged c3 carbon of the sugar ring . in addition , the mm model better reproduced experimental data for selected valence and dihedral angles in duplex dna . in the empirical versus potential energy surface , the flattening of the landscape was achieved by mitigating the electrostatic interactions . thus , adjustments of the partial atomic charges lead to significant improvements in the conformational properties in the context of the gas phase model compound qm data and the condensed phase duplex dna data . time series and probability distribution functions from the md simulation of the ecori dodecamer for and backbone torsions , as well as for the sugar puckering obtained for the drude parameters optimized based on model compound 2 . due to the changes in dna backbone electrostatics it was necessary to reevaluate the hydration of the phosphodiester backbone . this was performed based on the ability of the force field to reproduce the hydration free energy as well as the qm minimum interactions energies with individual water molecules using dmp as the model compound ( figure 1 ) . to estimate g2 , the hydration free energy of hdmp , we start with the known hydration free energy of a closely related compound , trimethylphosphate ( tmp ) , which has a hydration free energy of 8.7 kcal / mol . the final value of the hydration free energy obtained using the optimized drude model parameters was 74 kcal / mol , in good agreement with the experimental estimate . as evidenced above for the base - na interactions , the proper balance of the nonbond portion of the force field is essential . based on the osmotic pressure calculations , optimization of selected table 3 shows the osmotic pressures produced by the final drude parameters along with experimental values and results from the additive c36 ff . given the overall good agreement between the drude and experimental data , the current parametrization provides a plausible description of osmotic properties of biologically relevant ionic solutions . our final test of the drude model is focused on details of the ionic structuring around the dna duplex . ionic distribution around dna was characterized by computing rdfs , with averaging over the central region of the dna duplexes . na / dna and cl / dna rdfs from the drude ( black ) and additive ( red ) md simulations of the ecori dodecamer computed based on the closest approach , as elaborated in the methods section . the agreement with the cc estimates is clearly better for the drude ff simulations , which predict 77% , 79% , and 75% of the dna residual charge to be neutralized by ions within the manning radius for ecori , 1dcv and junfos , respectively . these results indicate that the drude model faithfully captures basic features of the interplay between dna duplexes and the surrounding ionic atmosphere , offering an improvement over the additive c36 ff , and further indicating the quality of the nonbonded parameters for dna and mobile ions . in the present study we addressed a number of critically important aspects of the development of the drude polarizable force field for dna . these issues included treatment of base - na interactions , proper treatment of the base intramolecular parameters , balancing dna electrostatic and hydration effects , and how they impact the overall dna stability and regulate subtler aspects of dna conformational behavior . such changes , however , required readjustment of selected lj parameters dictating interactions between dna and water , as well as dna and mobile ions . these modifications were based on simultaneous targeting a range of experimental and qm data , such as the hydration free energy for dmp and the qm gas - phase interactions of dmp with water molecules , qm data on small model compounds representative of the bases and dmp- interacting with na ion , and experimental data on osmotic pressure measurements . a critical test of the ffs ability to capture basic features of the distribution of mobile ions around dna involved comparing predictions from counterion condensation theory with those based on computing ion / dna rdfs obtained from md simulations . our results indicate that drude simulations generate a physically plausible picture of ionic distribution around dna , reproducing the theoretical estimates of the fraction of neutralized residual dna charge by the condensed ions . notably , the drude results show an improvement over the c36 ff , indicating the improved physical description of the forces dictating the ionic solvation of dna due to the explicit treatment of electronic polarizability . in summary , we focused on balancing the electrostatics and hydration of the dna backbone by optimizing selected drude parameters for interactions between dna , mobile ions and water molecules . force field validation presented in this study , together with the earlier demonstrated ability of the force field to adequately model all aspects of the b form dna , as well as to stabilize the a form in low water activity environment , indicate that the current drude polarizable model for dna is suitable for computational studies of various dna molecules under varying conditions on the 100 ns time scale . in addition , the availability of drude polarizable parameters for proteins , lipids , and carbohydrates will allow for simulation studies of heterogeneous biological systems .

computed tomography angiography ( cta ) is a rapidly evolving technique that has the ability to non - invasively and accurately detect significant coronary artery stenosis and coronary atherosclerotic plaque.1 - 5 a potentially interesting application of cta would be the identification of patients or lesions that have an increased likelihood of plaque rupture leading to acute coronary events . several previous studies have identified specific plaque characteristics which are frequently observed with cta in patients presenting with acute coronary syndrome ( acs).6 - 8 among these characteristics , a spotty pattern of calcifications has been related to the presence of acs.6 - 8 indeed , preliminary data with cta demonstrated that culprit lesions in patients with acs had a higher prevalence of smaller spotty calcifications than target lesions in patient with stable complaints.9 moreover , the presence of spotty calcifications on cta were associated with a higher likelihood of developing acs.8 more detailed information on plaque characteristics can be obtained by intravascular ultrasound with radiofrequency backscatter analysis ( ivus - vh).10,11 this technique was developed to improve grayscale intravascular ultrasound tissue characterization and provide detailed quantitative information on plaque composition in vivo.12,13 additionally , the presence of thin cap fibroatheroma ( tcfa ) on ivus - vh was demonstrated to be independently predictive of major cardiovascular events during follow - up.14 however , the relation between calcification patterns on cta and plaque characteristics on ivus - vh has not been previously reported . furthermore , in previous studies , the definition of spotty calcification on cta has been variable . indeed , definitions for spotty calcification on cta have ranged from presence of any calcified material embedded in non - calcified plaque to a specific threshold regarding the size of calcification . subsequently , due to the inconsistency of definitions , comparability of spotty calcifications on cta between investigations is hampered . therefore , the purpose of the study was to perform a systematic evaluation of plaques with different calcification patterns on cta and relate this to plaque characteristics on ivus - vh . a total of 108 patients were included in this evaluation who underwent cta prior to invasive coronary angiography with ivus - vh of 1 to 3 vessels , as part of clinical protocol . for this retrospective evaluation , consecutive patients were selected as part of an ongoing registry addressing the relative merits of cta in relation to other imaging techniques.15 patient with chest pain were referred for imaging for the non - invasive evaluation of coronary artery disease ( cad ) , according to clinical protocol . thereafter , patients were referred for invasive coronary angiography in combination with ivus - vh based on the patient s clinical presentation and/or imaging results to further evaluate the extent and severity of cad . during invasive coronary angiography , clinical history was evaluated between the cta examination and invasive coronary angiography to ensure that neither acute coronary syndromes nor worsening of angina occurred between examinations . exclusion criteria for cta examination were : ( i ) ( supra ) ventricular arrhythmias , ( ii ) renal failure ( glomerular filtration rate < 30 ml / minute ) , ( iii ) known allergy to iodine contrast material , ( iv ) severe claustrophobia , ( v ) pregnancy . patient data were prospectively collected in the departmental cardiology information system ( epd - vision , leiden university medical center , leiden , the netherlands ) and retrospectively analyzed . our institutional review board does not require its approval and written informed consent for retrospective technical analysis of data , as was the case for this study . in each patient , the presence of risk factors for cad and medication ( use of aspirin , statins , beta - blocking medication and angiotensin - converting enzyme ( ace ) inhibitors ) were recorded . cta examination was performed using either a 64-row helical scanner ( aquilion 64 , toshiba medical systems , toshiba medical systems , otawara , japan ) or a 320-row volumetric scanner ( aquilion one , toshiba medical systems , otawara , japan ) . if the patient s heart rate was 65 beats / minute and no contra - indications existed , beta - blocking medication ( metoprolol 50 or 100 mg , single oral dose , 1 hour prior to examination ) was administered . scan parameters for the 64-row contrast enhanced scan were : 400 ms gantry rotation time , collimation of 64 0.5 mm , tube voltage of 100 - 135 kv and tube current of 250 - 350 ma , depending on body posture . non - ionic contrast material ( iomeron 400 , bracco , milan , italy or ultravist 370 , bayer schering pharma ag berlin , germany ) was administered with an amount of 80 - 110 ml followed by a saline flush with a flow rate of 5 ml / second . datasets were reconstructed from the retrospectively gated raw data , the best phase was reconstructed with an interval of 0.3 mm . using a single test slice reconstructed throughout the various phases of the heart cycle , other suitable r - r intervals were examined for additional reconstructions . concerning the 320-row contrast enhanced scan ; imaging was performed in a single volume using prospective triggering with exposure interval depending on the heart rate . if the heart rate was 65 beats / minute , the phase window was set 65%-85% of r - r interval , if the heart rate was stable and < 60 beats / minute the phase window was set at 70%-80% of r - r interval . scan parameters were : 350 ms gantry rotation time , 320 0.5 mm collimation , 100 - 135 kv tube voltage and a tube current of 400 - 580 ma , depending on body mass index ( bmi ) . overall , 60 - 90 ml contrast material ( iomeron 400 , bracco , milan , italy ) was administered with a rate of 5 - 6 ml / second followed by a saline flush . data acquisition was performed during an inspiratory breath hold of approximately 4 - 10 seconds . subsequently , images were reconstructed in the best phase of the r - r interval and transferred to a remote workstation for post - processing . cta datasets were evaluated using dedicated software ( vitrea 2.0 or vitrea fx 1.1 vital images , minnetonka , mn , usa ) . analysis was performed with the use of available post - processing tools such as cross - sectional axial slices and multiplanar reconstructions . one experienced reader , blinded to the ivus - vh results , evaluated the cta datasets . the coronary arteries were divided into 17 segments according to the modified american heart association classification.16 each segment was evaluated for the presence of any atherosclerotic plaque . structures > 1 mm within and/or adjacent to the coronary artery lumen , which could be clearly distinguished from the vessel lumen , were defined as atherosclerotic plaque.3 calcification patterns in plaques on cta were classified morphologically as non - calcified , spotty calcifications or dense calcifications . non - calcified plaques were defined as a plaque with low ct attenuation located in the vessel wall in at least two independent image planes and clearly distinguishable from the contrast - enhanced lumen and pericardial tissue without any calcification.17 plaques with spotty calcifications were defined as follows : length ( extent in the longitudinal direction of the vessel ) of the calcification < 3/2 of vessel diameter and width ( extent of the calcification perpendicular to the longitudinal direction of the vessel ) of the calcification < 2/3 of vessel diameter , as previously described ( figure 1).17 plaques with spotty calcification were further differentiated according to their length ( extent of the calcification parallel to the longitudinal direction of the vessel on curved multiplanar reconstruction ) into small spotty ( < 1 mm ) , intermediate spotty ( 1 - 3 mm ) , and large spotty calcifications ( 3 mm ) as measured with caliper function ( for illustration see figure 2).18 dense calcifications were defined as a plaque with high ct density , completely calcified and with calcifications present bilateral on cross - sectional axial slices.figure 1example of measurement of spotty calcification . plaques with spotty calcifications were defined as follows : length ( extent in the longitudinal direction of the vessel ) of the calcification < 3/2 of vessel diameter and width ( extent of the calcification perpendicular to the longitudinal direction of the vessel ) of the calcification < 2/3 of vessel diameter . a , measurement of vessel diameter ( blue line ) perpendicular to long vessel axis ( dashed white line ) . b , measurement of length of calcification ( blue line ) parallel to long vessel axis ( dashed white line ) . c , measurement of width of calcification ( blue line ) perpendicular to long vessel axis ( dashed line)figure 2illustration of the different types of spotty calcification . a , small spotty calcification ( < 1 mm ) . c , large spotty calcification ( > 3 mm ) example of measurement of spotty calcification . plaques with spotty calcifications were defined as follows : length ( extent in the longitudinal direction of the vessel ) of the calcification < 3/2 of vessel diameter and width ( extent of the calcification perpendicular to the longitudinal direction of the vessel ) of the calcification < 2/3 of vessel diameter . a , measurement of vessel diameter ( blue line ) perpendicular to long vessel axis ( dashed white line ) . b , measurement of length of calcification ( blue line ) parallel to long vessel axis ( dashed white line ) . c , measurement of width of calcification ( blue line ) perpendicular to long vessel axis ( dashed line ) illustration of the different types of spotty calcification . a , small spotty calcification ( < 1 mm ) . c , large spotty calcification ( > 3 mm ) conventional invasive coronary angiography was performed according to standard protocols . ivus examinations were acquired with a 20 mhz , 2.9 f phased - array ivus catheter , ( eagle eye , volcano corporation , rancho cordova , ca , usa ) and a dedicated ivus - console ( volcano corporation , rancho cordova , ca , usa ) after intracoronary administration of nitroglycerine . under fluoroscopic guidance , the ivus catheter was introduced distally in the coronary artery and a motorized automated pullback with a continuous speed of 0.5 mm / second was used until the catheter reached the guiding catheter . ivus - vh analysis was performed by two experienced observers blinded to baseline patient characteristics and cta findings with the use of dedicated software ( qcu - cms , version 4.59 , medis medical imaging systems , leiden , the netherlands ) . first , contour detection ( lumen and media interface ) was performed manually with the use of cross - sectional views to provide compositional output.19 secondly , on a frame - by - frame basis , the four different tissue components were differentiated and labeled with four different color - codes ( fibrotic tissue was labeled in dark green , fibro - fatty tissue in light green , necrotic core in red and dense calcium in white ) , as validated previously.10 the mean percentage of each plaque component was obtained in the full length of the lesion observed on the cta examination . a lesion on intravascular ultrasound imaging was defined as at least three consecutive frames with a plaque burden of at least 40%.20 in addition , the presence of the thin cap fibroatheroma ( tcfa ) was evaluated on a per - lesion basis . tcfa was defined as a lesion with a plaque burden 40% , presence of > 10% confluent necrotic core on three consecutive frames and necrotic core in contact with the lumen for along the lumen circumference.14,21,22 high - risk plaque characteristics on ivus - vh were defined by % of necrotic core and presence of tcfa . to ensure that identical plaques were assessed by cta and ivus - vh , coronary ostia and side branches were used as landmarks and distances from the landmarks to the target lesions were measured ( figure 3 ) . on ivus - vh , longitudinal reconstructed ecg triggered datasets were used to measure the difference between corresponding plaque and landmarks . after alignment of plaques on cta and ivus - vh , we identified matching slices between the two modalities and could determine the proximal and distal end of a lesion . examples of different calcification patterns on cta with corresponding ivus - vh images are demonstrated in figures 4 and 5.figure 3example of anatomical matching of computed tomography angiography ( cta ) with intravascular ultrasound with radiofrequency backscatter analysis ( ivus - vh ) datasets . a , straightened multiplanar reconstruction of left anterior descending coronary artery in which various landmarks have been identified ( left circumflex artery ( lcx ) , first and second diagonal branch ( d1 and d2 ) . b and c , corresponding grayscale intravascular ultrasound and ivus - vh datasets in longitudinal view in which identical landmarks have been matched to cta dataset ( yellow dashed line ) . corresponding cross - sectional view of minimal lumen area on cta ( d ) and ivus - vh ( e , f)figure 4example of small spotty calcifications as assessed on computed tomography angiography ( cta ) with high - risk plaque characteristics on intravascular ultrasound with radiofrequency backscatter analysis ( ivus - vh ) . a , multiplanar reconstruction of the right coronary artery ( rca ) demonstrating a plaque with non - calcified elements and spotty calcifications ( arrow ) . b , enlargement of the same plaque demonstrating a spotty calcification with a length of 0.7 mm . c , longitudinal reconstructed ecg triggered ivus - vh run with superimposed color coded map . the plaque ( white arrow ) corresponds to the same plaque on cta demonstrating a large necrotic core and multiple small spotty calcifications . d , cross - sectional axial image of the same plaque demonstrating calcifications ( white ) and a large necrotic core ( red ) indicating presence of a thin cap fibroatheroma ( tcfa)figure 5example of a plaque with dense calcifications on computed tomography angiography ( cta ) with corresponding grayscale and intravascular ultrasound with radiofrequency backscatter analysis ( ivus - vh ) images . a , curved multiplanar reconstruction of the right coronary artery ( rca ) demonstrating a plaque with dense calcifications ( arrow ) . inlay showing cross - sectional axial image of the same plaque with calcifications present bilateral of the coronary artery . b , enlargement of the same plaque reporting the length of the calcification ( 4.5 mm ) . c , grayscale ivus image demonstrating a heavily calcified plaque with corresponding ivus - vh image ( d ) showing a plaque with heavy calcifications ( white ) example of anatomical matching of computed tomography angiography ( cta ) with intravascular ultrasound with radiofrequency backscatter analysis ( ivus - vh ) datasets . a , straightened multiplanar reconstruction of left anterior descending coronary artery in which various landmarks have been identified ( left circumflex artery ( lcx ) , first and second diagonal branch ( d1 and d2 ) . b and c , corresponding grayscale intravascular ultrasound and ivus - vh datasets in longitudinal view in which identical landmarks have been matched to cta dataset ( yellow dashed line ) . corresponding cross - sectional view of minimal lumen area on cta ( d ) and ivus - vh ( e , f ) example of small spotty calcifications as assessed on computed tomography angiography ( cta ) with high - risk plaque characteristics on intravascular ultrasound with radiofrequency backscatter analysis ( ivus - vh ) . a , multiplanar reconstruction of the right coronary artery ( rca ) demonstrating a plaque with non - calcified elements and spotty calcifications ( arrow ) . b , enlargement of the same plaque demonstrating a spotty calcification with a length of 0.7 mm . c , longitudinal reconstructed ecg triggered ivus - vh run with superimposed color coded map . the plaque ( white arrow ) corresponds to the same plaque on cta demonstrating a large necrotic core and multiple small spotty calcifications . d , cross - sectional axial image of the same plaque demonstrating calcifications ( white ) and a large necrotic core ( red ) indicating presence of a thin cap fibroatheroma ( tcfa ) example of a plaque with dense calcifications on computed tomography angiography ( cta ) with corresponding grayscale and intravascular ultrasound with radiofrequency backscatter analysis ( ivus - vh ) images . a , curved multiplanar reconstruction of the right coronary artery ( rca ) demonstrating a plaque with dense calcifications ( arrow ) . inlay showing cross - sectional axial image of the same plaque with calcifications present bilateral of the coronary artery . b , enlargement of the same plaque reporting the length of the calcification ( 4.5 mm ) . c , grayscale ivus image demonstrating a heavily calcified plaque with corresponding ivus - vh image ( d ) showing a plaque with heavy calcifications ( white ) plaques available on both cta and ivus - vh were included in the lesion based analysis . plaque composition ( % fibrotic , fibro - fatty , necrotic core , and dense calcium ) and presence of tcfa was compared between lesions with different calcification patterns on cta ( non - calcified , small spotty calcifications , intermediate spotty calcifications , large spotty calcifications , and dense calcifications ) . if normally distributed , continuous values were expressed as means ( standard deviation ) and differences in plaque composition and type were assessed using an analysis of variance ( anova ) . if not normally distributed , values were expressed as medians ( interquartile range ( iqr ) ) and differences in plaque composition and type were assessed using the kruskall - wallis test . to account for possible clustering of coronary artery plaques within patients , the generalized estimating equation ( gee ) method was applied to evaluate the differences in plaque characteristics between the groups of different calcification patterns on cta ( non - calcified , small spotty calcifications , intermediate spotty calcifications , large spotty calcifications , and dense calcium ) . this was performed with proc genmod with a binominal distribution for the outcome variable , the link function specified as a logistical ( presence of tcfa ) or a linear ( % fibrotic , fibro - fatty , necrotic core , and dense calcium ) distribution and patients as separate subjects . due to lack of power a total of 108 patients were included in this evaluation who underwent cta prior to invasive coronary angiography with ivus - vh of 1 to 3 vessels , as part of clinical protocol . for this retrospective evaluation , consecutive patients were selected as part of an ongoing registry addressing the relative merits of cta in relation to other imaging techniques.15 patient with chest pain were referred for imaging for the non - invasive evaluation of coronary artery disease ( cad ) , according to clinical protocol . thereafter , patients were referred for invasive coronary angiography in combination with ivus - vh based on the patient s clinical presentation and/or imaging results to further evaluate the extent and severity of cad . during invasive coronary angiography , clinical history was evaluated between the cta examination and invasive coronary angiography to ensure that neither acute coronary syndromes nor worsening of angina occurred between examinations . exclusion criteria for cta examination were : ( i ) ( supra ) ventricular arrhythmias , ( ii ) renal failure ( glomerular filtration rate < 30 ml / minute ) , ( iii ) known allergy to iodine contrast material , ( iv ) severe claustrophobia , ( v ) pregnancy . patient data were prospectively collected in the departmental cardiology information system ( epd - vision , leiden university medical center , leiden , the netherlands ) and retrospectively analyzed . our institutional review board does not require its approval and written informed consent for retrospective technical analysis of data , as was the case for this study . in each patient , the presence of risk factors for cad and medication ( use of aspirin , statins , beta - blocking medication and angiotensin - converting enzyme ( ace ) inhibitors ) were recorded . cta examination was performed using either a 64-row helical scanner ( aquilion 64 , toshiba medical systems , toshiba medical systems , otawara , japan ) or a 320-row volumetric scanner ( aquilion one , toshiba medical systems , otawara , japan ) . if the patient s heart rate was 65 beats / minute and no contra - indications existed , beta - blocking medication ( metoprolol 50 or 100 mg , single oral dose , 1 hour prior to examination ) was administered . scan parameters for the 64-row contrast enhanced scan were : 400 ms gantry rotation time , collimation of 64 0.5 mm , tube voltage of 100 - 135 kv and tube current of 250 - 350 ma , depending on body posture . non - ionic contrast material ( iomeron 400 , bracco , milan , italy or ultravist 370 , bayer schering pharma ag berlin , germany ) was administered with an amount of 80 - 110 ml followed by a saline flush with a flow rate of 5 ml / second . datasets were reconstructed from the retrospectively gated raw data , the best phase was reconstructed with an interval of 0.3 mm . using a single test slice reconstructed throughout the various phases of the heart cycle , other suitable r - r intervals were examined for additional reconstructions . concerning the 320-row contrast enhanced scan ; imaging was performed in a single volume using prospective triggering with exposure interval depending on the heart rate . if the heart rate was 65 beats / minute , the phase window was set 65%-85% of r - r interval , if the heart rate was stable and < 60 beats / minute the phase window was set at 70%-80% of r - r interval . scan parameters were : 350 ms gantry rotation time , 320 0.5 mm collimation , 100 - 135 kv tube voltage and a tube current of 400 - 580 ma , depending on body mass index ( bmi ) . overall , 60 - 90 ml contrast material ( iomeron 400 , bracco , milan , italy ) was administered with a rate of 5 - 6 ml / second followed by a saline flush . data acquisition was performed during an inspiratory breath hold of approximately 4 - 10 seconds . subsequently , images were reconstructed in the best phase of the r - r interval and transferred to a remote workstation for post - processing . cta datasets were evaluated using dedicated software ( vitrea 2.0 or vitrea fx 1.1 vital images , minnetonka , mn , usa ) . analysis was performed with the use of available post - processing tools such as cross - sectional axial slices and multiplanar reconstructions . one experienced reader , blinded to the ivus - vh results , evaluated the cta datasets . the coronary arteries were divided into 17 segments according to the modified american heart association classification.16 each segment was evaluated for the presence of any atherosclerotic plaque . structures > 1 mm within and/or adjacent to the coronary artery lumen , which could be clearly distinguished from the vessel lumen , were defined as atherosclerotic plaque.3 calcification patterns in plaques on cta were classified morphologically as non - calcified , spotty calcifications or dense calcifications . non - calcified plaques were defined as a plaque with low ct attenuation located in the vessel wall in at least two independent image planes and clearly distinguishable from the contrast - enhanced lumen and pericardial tissue without any calcification.17 plaques with spotty calcifications were defined as follows : length ( extent in the longitudinal direction of the vessel ) of the calcification < 3/2 of vessel diameter and width ( extent of the calcification perpendicular to the longitudinal direction of the vessel ) of the calcification < 2/3 of vessel diameter , as previously described ( figure 1).17 plaques with spotty calcification were further differentiated according to their length ( extent of the calcification parallel to the longitudinal direction of the vessel on curved multiplanar reconstruction ) into small spotty ( < 1 mm ) , intermediate spotty ( 1 - 3 mm ) , and large spotty calcifications ( 3 mm ) as measured with caliper function ( for illustration see figure 2).18 dense calcifications were defined as a plaque with high ct density , completely calcified and with calcifications present bilateral on cross - sectional axial slices.figure 1example of measurement of spotty calcification . plaques with spotty calcifications were defined as follows : length ( extent in the longitudinal direction of the vessel ) of the calcification < 3/2 of vessel diameter and width ( extent of the calcification perpendicular to the longitudinal direction of the vessel ) of the calcification < 2/3 of vessel diameter . a , measurement of vessel diameter ( blue line ) perpendicular to long vessel axis ( dashed white line ) . b , measurement of length of calcification ( blue line ) parallel to long vessel axis ( dashed white line ) . c , measurement of width of calcification ( blue line ) perpendicular to long vessel axis ( dashed line)figure 2illustration of the different types of spotty calcification . a , small spotty calcification ( < 1 mm ) . c , large spotty calcification ( > 3 mm ) example of measurement of spotty calcification . plaques with spotty calcifications were defined as follows : length ( extent in the longitudinal direction of the vessel ) of the calcification < 3/2 of vessel diameter and width ( extent of the calcification perpendicular to the longitudinal direction of the vessel ) of the calcification < 2/3 of vessel diameter . a , measurement of vessel diameter ( blue line ) perpendicular to long vessel axis ( dashed white line ) . b , measurement of length of calcification ( blue line ) parallel to long vessel axis ( dashed white line ) . c , measurement of width of calcification ( blue line ) perpendicular to long vessel axis ( dashed line ) illustration of the different types of spotty calcification . a , small spotty calcification ( < 1 mm ) . cta examination was performed using either a 64-row helical scanner ( aquilion 64 , toshiba medical systems , toshiba medical systems , otawara , japan ) or a 320-row volumetric scanner ( aquilion one , toshiba medical systems , otawara , japan ) . if the patient s heart rate was 65 beats / minute and no contra - indications existed , beta - blocking medication ( metoprolol 50 or 100 mg , single oral dose , 1 hour prior to examination ) was administered . scan parameters for the 64-row contrast enhanced scan were : 400 ms gantry rotation time , collimation of 64 0.5 mm , tube voltage of 100 - 135 kv and tube current of 250 - 350 ma , depending on body posture . non - ionic contrast material ( iomeron 400 , bracco , milan , italy or ultravist 370 , bayer schering pharma ag berlin , germany ) was administered with an amount of 80 - 110 ml followed by a saline flush with a flow rate of 5 ml / second . datasets were reconstructed from the retrospectively gated raw data , the best phase was reconstructed with an interval of 0.3 mm . using a single test slice reconstructed throughout the various phases of the heart cycle , other suitable r - r intervals were examined for additional reconstructions . concerning the 320-row contrast enhanced scan ; imaging was performed in a single volume using prospective triggering with exposure interval depending on the heart rate . if the heart rate was 65 beats / minute , the phase window was set 65%-85% of r - r interval , if the heart rate was stable and < 60 beats / minute the phase window was set at 70%-80% of r - r interval . scan parameters were : 350 ms gantry rotation time , 320 0.5 mm collimation , 100 - 135 kv tube voltage and a tube current of 400 - 580 ma , depending on body mass index ( bmi ) . overall , 60 - 90 ml contrast material ( iomeron 400 , bracco , milan , italy ) was administered with a rate of 5 - 6 ml / second followed by a saline flush . data acquisition was performed during an inspiratory breath hold of approximately 4 - 10 seconds . subsequently , images were reconstructed in the best phase of the r - r interval and transferred to a remote workstation for post - processing . cta datasets were evaluated using dedicated software ( vitrea 2.0 or vitrea fx 1.1 vital images , minnetonka , mn , usa ) . analysis was performed with the use of available post - processing tools such as cross - sectional axial slices and multiplanar reconstructions . one experienced reader , blinded to the ivus - vh results , evaluated the cta datasets . the coronary arteries were divided into 17 segments according to the modified american heart association classification.16 each segment was evaluated for the presence of any atherosclerotic plaque . structures > 1 mm within and/or adjacent to the coronary artery lumen , which could be clearly distinguished from the vessel lumen , were defined as atherosclerotic plaque.3 calcification patterns in plaques on cta were classified morphologically as non - calcified , spotty calcifications or dense calcifications . non - calcified plaques were defined as a plaque with low ct attenuation located in the vessel wall in at least two independent image planes and clearly distinguishable from the contrast - enhanced lumen and pericardial tissue without any calcification.17 plaques with spotty calcifications were defined as follows : length ( extent in the longitudinal direction of the vessel ) of the calcification < 3/2 of vessel diameter and width ( extent of the calcification perpendicular to the longitudinal direction of the vessel ) of the calcification < 2/3 of vessel diameter , as previously described ( figure 1).17 plaques with spotty calcification were further differentiated according to their length ( extent of the calcification parallel to the longitudinal direction of the vessel on curved multiplanar reconstruction ) into small spotty ( < 1 mm ) , intermediate spotty ( 1 - 3 mm ) , and large spotty calcifications ( 3 mm ) as measured with caliper function ( for illustration see figure 2).18 dense calcifications were defined as a plaque with high ct density , completely calcified and with calcifications present bilateral on cross - sectional axial slices.figure 1example of measurement of spotty calcification . plaques with spotty calcifications were defined as follows : length ( extent in the longitudinal direction of the vessel ) of the calcification < 3/2 of vessel diameter and width ( extent of the calcification perpendicular to the longitudinal direction of the vessel ) of the calcification < 2/3 of vessel diameter . a , measurement of vessel diameter ( blue line ) perpendicular to long vessel axis ( dashed white line ) . b , measurement of length of calcification ( blue line ) parallel to long vessel axis ( dashed white line ) . c , measurement of width of calcification ( blue line ) perpendicular to long vessel axis ( dashed line)figure 2illustration of the different types of spotty calcification . a , small spotty calcification ( < 1 mm ) . c , large spotty calcification ( > 3 mm ) example of measurement of spotty calcification . plaques with spotty calcifications were defined as follows : length ( extent in the longitudinal direction of the vessel ) of the calcification < 3/2 of vessel diameter and width ( extent of the calcification perpendicular to the longitudinal direction of the vessel ) of the calcification < 2/3 of vessel diameter . a , measurement of vessel diameter ( blue line ) perpendicular to long vessel axis ( dashed white line ) . b , measurement of length of calcification ( blue line ) parallel to long vessel axis ( dashed white line ) . c , measurement of width of calcification ( blue line ) perpendicular to long vessel axis ( dashed line ) illustration of the different types of spotty calcification . a , small spotty calcification ( < 1 mm ) . ivus examinations were acquired with a 20 mhz , 2.9 f phased - array ivus catheter , ( eagle eye , volcano corporation , rancho cordova , ca , usa ) and a dedicated ivus - console ( volcano corporation , rancho cordova , ca , usa ) after intracoronary administration of nitroglycerine . under fluoroscopic guidance , the ivus catheter was introduced distally in the coronary artery and a motorized automated pullback with a continuous speed of 0.5 mm / second was used until the catheter reached the guiding catheter . ivus - vh analysis was performed by two experienced observers blinded to baseline patient characteristics and cta findings with the use of dedicated software ( qcu - cms , version 4.59 , medis medical imaging systems , leiden , the netherlands ) . first , contour detection ( lumen and media interface ) was performed manually with the use of cross - sectional views to provide compositional output.19 secondly , on a frame - by - frame basis , the four different tissue components were differentiated and labeled with four different color - codes ( fibrotic tissue was labeled in dark green , fibro - fatty tissue in light green , necrotic core in red and dense calcium in white ) , as validated previously.10 the mean percentage of each plaque component was obtained in the full length of the lesion observed on the cta examination . a lesion on intravascular ultrasound imaging was defined as at least three consecutive frames with a plaque burden of at least 40%.20 in addition , the presence of the thin cap fibroatheroma ( tcfa ) was evaluated on a per - lesion basis . tcfa was defined as a lesion with a plaque burden 40% , presence of > 10% confluent necrotic core on three consecutive frames and necrotic core in contact with the lumen for along the lumen circumference.14,21,22 high - risk plaque characteristics on ivus - vh were defined by % of necrotic core and presence of tcfa . to ensure that identical plaques were assessed by cta and ivus - vh , coronary ostia and side branches were used as landmarks and distances from the landmarks to the target lesions were measured ( figure 3 ) . on ivus - vh , longitudinal reconstructed ecg triggered datasets were used to measure the difference between corresponding plaque and landmarks . after alignment of plaques on cta and ivus - vh , we identified matching slices between the two modalities and could determine the proximal and distal end of a lesion . examples of different calcification patterns on cta with corresponding ivus - vh images are demonstrated in figures 4 and 5.figure 3example of anatomical matching of computed tomography angiography ( cta ) with intravascular ultrasound with radiofrequency backscatter analysis ( ivus - vh ) datasets . a , straightened multiplanar reconstruction of left anterior descending coronary artery in which various landmarks have been identified ( left circumflex artery ( lcx ) , first and second diagonal branch ( d1 and d2 ) . b and c , corresponding grayscale intravascular ultrasound and ivus - vh datasets in longitudinal view in which identical landmarks have been matched to cta dataset ( yellow dashed line ) . corresponding cross - sectional view of minimal lumen area on cta ( d ) and ivus - vh ( e , f)figure 4example of small spotty calcifications as assessed on computed tomography angiography ( cta ) with high - risk plaque characteristics on intravascular ultrasound with radiofrequency backscatter analysis ( ivus - vh ) . a , multiplanar reconstruction of the right coronary artery ( rca ) demonstrating a plaque with non - calcified elements and spotty calcifications ( arrow ) . b , enlargement of the same plaque demonstrating a spotty calcification with a length of 0.7 mm . c , longitudinal reconstructed ecg triggered ivus - vh run with superimposed color coded map . the plaque ( white arrow ) corresponds to the same plaque on cta demonstrating a large necrotic core and multiple small spotty calcifications . d , cross - sectional axial image of the same plaque demonstrating calcifications ( white ) and a large necrotic core ( red ) indicating presence of a thin cap fibroatheroma ( tcfa)figure 5example of a plaque with dense calcifications on computed tomography angiography ( cta ) with corresponding grayscale and intravascular ultrasound with radiofrequency backscatter analysis ( ivus - vh ) images . a , curved multiplanar reconstruction of the right coronary artery ( rca ) demonstrating a plaque with dense calcifications ( arrow ) . inlay showing cross - sectional axial image of the same plaque with calcifications present bilateral of the coronary artery . b , enlargement of the same plaque reporting the length of the calcification ( 4.5 mm ) . c , grayscale ivus image demonstrating a heavily calcified plaque with corresponding ivus - vh image ( d ) showing a plaque with heavy calcifications ( white ) example of anatomical matching of computed tomography angiography ( cta ) with intravascular ultrasound with radiofrequency backscatter analysis ( ivus - vh ) datasets . a , straightened multiplanar reconstruction of left anterior descending coronary artery in which various landmarks have been identified ( left circumflex artery ( lcx ) , first and second diagonal branch ( d1 and d2 ) . b and c , corresponding grayscale intravascular ultrasound and ivus - vh datasets in longitudinal view in which identical landmarks have been matched to cta dataset ( yellow dashed line ) . corresponding cross - sectional view of minimal lumen area on cta ( d ) and ivus - vh ( e , f ) example of small spotty calcifications as assessed on computed tomography angiography ( cta ) with high - risk plaque characteristics on intravascular ultrasound with radiofrequency backscatter analysis ( ivus - vh ) . a , multiplanar reconstruction of the right coronary artery ( rca ) demonstrating a plaque with non - calcified elements and spotty calcifications ( arrow ) . b , enlargement of the same plaque demonstrating a spotty calcification with a length of 0.7 mm . c , longitudinal reconstructed ecg triggered ivus - vh run with superimposed color coded map . the plaque ( white arrow ) corresponds to the same plaque on cta demonstrating a large necrotic core and multiple small spotty calcifications . d , cross - sectional axial image of the same plaque demonstrating calcifications ( white ) and a large necrotic core ( red ) indicating presence of a thin cap fibroatheroma ( tcfa ) example of a plaque with dense calcifications on computed tomography angiography ( cta ) with corresponding grayscale and intravascular ultrasound with radiofrequency backscatter analysis ( ivus - vh ) images . a , curved multiplanar reconstruction of the right coronary artery ( rca ) demonstrating a plaque with dense calcifications ( arrow ) . inlay showing cross - sectional axial image of the same plaque with calcifications present bilateral of the coronary artery . b , enlargement of the same plaque reporting the length of the calcification ( 4.5 mm ) . c , grayscale ivus image demonstrating a heavily calcified plaque with corresponding ivus - vh image ( d ) showing a plaque with heavy calcifications ( white ) plaques available on both cta and ivus - vh were included in the lesion based analysis . plaque composition ( % fibrotic , fibro - fatty , necrotic core , and dense calcium ) and presence of tcfa was compared between lesions with different calcification patterns on cta ( non - calcified , small spotty calcifications , intermediate spotty calcifications , large spotty calcifications , and dense calcifications ) . if normally distributed , continuous values were expressed as means ( standard deviation ) and differences in plaque composition and type were assessed using an analysis of variance ( anova ) . if not normally distributed , values were expressed as medians ( interquartile range ( iqr ) ) and differences in plaque composition and type were assessed using the kruskall - wallis test . to account for possible clustering of coronary artery plaques within patients , the generalized estimating equation ( gee ) method was applied to evaluate the differences in plaque characteristics between the groups of different calcification patterns on cta ( non - calcified , small spotty calcifications , intermediate spotty calcifications , large spotty calcifications , and dense calcium ) . this was performed with proc genmod with a binominal distribution for the outcome variable , the link function specified as a logistical ( presence of tcfa ) or a linear ( % fibrotic , fibro - fatty , necrotic core , and dense calcium ) distribution and patients as separate subjects . due to lack of power ivus examinations were acquired with a 20 mhz , 2.9 f phased - array ivus catheter , ( eagle eye , volcano corporation , rancho cordova , ca , usa ) and a dedicated ivus - console ( volcano corporation , rancho cordova , ca , usa ) after intracoronary administration of nitroglycerine . under fluoroscopic guidance , the ivus catheter was introduced distally in the coronary artery and a motorized automated pullback with a continuous speed of 0.5 mm / second was used until the catheter reached the guiding catheter . ivus - vh analysis was performed by two experienced observers blinded to baseline patient characteristics and cta findings with the use of dedicated software ( qcu - cms , version 4.59 , medis medical imaging systems , leiden , the netherlands ) . first , contour detection ( lumen and media interface ) was performed manually with the use of cross - sectional views to provide compositional output.19 secondly , on a frame - by - frame basis , the four different tissue components were differentiated and labeled with four different color - codes ( fibrotic tissue was labeled in dark green , fibro - fatty tissue in light green , necrotic core in red and dense calcium in white ) , as validated previously.10 the mean percentage of each plaque component was obtained in the full length of the lesion observed on the cta examination . a lesion on intravascular ultrasound imaging was defined as at least three consecutive frames with a plaque burden of at least 40%.20 in addition , the presence of the thin cap fibroatheroma ( tcfa ) was evaluated on a per - lesion basis . tcfa was defined as a lesion with a plaque burden 40% , presence of > 10% confluent necrotic core on three consecutive frames and necrotic core in contact with the lumen for along the lumen circumference.14,21,22 high - risk plaque characteristics on ivus - vh were defined by % of necrotic core and presence of tcfa . to ensure that identical plaques were assessed by cta and ivus - vh , coronary ostia and side branches were used as landmarks and distances from the landmarks to the target lesions were measured ( figure 3 ) . on ivus - vh , longitudinal reconstructed ecg triggered datasets were used to measure the difference between corresponding plaque and landmarks . after alignment of plaques on cta and ivus - vh , we identified matching slices between the two modalities and could determine the proximal and distal end of a lesion . examples of different calcification patterns on cta with corresponding ivus - vh images are demonstrated in figures 4 and 5.figure 3example of anatomical matching of computed tomography angiography ( cta ) with intravascular ultrasound with radiofrequency backscatter analysis ( ivus - vh ) datasets . a , straightened multiplanar reconstruction of left anterior descending coronary artery in which various landmarks have been identified ( left circumflex artery ( lcx ) , first and second diagonal branch ( d1 and d2 ) . b and c , corresponding grayscale intravascular ultrasound and ivus - vh datasets in longitudinal view in which identical landmarks have been matched to cta dataset ( yellow dashed line ) . corresponding cross - sectional view of minimal lumen area on cta ( d ) and ivus - vh ( e , f)figure 4example of small spotty calcifications as assessed on computed tomography angiography ( cta ) with high - risk plaque characteristics on intravascular ultrasound with radiofrequency backscatter analysis ( ivus - vh ) . a , multiplanar reconstruction of the right coronary artery ( rca ) demonstrating a plaque with non - calcified elements and spotty calcifications ( arrow ) . b , enlargement of the same plaque demonstrating a spotty calcification with a length of 0.7 mm . c , longitudinal reconstructed ecg triggered ivus - vh run with superimposed color coded map . the plaque ( white arrow ) corresponds to the same plaque on cta demonstrating a large necrotic core and multiple small spotty calcifications . d , cross - sectional axial image of the same plaque demonstrating calcifications ( white ) and a large necrotic core ( red ) indicating presence of a thin cap fibroatheroma ( tcfa)figure 5example of a plaque with dense calcifications on computed tomography angiography ( cta ) with corresponding grayscale and intravascular ultrasound with radiofrequency backscatter analysis ( ivus - vh ) images . a , curved multiplanar reconstruction of the right coronary artery ( rca ) demonstrating a plaque with dense calcifications ( arrow ) . inlay showing cross - sectional axial image of the same plaque with calcifications present bilateral of the coronary artery . b , enlargement of the same plaque reporting the length of the calcification ( 4.5 mm ) . c , grayscale ivus image demonstrating a heavily calcified plaque with corresponding ivus - vh image ( d ) showing a plaque with heavy calcifications ( white ) example of anatomical matching of computed tomography angiography ( cta ) with intravascular ultrasound with radiofrequency backscatter analysis ( ivus - vh ) datasets . a , straightened multiplanar reconstruction of left anterior descending coronary artery in which various landmarks have been identified ( left circumflex artery ( lcx ) , first and second diagonal branch ( d1 and d2 ) . b and c , corresponding grayscale intravascular ultrasound and ivus - vh datasets in longitudinal view in which identical landmarks have been matched to cta dataset ( yellow dashed line ) . corresponding cross - sectional view of minimal lumen area on cta ( d ) and ivus - vh ( e , f ) example of small spotty calcifications as assessed on computed tomography angiography ( cta ) with high - risk plaque characteristics on intravascular ultrasound with radiofrequency backscatter analysis ( ivus - vh ) . a , multiplanar reconstruction of the right coronary artery ( rca ) demonstrating a plaque with non - calcified elements and spotty calcifications ( arrow ) . b , enlargement of the same plaque demonstrating a spotty calcification with a length of 0.7 mm . c , longitudinal reconstructed ecg triggered ivus - vh run with superimposed color coded map . the plaque ( white arrow ) corresponds to the same plaque on cta demonstrating a large necrotic core and multiple small spotty calcifications . d , cross - sectional axial image of the same plaque demonstrating calcifications ( white ) and a large necrotic core ( red ) indicating presence of a thin cap fibroatheroma ( tcfa ) example of a plaque with dense calcifications on computed tomography angiography ( cta ) with corresponding grayscale and intravascular ultrasound with radiofrequency backscatter analysis ( ivus - vh ) images . a , curved multiplanar reconstruction of the right coronary artery ( rca ) demonstrating a plaque with dense calcifications ( arrow ) . inlay showing cross - sectional axial image of the same plaque with calcifications present bilateral of the coronary artery . b , enlargement of the same plaque reporting the length of the calcification ( 4.5 mm ) . c , grayscale ivus image demonstrating a heavily calcified plaque with corresponding ivus - vh image ( d ) showing a plaque with heavy calcifications ( white ) plaques available on both cta and ivus - vh were included in the lesion based analysis . plaque composition ( % fibrotic , fibro - fatty , necrotic core , and dense calcium ) and presence of tcfa was compared between lesions with different calcification patterns on cta ( non - calcified , small spotty calcifications , intermediate spotty calcifications , large spotty calcifications , and dense calcifications ) . if normally distributed , continuous values were expressed as means ( standard deviation ) and differences in plaque composition and type were assessed using an analysis of variance ( anova ) . if not normally distributed , values were expressed as medians ( interquartile range ( iqr ) ) and differences in plaque composition and type were assessed using the kruskall - wallis test . to account for possible clustering of coronary artery plaques within patients , the generalized estimating equation ( gee ) method was applied to evaluate the differences in plaque characteristics between the groups of different calcification patterns on cta ( non - calcified , small spotty calcifications , intermediate spotty calcifications , large spotty calcifications , and dense calcium ) . this was performed with proc genmod with a binominal distribution for the outcome variable , the link function specified as a logistical ( presence of tcfa ) or a linear ( % fibrotic , fibro - fatty , necrotic core , and dense calcium ) distribution and patients as separate subjects . out of a total of 108 patients , 33 patients ( 31% ) were scanned with a 64-row cta scanner and 75 patients ( 69% ) with a 320-row cta scanner . baseline patient characteristics are provided in table 1 . in summary , 80 patients ( 74% ) were male and mean age was 57 10 years . ivus - vh examination was successfully performed in 264 vessels ( 81% ) of the available 324 vessels without complications ( right coronary artery ( rca ) , n = 88 ; left anterior descending coronary artery ( lad ) , n = 98 ; left circumflex coronary artery ( lcx ) , n = 78 ) . in the remaining vessels , ivus imaging could not be performed due to severe vessel tortuousity , severe stenosis , vessel occlusion or due to time - constraints in the cathlab.table 1baseline patient characteristicsnumber of patients108age ( years)57 10male gender80 ( 74%)stable angina / suspected acute coronary syndrome38/70hypertension63 ( 58%)hypercholesterolemia55 ( 51%)diabetes29 ( 27%)smoking43 ( 40%)family history of cad49 ( 45%)obesity ( 30 kg / m)21 ( 19%)medication at time of referral aspirin61 ( 57% ) statins69 ( 64% ) ace inhibitor53 ( 49% ) beta - blockers58 ( 54%)previous myocardial infarction22 ( 20%)previous pci29 ( 27%)data are absolute values , percentages or means standard deviationcad , coronary artery disease ; ace , angiotensin - converting enzyme ; pci , percutaneous coronary intervention defined as systolic blood pressure 140 mmhg or diastolic blood pressure 90 mmhg or the use of antihypertensive medicationserum total cholesterol 230 mg / dl or serum triglycerides 200 mg / dl or treatment with lipid lowering drugs baseline patient characteristics data are absolute values , percentages or means standard deviation cad , coronary artery disease ; ace , angiotensin - converting enzyme ; pci , percutaneous coronary intervention defined as systolic blood pressure 140 mmhg or diastolic blood pressure 90 mmhg or the use of antihypertensive medication serum total cholesterol 230 mg / dl or serum triglycerides 200 mg / dl or treatment with lipid lowering drugs in total , 799 plaques were demonstrated on cta and in 300 plaques ( 38% ) ivus - vh was also available . of the 300 plaques identified on cta , 78 plaques ( 26% ) were non - calcified , 39 plaques ( 13% ) had small spotty calcifications ( < 1 mm ) , 96 plaques ( 32% ) had intermediate spotty calcifications ( 1 - 3 mm ) , 54 plaques ( 18% ) had large spotty calcifications ( 3 mm ) , and 33 plaques ( 11% ) had dense calcifications . on ivus - vh , the average plaque length analyzed was 26 18 mm . the most prevalent plaque component was fibrotic tissue ( 58% , iqr 50%-63% ) , followed by necrotic core ( 15% , iqr 10%-21% ) , fibro - fatty tissue ( 13% , iqr 8%-21% ) , and dense calcium ( 6% , iqr 3%-10% ) . the results comparing calcification patterns on cta against relative plaque composition on ivus - vh are reported in table 2 . small spotty calcifications contained less fibro - fatty tissue ( 9% , iqr 6%-15% ) as compared to non - calcified plaques ( 12% , iqr 8%-22% ) , intermediate spotty calcifications ( 13% , iqr 7%21% ) , large spotty calcifications ( 15% , iqr 9%-25% ) , and dense calcifications ( 20% , iqr 13%-25% , p the more calcified plaques contained significantly more dense calcium on ivus - vh than the non - calcified plaques ( 9% , iqr 6%-11% vs 3% , iqr 1%-6% , p < more importantly , necrotic core was significantly higher in plaques with small spotty calcifications ( 20% , iqr 12%-24% ) as compared to non - calcified plaques ( 13% , iqr 6%-20% , p = .006 ) . in addition , there was a trend for a higher percentage of necrotic core in plaques with small spotty calcifications than in plaques with intermediate spotty calcifications ( p = .053 ) . moreover , as demonstrated in figure 6 , plaques with small spotty calcifications were demonstrated to have a high percentage of tcfa ( 31% ) as compared to large spotty calcifications ( 9% ) and dense calcifications ( 6% , p < , there was a trend for plaques with small spotty calcifications to have a higher percentage of tcfa than plaques with intermediate spotty calcifications ( 17% , p = .073 ) . however , no significant difference was demonstrated between the percentage of tcfa in plaques with small spotty calcifications and non - calcified plaques ( p = .37).table 2plaque composition on intravascular ultrasound with radiofrequency backscatter analysis ( ivus - vh ) is reported in relation to the different calcification patterns on computed tomography angiography ( cta)non - calcifiedsmall spotty ( < 1 mm)intermediate spotty ( 1 - 3 mm)large spotty ( 3 mm)dense calcificationsglobal p value% fibrotic61 ( 5568)56 ( 5063)58 ( 5064)54 ( 4959)54 ( 4959)<.001% fibro - fatty12 ( 822)*9 ( 615)13 ( 721)*15 ( 925)*20 ( 1325)*<.001% necrotic core13 ( 620)*20 ( 1224)14 ( 921)**17 ( 1321)14 ( 1222).003% dense calcium3 ( 16)*7 ( 413)6 ( 310)9 ( 613)9 ( 611)<.001data in parentheses are percentages with 95% confidence intervals * p value < .05 , individual groups compared to plaques with small spotty calcifications as reference group * * p value = .053 , individual group compared to plaques with small spotty calcifications as reference groupfigure 6bar graph represents the percentage of thin cap fibroatheroma ( tcfa ) in relation to the different calcification patterns on computed tomography angiography ( cta ) . as is shown , in particular plaques with small spotty calcifications on cta contain the highest percentage of tcfa as compared to large spotty calcifications and dense calcium . * small spotty calcifications did not have more tcfa than non - calcified plaques ( p = ns ) plaque composition on intravascular ultrasound with radiofrequency backscatter analysis ( ivus - vh ) is reported in relation to the different calcification patterns on computed tomography angiography ( cta ) data in parentheses are percentages with 95% confidence intervals * p value < .05 , individual groups compared to plaques with small spotty calcifications as reference group * * p value = .053 , individual group compared to plaques with small spotty calcifications as reference group bar graph represents the percentage of thin cap fibroatheroma ( tcfa ) in relation to the different calcification patterns on computed tomography angiography ( cta ) . as is shown , in particular plaques with small spotty calcifications on cta contain the highest percentage of tcfa as compared to large spotty calcifications and dense calcium . * small spotty calcifications did not have more tcfa than non - calcified plaques ( p = ns ) in total , 799 plaques were demonstrated on cta and in 300 plaques ( 38% ) ivus - vh was also available . of the 300 plaques identified on cta , 78 plaques ( 26% ) were non - calcified , 39 plaques ( 13% ) had small spotty calcifications ( < 1 mm ) , 96 plaques ( 32% ) had intermediate spotty calcifications ( 1 - 3 mm ) , 54 plaques ( 18% ) had large spotty calcifications ( 3 mm ) , and 33 plaques ( 11% ) had dense calcifications . on ivus - vh , the average plaque length analyzed was 26 18 mm . the most prevalent plaque component was fibrotic tissue ( 58% , iqr 50%-63% ) , followed by necrotic core ( 15% , iqr 10%-21% ) , fibro - fatty tissue ( 13% , iqr 8%-21% ) , and dense calcium ( 6% , iqr 3%-10% ) . the results comparing calcification patterns on cta against relative plaque composition on ivus - vh are reported in table 2 . small spotty calcifications contained less fibro - fatty tissue ( 9% , iqr 6%-15% ) as compared to non - calcified plaques ( 12% , iqr 8%-22% ) , intermediate spotty calcifications ( 13% , iqr 7%21% ) , large spotty calcifications ( 15% , iqr 9%-25% ) , and dense calcifications ( 20% , iqr 13%-25% , p < .05 ) . in line with this observation , the more calcified plaques contained significantly more dense calcium on ivus - vh than the non - calcified plaques ( 9% , iqr 6%-11% vs 3% , iqr 1%-6% , p < .001 ) . more importantly , necrotic core was significantly higher in plaques with small spotty calcifications ( 20% , iqr 12%-24% ) as compared to non - calcified plaques ( 13% , iqr 6%-20% , p = .006 ) . in addition , there was a trend for a higher percentage of necrotic core in plaques with small spotty calcifications than in plaques with intermediate spotty calcifications ( p = .053 ) . moreover , as demonstrated in figure 6 , plaques with small spotty calcifications were demonstrated to have a high percentage of tcfa ( 31% ) as compared to large spotty calcifications ( 9% ) and dense calcifications ( 6% , p < in addition , there was a trend for plaques with small spotty calcifications to have a higher percentage of tcfa than plaques with intermediate spotty calcifications ( 17% , p = .073 ) . however , no significant difference was demonstrated between the percentage of tcfa in plaques with small spotty calcifications and non - calcified plaques ( p = .37).table 2plaque composition on intravascular ultrasound with radiofrequency backscatter analysis ( ivus - vh ) is reported in relation to the different calcification patterns on computed tomography angiography ( cta)non - calcifiedsmall spotty ( < 1 mm)intermediate spotty ( 1 - 3 mm)large spotty ( 3 mm)dense calcificationsglobal p value% fibrotic61 ( 5568)56 ( 5063)58 ( 5064)54 ( 4959)54 ( 4959)<.001% fibro - fatty12 ( 822)*9 ( 615)13 ( 721)*15 ( 925)*20 ( 1325)*<.001% necrotic core13 ( 620)*20 ( 1224)14 ( 921)**17 ( 1321)14 ( 1222).003% dense calcium3 ( 16)*7 ( 413)6 ( 310)9 ( 613)9 ( 611)<.001data in parentheses are percentages with 95% confidence intervals * p value < .05 , individual groups compared to plaques with small spotty calcifications as reference group * * p value = .053 , individual group compared to plaques with small spotty calcifications as reference groupfigure 6bar graph represents the percentage of thin cap fibroatheroma ( tcfa ) in relation to the different calcification patterns on computed tomography angiography ( cta ) . as is shown , in particular plaques with small spotty calcifications on cta contain the highest percentage of tcfa as compared to large spotty calcifications and dense calcium . * small spotty calcifications did not have more tcfa than non - calcified plaques ( p = ns ) plaque composition on intravascular ultrasound with radiofrequency backscatter analysis ( ivus - vh ) is reported in relation to the different calcification patterns on computed tomography angiography ( cta ) data in parentheses are percentages with 95% confidence intervals * p value < .05 , individual groups compared to plaques with small spotty calcifications as reference group * * p value = .053 , individual group compared to plaques with small spotty calcifications as reference group bar graph represents the percentage of thin cap fibroatheroma ( tcfa ) in relation to the different calcification patterns on computed tomography angiography ( cta ) . as is shown , in particular plaques with small spotty calcifications on cta contain the highest percentage of tcfa as compared to large spotty calcifications and dense calcium . * small spotty calcifications did not have more tcfa than non - calcified plaques ( p = ns ) this study systematically evaluated the relation between different calcification patterns on cta and plaque characteristics on invasive ivus - vh . it was demonstrated that plaques with spotty calcifications , in particular the smaller spotty calcifications ( < 1 mm ) had plaque characteristics deemed high - risk on ivus - vh , as defined by a higher % of necrotic core and presence of tcfa . interestingly , the present findings are in line with histopathological studies which have reported that lesions associated with acute coronary events are often not heavily calcified.20,21,23 burke et al21 studied the degree of calcification in serial sections of coronary arteries in sudden coronary death cases and observed that plaque ruptures showed relatively little calcification and that most acute plaque ruptures resulting in sudden death occurred in areas of only mild speckled calcification . furthermore , smaller calcium deposits on grayscale ivus have been related to more unstable clinical presentation such as unstable angina and myocardial infarction . beckman et al24 measured the arc of calcium in lesions of 78 patients and found that the average arc of calcium was greatest ( 32 7 ) in patients with stable angina , less ( 15 4 ) in patients with unstable angina , and least ( 10 5 ) in patients with acute myocardial infarction ( p < .014 ) . in line with these findings , van der hoeven et al18 studied 60 patients with acute myocardial infarction on grayscale ivus and demonstrated that small calcified spots ( arc < 45 , length < 1.5 mm ) were more common in the culprit lesions as compared to the adjacent distal and proximal segments . accordingly , ehara et al25 confirmed the findings in a larger patient population of 178 patients with grayscale ivus . the authors found that not only the frequency of calcium deposits was significantly different between patients with stable , unstable angina , and myocardial infarction but also calcium deposits were significantly longer in patients with stable angina ( mean length of 4.3 3.2 mm ) as compared to patients with unstable angina ( mean length of 1.9 1.8 ) and myocardial infarction ( mean length of 2.2 1.6 ) . thus , these findings support the concept that there may be a significant difference in the pattern of coronary calcifications , particularly with respect to size and length of the deposits , among patients with stable and unstable angina . non - invasively , calcium scoring with electron beam computed tomography ( ebct ) showed similar findings . although a higher calcium score has been related to larger plaque areas and worse prognosis,26 when comparing coronary calcium scores of patients with stable angina to patients with acute myocardial infarction , significant differences were observed.27 extensive calcium was more often present in the coronary arteries of patients with chronic stable angina , whereas patients with acute myocardial infarction demonstrated a more diffuse pattern of calcifications . more interestingly , using contrast - enhanced cta , the presence of spotty calcifications were frequently present in lesions of patients with acs but rarely observed in culprit lesions of patients with stable cad.9 similarly , motoyama et al7,8 demonstrated both retrospectively and prospectively with cta that non - calcified plaques with spotty calcifications and positive remodeling were associated with a higher likelihood of the development of acs however , unlike this study , the aforementioned findings were not related to invasive observations . interestingly , a previous study by our institution evaluated the association between plaque composition on cta and the presence of high - risk features on ivus - vh in a smaller population of 50 patients.28 it was demonstrated that the more high - risk plaques ( tcfa ) were most often present in mixed plaques ( 32% ) on cta . although similar to the current findings , the % of tcfa in mixed plaques was higher in the previous evaluation than in the current study ( 31% ) . this could probably be explained by difference in the size and baseline characteristics of the patient population . in addition , the amount of dense calcium in the current study is somewhat lower than reported in other studies . however , this could be explained by the inclusion of a substantial number ( 65% ) of patients presenting with acute coronary syndrome , known to have substantially less calcified arteries . interestingly , earlier investigations have used various definitions of spotty calcifications on cta . for instance , some investigations described spotty calcification as any calcified material embedded within a non - calcified plaque , whereas other use a threshold of calcified material less than 3 mm in size.7,9,29 however , a comprehensive approach for the evaluation of the extent of the spotty calcifications on cta is preferred , thereby improving reproducibility and use in risk stratification . the strength of the current study is that a systematic approach for the evaluation of spotty calcifications was applied , demonstrating that indeed the smaller spotty calcifications were related to the plaque characteristics deemed more high - risk such as necrotic core and presence of tcfa . importantly , the findings are clinically relevant considering that cta is increasingly used for the assessment of cad and the use of this technique is extended from assessment of stenosis to the evaluation of the vessel wall . nevertheless , even with the latest generation cta scanners , exact identification of the lipid core and thin fibrous cap is not feasible at the moment . therefore , assessment of spotty calcification on cta may be beneficial for individualized risk stratification in combination with other high - risk features such as cardiovascular risk factors and biomarkers . perhaps , cta has a potential application in the context of identifying the vulnerable patient at risk for acs . however , the clinical and prognostic impact of these findings has to be evaluated in future prospective studies . first , due to the limitations of intravascular ultrasound , not all plaques on cta were also evaluated by ivus - vh . in addition , detection of the thin fibrous cap ( < 65 m ) is not yet feasible as ivus - vh has a limited radial resolution of only 100 m . furthermore , it has been suggested that dense calcium on ivus - vh is related to artifacts in the form of a halo of necrotic core surrounding dense calcium . potentially , incorporating other cta variables such as degree of stenosis and overall plaque burden , may allow for better risk stratification . in addition , this study performed a retrospective analysis of data possibly introducing a bias with regards to lesion selection . future evaluations examining on the relationship between calcification patterns on cta and ivus - vh characteristics should be performed in a more prospective manner . moreover , reproducibility information on the measurement of calcification on cta was not provided . however , recently , dose - saving algorithms and prospective ecg triggering were introduced to substantially reduce radiation dose.30 the current study demonstrated that plaques with small spotty calcifications on cta were related to plaque characteristics deemed more high - risk on invasive ivus - vh .

backgroundthe purpose of the study was to systematically compare calcification patterns in plaques on computed tomography angiography ( cta ) with plaque characteristics on intravascular ultrasound with radiofrequency backscatter analysis ( ivus - vh).methods and resultsin total , 108 patients underwent cta and ivus - vh . on cta , calcification patterns in plaques were classified as non - calcified , spotty or dense calcifications . plaques with spotty calcifications were differentiated into small spotty ( < 1 mm ) , intermediate spotty ( 1 - 3 mm ) and large spotty calcifications ( 3 mm ) . plaque characteristics deemed more high - risk on ivus - vh were defined by % necrotic core ( nc ) and presence of thin cap fibroatheroma ( tcfa ) . overall , 300 plaques were identified both on cta and ivus - vh . % nc core was significantly higher in plaques with small spotty calcifications as compared to non - calcified plaques ( 20% vs 13% , p = .006 ) . in addition , there was a trend for a higher % nc in plaques with small spotty calcifications than in plaques with intermediate spotty calcifications ( 20% vs 14% , p = .053 ) . plaques with small spotty calcifications had the highest % tcfa as compared to large spotty and dense calcifications ( 31% vs 9% and 31% vs 6% , p < .05).conclusionplaques with small spotty calcifications on cta were related to plaque characteristics deemed more high - risk on ivus - vh . therefore , cta may be valuable in the assessment of the vulnerable plaque .

Introduction Methods Patient Population and Study Protocol CTA Image acquisition Image analysis Invasive IVUS-VH Image acquisition Image analysis Statistical analysis Results Baseline CTA and IVUS-VH Results Comparison of Calcification Patterns on CTA and Plaque Composition on IVUS-VH Discussion Limitations Conclusion

several previous studies have identified specific plaque characteristics which are frequently observed with cta in patients presenting with acute coronary syndrome ( acs).6 - 8 among these characteristics , a spotty pattern of calcifications has been related to the presence of acs.6 - 8 indeed , preliminary data with cta demonstrated that culprit lesions in patients with acs had a higher prevalence of smaller spotty calcifications than target lesions in patient with stable complaints.9 moreover , the presence of spotty calcifications on cta were associated with a higher likelihood of developing acs.8 more detailed information on plaque characteristics can be obtained by intravascular ultrasound with radiofrequency backscatter analysis ( ivus - vh).10,11 this technique was developed to improve grayscale intravascular ultrasound tissue characterization and provide detailed quantitative information on plaque composition in vivo.12,13 additionally , the presence of thin cap fibroatheroma ( tcfa ) on ivus - vh was demonstrated to be independently predictive of major cardiovascular events during follow - up.14 however , the relation between calcification patterns on cta and plaque characteristics on ivus - vh has not been previously reported . structures > 1 mm within and/or adjacent to the coronary artery lumen , which could be clearly distinguished from the vessel lumen , were defined as atherosclerotic plaque.3 calcification patterns in plaques on cta were classified morphologically as non - calcified , spotty calcifications or dense calcifications . non - calcified plaques were defined as a plaque with low ct attenuation located in the vessel wall in at least two independent image planes and clearly distinguishable from the contrast - enhanced lumen and pericardial tissue without any calcification.17 plaques with spotty calcifications were defined as follows : length ( extent in the longitudinal direction of the vessel ) of the calcification < 3/2 of vessel diameter and width ( extent of the calcification perpendicular to the longitudinal direction of the vessel ) of the calcification < 2/3 of vessel diameter , as previously described ( figure 1).17 plaques with spotty calcification were further differentiated according to their length ( extent of the calcification parallel to the longitudinal direction of the vessel on curved multiplanar reconstruction ) into small spotty ( < 1 mm ) , intermediate spotty ( 1 - 3 mm ) , and large spotty calcifications ( 3 mm ) as measured with caliper function ( for illustration see figure 2).18 dense calcifications were defined as a plaque with high ct density , completely calcified and with calcifications present bilateral on cross - sectional axial slices.figure 1example of measurement of spotty calcification . corresponding cross - sectional view of minimal lumen area on cta ( d ) and ivus - vh ( e , f)figure 4example of small spotty calcifications as assessed on computed tomography angiography ( cta ) with high - risk plaque characteristics on intravascular ultrasound with radiofrequency backscatter analysis ( ivus - vh ) . d , cross - sectional axial image of the same plaque demonstrating calcifications ( white ) and a large necrotic core ( red ) indicating presence of a thin cap fibroatheroma ( tcfa)figure 5example of a plaque with dense calcifications on computed tomography angiography ( cta ) with corresponding grayscale and intravascular ultrasound with radiofrequency backscatter analysis ( ivus - vh ) images . corresponding cross - sectional view of minimal lumen area on cta ( d ) and ivus - vh ( e , f ) example of small spotty calcifications as assessed on computed tomography angiography ( cta ) with high - risk plaque characteristics on intravascular ultrasound with radiofrequency backscatter analysis ( ivus - vh ) . d , cross - sectional axial image of the same plaque demonstrating calcifications ( white ) and a large necrotic core ( red ) indicating presence of a thin cap fibroatheroma ( tcfa ) example of a plaque with dense calcifications on computed tomography angiography ( cta ) with corresponding grayscale and intravascular ultrasound with radiofrequency backscatter analysis ( ivus - vh ) images . non - calcified plaques were defined as a plaque with low ct attenuation located in the vessel wall in at least two independent image planes and clearly distinguishable from the contrast - enhanced lumen and pericardial tissue without any calcification.17 plaques with spotty calcifications were defined as follows : length ( extent in the longitudinal direction of the vessel ) of the calcification < 3/2 of vessel diameter and width ( extent of the calcification perpendicular to the longitudinal direction of the vessel ) of the calcification < 2/3 of vessel diameter , as previously described ( figure 1).17 plaques with spotty calcification were further differentiated according to their length ( extent of the calcification parallel to the longitudinal direction of the vessel on curved multiplanar reconstruction ) into small spotty ( < 1 mm ) , intermediate spotty ( 1 - 3 mm ) , and large spotty calcifications ( 3 mm ) as measured with caliper function ( for illustration see figure 2).18 dense calcifications were defined as a plaque with high ct density , completely calcified and with calcifications present bilateral on cross - sectional axial slices.figure 1example of measurement of spotty calcification . non - calcified plaques were defined as a plaque with low ct attenuation located in the vessel wall in at least two independent image planes and clearly distinguishable from the contrast - enhanced lumen and pericardial tissue without any calcification.17 plaques with spotty calcifications were defined as follows : length ( extent in the longitudinal direction of the vessel ) of the calcification < 3/2 of vessel diameter and width ( extent of the calcification perpendicular to the longitudinal direction of the vessel ) of the calcification < 2/3 of vessel diameter , as previously described ( figure 1).17 plaques with spotty calcification were further differentiated according to their length ( extent of the calcification parallel to the longitudinal direction of the vessel on curved multiplanar reconstruction ) into small spotty ( < 1 mm ) , intermediate spotty ( 1 - 3 mm ) , and large spotty calcifications ( 3 mm ) as measured with caliper function ( for illustration see figure 2).18 dense calcifications were defined as a plaque with high ct density , completely calcified and with calcifications present bilateral on cross - sectional axial slices.figure 1example of measurement of spotty calcification . corresponding cross - sectional view of minimal lumen area on cta ( d ) and ivus - vh ( e , f)figure 4example of small spotty calcifications as assessed on computed tomography angiography ( cta ) with high - risk plaque characteristics on intravascular ultrasound with radiofrequency backscatter analysis ( ivus - vh ) . d , cross - sectional axial image of the same plaque demonstrating calcifications ( white ) and a large necrotic core ( red ) indicating presence of a thin cap fibroatheroma ( tcfa)figure 5example of a plaque with dense calcifications on computed tomography angiography ( cta ) with corresponding grayscale and intravascular ultrasound with radiofrequency backscatter analysis ( ivus - vh ) images . corresponding cross - sectional view of minimal lumen area on cta ( d ) and ivus - vh ( e , f ) example of small spotty calcifications as assessed on computed tomography angiography ( cta ) with high - risk plaque characteristics on intravascular ultrasound with radiofrequency backscatter analysis ( ivus - vh ) . d , cross - sectional axial image of the same plaque demonstrating calcifications ( white ) and a large necrotic core ( red ) indicating presence of a thin cap fibroatheroma ( tcfa ) example of a plaque with dense calcifications on computed tomography angiography ( cta ) with corresponding grayscale and intravascular ultrasound with radiofrequency backscatter analysis ( ivus - vh ) images . examples of different calcification patterns on cta with corresponding ivus - vh images are demonstrated in figures 4 and 5.figure 3example of anatomical matching of computed tomography angiography ( cta ) with intravascular ultrasound with radiofrequency backscatter analysis ( ivus - vh ) datasets . corresponding cross - sectional view of minimal lumen area on cta ( d ) and ivus - vh ( e , f)figure 4example of small spotty calcifications as assessed on computed tomography angiography ( cta ) with high - risk plaque characteristics on intravascular ultrasound with radiofrequency backscatter analysis ( ivus - vh ) . d , cross - sectional axial image of the same plaque demonstrating calcifications ( white ) and a large necrotic core ( red ) indicating presence of a thin cap fibroatheroma ( tcfa)figure 5example of a plaque with dense calcifications on computed tomography angiography ( cta ) with corresponding grayscale and intravascular ultrasound with radiofrequency backscatter analysis ( ivus - vh ) images . corresponding cross - sectional view of minimal lumen area on cta ( d ) and ivus - vh ( e , f ) example of small spotty calcifications as assessed on computed tomography angiography ( cta ) with high - risk plaque characteristics on intravascular ultrasound with radiofrequency backscatter analysis ( ivus - vh ) . d , cross - sectional axial image of the same plaque demonstrating calcifications ( white ) and a large necrotic core ( red ) indicating presence of a thin cap fibroatheroma ( tcfa ) example of a plaque with dense calcifications on computed tomography angiography ( cta ) with corresponding grayscale and intravascular ultrasound with radiofrequency backscatter analysis ( ivus - vh ) images . of the 300 plaques identified on cta , 78 plaques ( 26% ) were non - calcified , 39 plaques ( 13% ) had small spotty calcifications ( < 1 mm ) , 96 plaques ( 32% ) had intermediate spotty calcifications ( 1 - 3 mm ) , 54 plaques ( 18% ) had large spotty calcifications ( 3 mm ) , and 33 plaques ( 11% ) had dense calcifications . small spotty calcifications contained less fibro - fatty tissue ( 9% , iqr 6%-15% ) as compared to non - calcified plaques ( 12% , iqr 8%-22% ) , intermediate spotty calcifications ( 13% , iqr 7%21% ) , large spotty calcifications ( 15% , iqr 9%-25% ) , and dense calcifications ( 20% , iqr 13%-25% , p the more calcified plaques contained significantly more dense calcium on ivus - vh than the non - calcified plaques ( 9% , iqr 6%-11% vs 3% , iqr 1%-6% , p < more importantly , necrotic core was significantly higher in plaques with small spotty calcifications ( 20% , iqr 12%-24% ) as compared to non - calcified plaques ( 13% , iqr 6%-20% , p = .006 ) . in addition , there was a trend for a higher percentage of necrotic core in plaques with small spotty calcifications than in plaques with intermediate spotty calcifications ( p = .053 ) . moreover , as demonstrated in figure 6 , plaques with small spotty calcifications were demonstrated to have a high percentage of tcfa ( 31% ) as compared to large spotty calcifications ( 9% ) and dense calcifications ( 6% , p < , there was a trend for plaques with small spotty calcifications to have a higher percentage of tcfa than plaques with intermediate spotty calcifications ( 17% , p = .073 ) . however , no significant difference was demonstrated between the percentage of tcfa in plaques with small spotty calcifications and non - calcified plaques ( p = .37).table 2plaque composition on intravascular ultrasound with radiofrequency backscatter analysis ( ivus - vh ) is reported in relation to the different calcification patterns on computed tomography angiography ( cta)non - calcifiedsmall spotty ( < 1 mm)intermediate spotty ( 1 - 3 mm)large spotty ( 3 mm)dense calcificationsglobal p value% fibrotic61 ( 5568)56 ( 5063)58 ( 5064)54 ( 4959)54 ( 4959)<.001% fibro - fatty12 ( 822)*9 ( 615)13 ( 721)*15 ( 925)*20 ( 1325)*<.001% necrotic core13 ( 620)*20 ( 1224)14 ( 921)**17 ( 1321)14 ( 1222).003% dense calcium3 ( 16)*7 ( 413)6 ( 310)9 ( 613)9 ( 611)<.001data in parentheses are percentages with 95% confidence intervals * p value < .05 , individual groups compared to plaques with small spotty calcifications as reference group * * p value = .053 , individual group compared to plaques with small spotty calcifications as reference groupfigure 6bar graph represents the percentage of thin cap fibroatheroma ( tcfa ) in relation to the different calcification patterns on computed tomography angiography ( cta ) . * small spotty calcifications did not have more tcfa than non - calcified plaques ( p = ns ) plaque composition on intravascular ultrasound with radiofrequency backscatter analysis ( ivus - vh ) is reported in relation to the different calcification patterns on computed tomography angiography ( cta ) data in parentheses are percentages with 95% confidence intervals * p value < .05 , individual groups compared to plaques with small spotty calcifications as reference group * * p value = .053 , individual group compared to plaques with small spotty calcifications as reference group bar graph represents the percentage of thin cap fibroatheroma ( tcfa ) in relation to the different calcification patterns on computed tomography angiography ( cta ) . of the 300 plaques identified on cta , 78 plaques ( 26% ) were non - calcified , 39 plaques ( 13% ) had small spotty calcifications ( < 1 mm ) , 96 plaques ( 32% ) had intermediate spotty calcifications ( 1 - 3 mm ) , 54 plaques ( 18% ) had large spotty calcifications ( 3 mm ) , and 33 plaques ( 11% ) had dense calcifications . small spotty calcifications contained less fibro - fatty tissue ( 9% , iqr 6%-15% ) as compared to non - calcified plaques ( 12% , iqr 8%-22% ) , intermediate spotty calcifications ( 13% , iqr 7%21% ) , large spotty calcifications ( 15% , iqr 9%-25% ) , and dense calcifications ( 20% , iqr 13%-25% , p < .05 ) . more importantly , necrotic core was significantly higher in plaques with small spotty calcifications ( 20% , iqr 12%-24% ) as compared to non - calcified plaques ( 13% , iqr 6%-20% , p = .006 ) . in addition , there was a trend for a higher percentage of necrotic core in plaques with small spotty calcifications than in plaques with intermediate spotty calcifications ( p = .053 ) . moreover , as demonstrated in figure 6 , plaques with small spotty calcifications were demonstrated to have a high percentage of tcfa ( 31% ) as compared to large spotty calcifications ( 9% ) and dense calcifications ( 6% , p < in addition , there was a trend for plaques with small spotty calcifications to have a higher percentage of tcfa than plaques with intermediate spotty calcifications ( 17% , p = .073 ) . however , no significant difference was demonstrated between the percentage of tcfa in plaques with small spotty calcifications and non - calcified plaques ( p = .37).table 2plaque composition on intravascular ultrasound with radiofrequency backscatter analysis ( ivus - vh ) is reported in relation to the different calcification patterns on computed tomography angiography ( cta)non - calcifiedsmall spotty ( < 1 mm)intermediate spotty ( 1 - 3 mm)large spotty ( 3 mm)dense calcificationsglobal p value% fibrotic61 ( 5568)56 ( 5063)58 ( 5064)54 ( 4959)54 ( 4959)<.001% fibro - fatty12 ( 822)*9 ( 615)13 ( 721)*15 ( 925)*20 ( 1325)*<.001% necrotic core13 ( 620)*20 ( 1224)14 ( 921)**17 ( 1321)14 ( 1222).003% dense calcium3 ( 16)*7 ( 413)6 ( 310)9 ( 613)9 ( 611)<.001data in parentheses are percentages with 95% confidence intervals * p value < .05 , individual groups compared to plaques with small spotty calcifications as reference group * * p value = .053 , individual group compared to plaques with small spotty calcifications as reference groupfigure 6bar graph represents the percentage of thin cap fibroatheroma ( tcfa ) in relation to the different calcification patterns on computed tomography angiography ( cta ) . * small spotty calcifications did not have more tcfa than non - calcified plaques ( p = ns ) plaque composition on intravascular ultrasound with radiofrequency backscatter analysis ( ivus - vh ) is reported in relation to the different calcification patterns on computed tomography angiography ( cta ) data in parentheses are percentages with 95% confidence intervals * p value < .05 , individual groups compared to plaques with small spotty calcifications as reference group * * p value = .053 , individual group compared to plaques with small spotty calcifications as reference group bar graph represents the percentage of thin cap fibroatheroma ( tcfa ) in relation to the different calcification patterns on computed tomography angiography ( cta ) . it was demonstrated that plaques with spotty calcifications , in particular the smaller spotty calcifications ( < 1 mm ) had plaque characteristics deemed high - risk on ivus - vh , as defined by a higher % of necrotic core and presence of tcfa . however , recently , dose - saving algorithms and prospective ecg triggering were introduced to substantially reduce radiation dose.30 the current study demonstrated that plaques with small spotty calcifications on cta were related to plaque characteristics deemed more high - risk on invasive ivus - vh .

neurofilaments ( nfs ) , the intermediate filaments of mature neurons , are among the most abundant proteins in brain . nfs expand the calibers of large myelinated axons to support nerve conduction and support dendrites of large motoneurons . unlike the intermediate filaments ( if ) of other cell types , cns neurofilaments are hetero - polymers composed of nfl , nfm , nfh and -internexin . nfm , nfl , and nfh subunits also differ from most other if proteins in being regulated by phosphorylation at many sites by multiple protein kinases . the complex hetero - polymeric structure and dynamically changing phosphate topography of nf proteins suggest that they might serve additional biological roles beyond static structural support of axon caliber . notably , nf proteins have been reported to be altered in certain neuropsychiatric states although a mechanistic understanding of their involvement is lacking . synapses have long been considered to be degradative sites for nf reaching terminals by axonal transport . a functional role for nf proteins within synapses , however , has rarely been considered although scattered observations hint at this possibility . for example , nf subunits can be transported to synaptic terminals in oligomeric form , including hetero - dimers . individual nf subunits or their fragments have been detected in synaptic fractions and bound to specific isolated synaptic proteins although contamination by axonal nf has not been excluded . a splice variant of the nmda receptor subunit nr1 has been reported to bind specifically through its cytoplasmic c - terminal domain to the nfl subunit in vitro and in non - neuronal cells transfected with nf proteins . evidence is lacking , however , for a direct interaction between nfl and nr1 in brain . nfm was shown to bind via its c - terminus to the third cytoplasmic loop of the dopamine d1 receptor ( d1r ) in vitro when both proteins were co - transfected in non - neuronal cells , which lowered d1r cell surface occupancy and sensitivity of the cells to d1r agonists . d1r is a primary site of action of stimulant drugs like cocaine and amphetamine and , interestingly , chronic exposure to drugs of abuse in humans and in animal models of drug addiction selectively lowers levels of nf proteins and alters their phosphorylation states . cocaine administration also activates extracellular signal- regulated kinase ( erk ) , a known nf protein kinase , by promoting its phosphorylation , which is coupled to increased nf phosphorylation . the functional significance of these cytoskeletal changes and their implications for synaptic function remain unclear . in this study , we used multiple independent approaches to establish that all four nf subunits of the mature cns are integral proteins of synapses , particularly the post - synaptic area . this synaptic population can be distinguished from nfs within the axonal cytoskeleton , thus excluding the possibility of artifactual nf contamination as a basis for their presence in synapses . we further observed that eliminating nf proteins from the cns profoundly disrupts synaptic plasticity without altering the structural integrity of synapses . finally , using a large panel of genetic mouse models in which individual nf subunits were selectively deleted , we establish in vivo a specific functional role of post - synaptic nfm subunits in modulating d1 receptor - mediated behavior . our findings indicate that distinctive pools of nf proteins within synapses perform roles in neurotransmission quite different from the conventional role of nf in axons . thus , they provide insight into the functional significance of selective nf subunit alterations observed in certain neuropsychiatric disorders , in addition to the better known alterations associated with neurodegenerative diseases . although nf subunits have diverse ligands , including several structural and receptor proteins enriched in synaptic terminals , it is not known whether or not nf proteins are actually stable constituents of synapses or simply axonal nf contaminants . to resolve this issue , we applied multiple complementary analytical approaches , beginning with investigations of nf subunits in synapses using immunogold electron microscopy . we first performed quantitative immunogold analyses on nfm in synaptic terminals of mouse striatum with monoclonal anti - nfm rmo44 using a post - embedding labeling procedure . in analyses of > 100 digitized micrographs using bioquant software , we quantified the densities of gold particles contained within the cytoplasm of pre - synaptic or post - synaptic bouton areas , respectively identified by clear synaptic vesicles or a post - synaptic density , and separately analyzed the dendrites adjacent to synaptic terminals . to assess non - specific labeling , we analyzed grids incubated without primary antibodies or with primary antibody pre - absorbed as a negative control ( figure 1e , f ) and , as a second negative control , we also compared immunogold labeling over synaptic mitochondria , which should lack specific labeling . comparisons of gold particles per unit area for these morphological structures ( figure 1a , b ) revealed a significant nfm enrichment in post - synaptic bouton areas compared to pre - terminal dendritic areas or pre - synaptic terminals . immunogold particle density for nfm in post - synaptic boutons was 8-fold higher ( p<0.0001 , mann - whitney test ) than that associated with mitochondria and 4 fold greater than that in adjacent pre - terminal regions of dendrites ( p<0.0001 , mann - whitney test ) . nfm immunogold density in pre - synaptic terminals , while 4 fold higher than non - specific mitochondrial labeling ( p<0.0001 , mann - whitney test ) was much lower than that in adjacent axon preterminal regions , contrasting with the nfm enrichment in postsynaptic boutons relative to pre - terminal dendritic areas . we then confirmed nfm enrichment in post - synaptic boutons by double - immunofluorescence analysis of primary cortical neurons derived from wild - type c57bl/6j mice at e17.5 days . neurons ( 21 div ) immunostained with mouse mab rmo-44 or anti - nfm rabbit polyclonal antibody displayed concentrations of nfm immunofluorescence that colocalized with the post - synaptic marker , psd95 , identified by the monoclonal antibody from sigma , and were apposed to presynaptic terminals labeled with antibodies to synaptophysin ( monoclonal , sigma ) or synapsin i ( polyclonal , sigma ) ( figure 1g nfm immunofluorescence in the post - synaptic terminal was stronger than in the pre - terminal dendrite . we could observe short 10 nm filaments in occasional synapses , which in some cases , were linearly decorated by gold particles after reaction with anti - nfm antibody in immunoem analyses ( figure 1j p ) . finally , we also confirmed the immuno - gold labeling of nfm was 150kda intact subunit and not the degradation products of this subunit by immunoblot analysis , which showed that full - length nfm is the major nfm - related protein species of synaptosomes ( supplementary figure s1 ) . we also performed pre - embedding immunogold em labeling to investigate the presence of all 4 nf subunits in synapses of wt mouse striatum ( supplementary figure s2a e ) and hippocampus ( supplementary figure s2f h ) . with each antibody , we analyzed , as negative controls , brain sections from mice lacking all nf proteins through knockout of -internexin , nfh and nfl ( triple ihl - tko mice , described in detail in figure 3 ) to assess the specific binding of antibodies to each nf subunit ( supplementary figure s2c d ) . under the conditions used in our labeling studies , morphometric analyses showed that each nf subunit antibody negligibly labeled ihl - tko striatum ( figure 2c d ) or hippocampus ( not shown ) in contrast to the strong decoration of the abundant nfs in myelinated axons of wt mice , which served as a positive control ( supplementary figure s2a b ) . morphometric analysis of gold particles associated with each of the four nf subunits , nfm ( supplementary figure s2e ) , nfl ( supplementary figure s2f ) , nfh ( supplementary figure s2 g ) and -internexin ( supplementary figure s2h ) , showed significantly higher labeling density in wild - type than in ihl - tko mice ( supplementary figure s2i ) ( p<0.0001 , mann - whitney test , mean sem , n=1026 ) . similar to nfm labeling , more labeling of nfl , nfh and -internexin was seen in post - synaptic areas than in pre - synaptic areas . to further establish the identity of nf assemblies in synapses , we performed immuno - gold double - labeling of nfl with nfm or nfl with nfh . the labeling of two subunits was clearly aligned along single filaments or associated with a loose filamentous web within the postsynaptic bouton ( figure 2a g ) . in further confirmation of the immunoem results , western blot profiles of fractions from 30% optiprep gradients demonstrated that all four nf subunits co - fractionate in significant quantities with vesicular fractions containing the synaptosomal marker psd95 ( figure 2h , fractions 914 ) . supporting the uniqueness of the nf subunit assemblies in synaptosomes , the ratio of -internexin to nfl in synaptosomes ( supplementary figure s3 ) from striatal tissue was significantly higher than that in triton - insoluble nf fractions ( p<0.05 , n=3 , student s t test ) ( figure 2i ) . moreover , we observed a significant difference in phosphorylation states of the long c - terminal tails of synaptic nfm and nfh compared to their counterparts in the total tissue ( mainly axonal ) nf population , as evidenced by significantly less phosphorylation of nfm ( p<0.01 , n=3 , student s t - test ) ( figure 2j ) and greater phosphorylation of nfh ( p<0.01 , n=3 , student s t - test ) ( figure 2k ) . we confirmed that the same pattern exists in hippocampal synaptosomes ( figure 2l n ) . when nfm was deleted from mice , the proportion of nfh relative to nfl increased 107% in the synaptic pool compared to the total pool ( p = 0.05 , n=3 , student s t test ) while the nfh to -internexin ratio showed a similar though non - significant trend ( p = 0.16 , student s t test , n=3)(figure 2o q ) . moreover , in the absence of nfm , phosphorylation of synaptic nfh increased relative to that of total ( mainly axonal ) nf ( p=0.02 , n=3 , student s t test ) . these results indicate a differential effect of nfm loss on axonal and synaptic nf pools . ihl - tko mice were further characterized and used to investigate the importance of nf proteins for synaptic function . in the absence of the three subunits from these mice , nfm was also undetectable in the cns ( figure 3a , b ) , including in synaptosomal preparations from hippocampus ( figure 3c ) . despite the complete absence of nf , g ) . in electrophysiological studies , we determined the input / output ( i / o ) responses of fepsp in the schaffer collateral pathway of the hippocampal slices prepared from control and ihl - tko animals . increasing stimulus intensity evoked robust i / o responses of fepsp in wild - type mice while the fepsp slope was significantly reduced in ihl - tko mice ( figure 3h ) . before tetanic stimulations , the baseline fepsp was recorded in 60 s intervals for 10 min with stimulation at an intensity equivalent to about 35% of the maximum evoked response . the tetanic stimulation evoked a typical ltp of fepsp in slices from wild - type mice . however , tetanic stimulation evoked a significantly reduced fepsp slopes in ihl - tko slices ( n = 12 slices/5 mice / group , p<0.001 , two - way anova with bonferroni s post hoc test ) ( figure 3i j ) . consistent with this result ihl - tko mice showed social interaction deficits using the 5-trial social memory test ( supplementary figure s4 ) , indicating that mice lacking nf proteins failed to develop normal social memory . we evaluated the effect of genetically deleting the nfm subunit on a classic d1 receptor - mediated behavior -- locomotor activity response to cocaine administration . baseline locomotor activity , calculated as total ambulatory counts over 14 hours of nighttime activity in 30-min segments , was 33% higher in mko mice than in wild - type ( wt ) controls ( 252173058 vs 188613525 , n=10 , p=0.19 , student s t test ) . to achieve sensitization to cocaine , we then injected the mice with saline on day one followed by daily cocaine injections ( 25 mg / kg ) for 3 days . activity measurements over the first 90 min after each administration showed that the repeated injections induced sensitization in both mouse groups , as evidenced by higher activity after the second cocaine injection compared with the first cocaine injection ( figure 4a ) . we obtained similar results after injecting amphetamine , another dopamine - releasing drug ( figure 4b ) . in analyses of the pooled data from 24 mko and 66 wild - type controls , onset of a motor response was faster ( within the first 10 min ) after the second cocaine injection than after the first injection in both mko ( 582 132 vs 1636 349 , n=24 , p=0.0024 , mann - whitney test ) and wt controls ( 237 29 vs 536 78 , n=66 , p = 0.0002 , mann - whitney test ) . additional wt and mko mice were later studied and the pooled data from 59 mko and 157 wt mice showed a significantly higher locomotor activity in mko mice than wt controls after cocaine injection on days 2 , 3 and 4 ( p<0.0001 , mann - whitney test ) ( figure 4a ) . we showed that these enhanced responses to stimulant drugs in mko mice were d1r - mediated by measuring stimulant drug responses in the presence of the specific d1r antagonist sch-23390 . on days 13 , mko and wild - type mice received 4 mg / kg amphetamine ; half of each group was also treated with 0.5 mg / kg sch-23390 for 30 min before amphetamine . on day 9 ( 6-day abstinence ) , mice were challenged with 1 mg / kg amphetamine ( no sch-23390 given ) . in the absence of sch-23390 , mko mice exhibited a higher degree of sensitization than wild - type controls ; however , in sch-23390-treated mko and wt mice , the sensitization responses were markedly attenuated to the same low level ( figure 4b ) . to determine whether or not these enhanced cocaine responses were selective for nfm among the four cns nf subunits , we also analyzed mice lacking either nfh , nfl , or -internexin . in contrast to mko mice , these three mouse lines exhibited normal responses to cocaine ( figure 4c ) . replacing half the normal level of nfm in mko ( mko ) mice by cross - breeding them with wild - type mice restored normal cocaine responses ( figure 4c ) , indicating that the effect on d1r - mediated locomotor behavior was related to nfm ( supplementary figure s5 ) and could be achieved at half the normal nfm gene dosage . in mice receiving daily cocaine injections for 7 days , p - erk immunoreactivity was stronger in neurons of mko mice than in wt controls ( supplementary figure s6 ) . hplc - electrochemical detection of dopamine , serotonin , and their metabolites revealed no differences in levels between wt and mko mice ( supplementary figure s7 ) , thus excluding a change in biogenic amine levels as an explanation for the enhancement of cocaine - induced locomotor activity in mko mice . in addition , total d1r levels in striatum , determined by quantitative immunoblot analysis , were not significantly affected by nfm deletion ( supplementary figure s8 ) . to investigate this relationship in neurons in vivo in the context of cocaine sensitization , we isolated striata from 12 mko and 12 wt mice injected daily for 7 days with saline or cocaine to induce sensitization and assesses d1r distribution within plasma membrane - enriched and endosome - enriched subcellular fractions . western blot analyses of the plasma membrane marker atpase or the endosome marker eea1 confirmed the substantial enrichment of plasma membranes and endosomes , respectively ( supplementary figure s9 ) . as predicted , cocaine treatment caused more d1r to shift from endosomes ( es ) to plasma membranes ( pm ) in mko mice as demonstrated by sch ligand binding ( p<0.05 , student s t- test ) ( figure 4d ) . to further establish the relationship of d1r - positive endosomes to nfm , we performed immuno - gold double labeling of d1r and nfm , which demonstrated co - localization of d1r and nfm in postsynaptic boutons ( figure 4e h ) . moreover , in synaptosomal fractions , we could show that co - immunoprecipitation of nfm and endosomal markers also pulls down d1r further indicating in vivo interaction between nfm and d1r - containing endosomes ( figure 4i ) . although nf subunits have diverse ligands , including several structural and receptor proteins enriched in synaptic terminals , it is not known whether or not nf proteins are actually stable constituents of synapses or simply axonal nf contaminants . to resolve this issue , we applied multiple complementary analytical approaches , beginning with investigations of nf subunits in synapses using immunogold electron microscopy . we first performed quantitative immunogold analyses on nfm in synaptic terminals of mouse striatum with monoclonal anti - nfm rmo44 using a post - embedding labeling procedure . in analyses of > 100 digitized micrographs using bioquant software , we quantified the densities of gold particles contained within the cytoplasm of pre - synaptic or post - synaptic bouton areas , respectively identified by clear synaptic vesicles or a post - synaptic density , and separately analyzed the dendrites adjacent to synaptic terminals . to assess non - specific labeling , we analyzed grids incubated without primary antibodies or with primary antibody pre - absorbed as a negative control ( figure 1e , f ) and , as a second negative control , we also compared immunogold labeling over synaptic mitochondria , which should lack specific labeling . comparisons of gold particles per unit area for these morphological structures ( figure 1a , b ) revealed a significant nfm enrichment in post - synaptic bouton areas compared to pre - terminal dendritic areas or pre - synaptic terminals . immunogold particle density for nfm in post - synaptic boutons was 8-fold higher ( p<0.0001 , mann - whitney test ) than that associated with mitochondria and 4 fold greater than that in adjacent pre - terminal regions of dendrites ( p<0.0001 , mann - whitney test ) . nfm immunogold density in pre - synaptic terminals , while 4 fold higher than non - specific mitochondrial labeling ( p<0.0001 , mann - whitney test ) was much lower than that in adjacent axon preterminal regions , contrasting with the nfm enrichment in postsynaptic boutons relative to pre - terminal dendritic areas . we then confirmed nfm enrichment in post - synaptic boutons by double - immunofluorescence analysis of primary cortical neurons derived from wild - type c57bl/6j mice at e17.5 days . neurons ( 21 div ) immunostained with mouse mab rmo-44 or anti - nfm rabbit polyclonal antibody displayed concentrations of nfm immunofluorescence that colocalized with the post - synaptic marker , psd95 , identified by the monoclonal antibody from sigma , and were apposed to presynaptic terminals labeled with antibodies to synaptophysin ( monoclonal , sigma ) or synapsin i ( polyclonal , sigma ) ( figure 1g nfm immunofluorescence in the post - synaptic terminal was stronger than in the pre - terminal dendrite . we could observe short 10 nm filaments in occasional synapses , which in some cases , were linearly decorated by gold particles after reaction with anti - nfm antibody in immunoem analyses ( figure 1j p ) . finally , we also confirmed the immuno - gold labeling of nfm was 150kda intact subunit and not the degradation products of this subunit by immunoblot analysis , which showed that full - length nfm is the major nfm - related protein species of synaptosomes ( supplementary figure s1 ) . we also performed pre - embedding immunogold em labeling to investigate the presence of all 4 nf subunits in synapses of wt mouse striatum ( supplementary figure s2a e ) and hippocampus ( supplementary figure s2f h ) . with each antibody , we analyzed , as negative controls , brain sections from mice lacking all nf proteins through knockout of -internexin , nfh and nfl ( triple ihl - tko mice , described in detail in figure 3 ) to assess the specific binding of antibodies to each nf subunit ( supplementary figure s2c d ) . under the conditions used in our labeling studies , morphometric analyses showed that each nf subunit antibody negligibly labeled ihl - tko striatum ( figure 2c d ) or hippocampus ( not shown ) in contrast to the strong decoration of the abundant nfs in myelinated axons of wt mice , which served as a positive control ( supplementary figure s2a b ) . morphometric analysis of gold particles associated with each of the four nf subunits , nfm ( supplementary figure s2e ) , nfl ( supplementary figure s2f ) , nfh ( supplementary figure s2 g ) and -internexin ( supplementary figure s2h ) , showed significantly higher labeling density in wild - type than in ihl - tko mice ( supplementary figure s2i ) ( p<0.0001 , mann - whitney test , mean sem , n=1026 ) . similar to nfm labeling , more labeling of nfl , nfh and -internexin was seen in post - synaptic areas than in pre - synaptic areas . to further establish the identity of nf assemblies in synapses , we performed immuno - gold double - labeling of nfl with nfm or nfl with nfh . the labeling of two subunits was clearly aligned along single filaments or associated with a loose filamentous web within the postsynaptic bouton ( figure 2a g ) . in further confirmation of the immunoem results , western blot profiles of fractions from 30% optiprep gradients demonstrated that all four nf subunits co - fractionate in significant quantities with vesicular fractions containing the synaptosomal marker psd95 ( figure 2h , fractions 914 ) . supporting the uniqueness of the nf subunit assemblies in synaptosomes , the ratio of -internexin to nfl in synaptosomes ( supplementary figure s3 ) from striatal tissue was significantly higher than that in triton - insoluble nf fractions ( p<0.05 , n=3 , student s t test ) ( figure 2i ) . moreover , we observed a significant difference in phosphorylation states of the long c - terminal tails of synaptic nfm and nfh compared to their counterparts in the total tissue ( mainly axonal ) nf population , as evidenced by significantly less phosphorylation of nfm ( p<0.01 , n=3 , student s t - test ) ( figure 2j ) and greater phosphorylation of nfh ( p<0.01 , n=3 , student s t - test ) ( figure 2k ) . we confirmed that the same pattern exists in hippocampal synaptosomes ( figure 2l n ) . when nfm was deleted from mice , the proportion of nfh relative to nfl increased 107% in the synaptic pool compared to the total pool ( p = 0.05 , n=3 , student s t test ) while the nfh to -internexin ratio showed a similar though non - significant trend ( p = 0.16 , student s t test , n=3)(figure 2o q ) . moreover , in the absence of nfm , phosphorylation of synaptic nfh increased relative to that of total ( mainly axonal ) nf ( p=0.02 , n=3 , student s t test ) . these results indicate a differential effect of nfm loss on axonal and synaptic nf pools . ihl - tko mice were further characterized and used to investigate the importance of nf proteins for synaptic function . in the absence of the three subunits from these mice , nfm was also undetectable in the cns ( figure 3a , b ) , including in synaptosomal preparations from hippocampus ( figure 3c ) . despite the complete absence of nf , g ) . in electrophysiological studies , we determined the input / output ( i / o ) responses of fepsp in the schaffer collateral pathway of the hippocampal slices prepared from control and ihl - tko animals . increasing stimulus intensity evoked robust i / o responses of fepsp in wild - type mice while the fepsp slope was significantly reduced in ihl - tko mice ( figure 3h ) . before tetanic stimulations , the baseline fepsp was recorded in 60 s intervals for 10 min with stimulation at an intensity equivalent to about 35% of the maximum evoked response . the tetanic stimulation evoked a typical ltp of fepsp in slices from wild - type mice . however , tetanic stimulation evoked a significantly reduced fepsp slopes in ihl - tko slices ( n = 12 slices/5 mice / group , p<0.001 , two - way anova with bonferroni s post hoc test ) ( figure 3i j ) . consistent with this result ihl - tko mice showed social interaction deficits using the 5-trial social memory test ( supplementary figure s4 ) , indicating that mice lacking nf proteins failed to develop normal social memory . we evaluated the effect of genetically deleting the nfm subunit on a classic d1 receptor - mediated behavior -- locomotor activity response to cocaine administration . baseline locomotor activity , calculated as total ambulatory counts over 14 hours of nighttime activity in 30-min segments , was 33% higher in mko mice than in wild - type ( wt ) controls ( 252173058 vs 188613525 , n=10 , p=0.19 , student s t test ) . to achieve sensitization to cocaine , we then injected the mice with saline on day one followed by daily cocaine injections ( 25 mg / kg ) for 3 days . activity measurements over the first 90 min after each administration showed that the repeated injections induced sensitization in both mouse groups , as evidenced by higher activity after the second cocaine injection compared with the first cocaine injection ( figure 4a ) . we obtained similar results after injecting amphetamine , another dopamine - releasing drug ( figure 4b ) . in analyses of the pooled data from 24 mko and 66 wild - type controls , onset of a motor response was faster ( within the first 10 min ) after the second cocaine injection than after the first injection in both mko ( 582 132 vs 1636 349 , n=24 , p=0.0024 , mann - whitney test ) and wt controls ( 237 29 vs 536 78 , n=66 , p = 0.0002 , mann - whitney test ) . additional wt and mko mice were later studied and the pooled data from 59 mko and 157 wt mice showed a significantly higher locomotor activity in mko mice than wt controls after cocaine injection on days 2 , 3 and 4 ( p<0.0001 , mann - whitney test ) ( figure 4a ) . we showed that these enhanced responses to stimulant drugs in mko mice were d1r - mediated by measuring stimulant drug responses in the presence of the specific d1r antagonist sch-23390 . on days 13 , mko and wild - type mice received 4 mg / kg amphetamine ; half of each group was also treated with 0.5 mg / kg sch-23390 for 30 min before amphetamine . on day 9 ( 6-day abstinence ) , mice were challenged with 1 mg / kg amphetamine ( no sch-23390 given ) . in the absence of sch-23390 , mko mice exhibited a higher degree of sensitization than wild - type controls ; however , in sch-23390-treated mko and wt mice , the sensitization responses were markedly attenuated to the same low level ( figure 4b ) . to determine whether or not these enhanced cocaine responses were selective for nfm among the four cns nf subunits , we also analyzed mice lacking either nfh , nfl , or -internexin . in contrast to mko mice , these three mouse lines exhibited normal responses to cocaine ( figure 4c ) . replacing half the normal level of nfm in mko ( mko ) mice by cross - breeding them with wild - type mice restored normal cocaine responses ( figure 4c ) , indicating that the effect on d1r - mediated locomotor behavior was related to nfm ( supplementary figure s5 ) and could be achieved at half the normal nfm gene dosage . in mice receiving daily cocaine injections for 7 days , p - erk immunoreactivity was stronger in neurons of mko mice than in wt controls ( supplementary figure s6 ) . hplc - electrochemical detection of dopamine , serotonin , and their metabolites revealed no differences in levels between wt and mko mice ( supplementary figure s7 ) , thus excluding a change in biogenic amine levels as an explanation for the enhancement of cocaine - induced locomotor activity in mko mice . in addition , total d1r levels in striatum , determined by quantitative immunoblot analysis , were not significantly affected by nfm deletion ( supplementary figure s8 ) . to investigate this relationship in neurons in vivo in the context of cocaine sensitization , we isolated striata from 12 mko and 12 wt mice injected daily for 7 days with saline or cocaine to induce sensitization and assesses d1r distribution within plasma membrane - enriched and endosome - enriched subcellular fractions . western blot analyses of the plasma membrane marker atpase or the endosome marker eea1 confirmed the substantial enrichment of plasma membranes and endosomes , respectively ( supplementary figure s9 ) . as predicted , cocaine treatment caused more d1r to shift from endosomes ( es ) to plasma membranes ( pm ) in mko mice as demonstrated by sch ligand binding ( p<0.05 , student s t- test ) ( figure 4d ) . to further establish the relationship of d1r - positive endosomes to nfm , we performed immuno - gold double labeling of d1r and nfm , which demonstrated co - localization of d1r and nfm in postsynaptic boutons ( figure 4e h ) . moreover , in synaptosomal fractions , we could show that co - immunoprecipitation of nfm and endosomal markers also pulls down d1r further indicating in vivo interaction between nfm and d1r - containing endosomes ( figure 4i ) . although nf proteins have been detected in synaptic fractions and bound to synaptic proteins in vitro , they are usually considered to be axonal nf contaminants and their presence , let alone their possible function , in synapses has been controversial . using multiple independent approaches , we have shown unequivocally for the first time that a unique population of nf proteins is present and functional in synapses and relatively abundant in the postsynaptic area relative to terminal dendritic branches . nf proteins within the synapses not only exhibit different relative subunit ratios than the ones in axons , but they are also differently phosphorylated , indicating that nf proteins in synapses may serve different functions from those in axons . the relatively high proportions of -internexin and lowered phosphorylation state of nfm in synaptic nf assemblies would be expected to confer greater plasticity to the cytoskeleton and allow increased interaction with binding partners . although nf proteins are present in both pre- and postsynaptic areas , 10 nm nf polymers are not abundant in synaptic areas compared to myelinated axons , estimated by conventional electron microscopy . using a rapid - freezing technique , landis and reese described infrequent 910 nm filaments in dendritic spines of mouse brain ( figure 1l ) but interpreted these structures as actin filaments due to their periodicity . nf also co - isolated with psd but have generally been regarded as contaminants . the infrequency of conventional long intermediate - sized polymers in synapses indicates that most nf subunits in synapses are probably in the form of oligomeric structures ( protofilaments or protofibrils ) . consistent with this interpretation , transport of nfm in non - filamentous , but oligomeric form , has been reported in vivo . it is also possible that nf polymers in synapses are more susceptible than axonal nf to disassembly and /or degradation . our findings that nf proteins within the synapses contain more -internexin and a less phosphorylated form of nfm support the notion that nf proteins within the synaptic cytoskeleton network are in a relatively immature state as in developing neurons , making the cytoskeletal structure more dynamic and susceptible to disassembly . because nfl and nfm monomers are inefficiently transported along axons , nf proteins in synapses are at least oligomeric in form and can be transported as such at least in axons , providing one possible basis for their postsynaptic location . it is also possible that nf proteins in postsynaptic boutons are synthesized locally because mrnas of nfl and -internexin together with mrnas of -actin and tubulin were reported to be present in dendrites and polyribosomes are preferentially localized under the base of dendritic spines . furthermore , bdnf - induced synaptic plasticity from hippocampal slices is blocked by inhibitors of protein synthesis and schaffer - collateral ca1 synapses that were isolated from their pre- and postsynaptic cell bodies still exhibited protein synthesis - dependent plasticity , suggesting a local dendritic source of protein synthesis . in this regard , evidence indicates that nf proteins identified in nerve terminals isolated from squid brain are generated by local protein synthesis . therefore , by either mechanism , assembly states and ratios of nf subunits could be regulated by the local concentration and phosphorylation of individual subunits . in our triple knockout mice lacking -internexin , nfh , and nfl , or a second mouse line lacking -internexin , nfh and nfm ( not shown ) , the content of nfm or nfl proteins in the cns , respectively , is negligible suggesting that single subunits are unlikely to be a functional form of nf proteins in synapses . collectively , our data suggest that a platform minimally of oligomeric nf assemblies or short 10 nm nf polymers may be needed for function at synapses . our gene deletion studies demonstrated for the first time that lack of nfm leads to changes of d1r - mediated ltp and d1r - mediated behavior . our findings are consistent with a model ( figure 5 ) in which nfm , within a synaptic cytoskeletal structure , anchors d1r - containing endosomes within synapses , thereby establishing a reservoir of receptors available for rapid recycling to the synaptic plasma membrane following dopamine agonist stimulation . without nfm , recycling back to the surface is accentuated favoring hypersensitivity to d1r agonists . nfm is one of two neurofilament subunits with exceptionally long carboxyl - terminal tail domains but it seems to play a much more important role than its larger counterpart , nfh , in regulating the structure and function of neurofilaments in axons . the diversity of roles for nfm in neuronal function is consistent with its complex regulation by phosphorylation , which involves second - messenger regulated kinase regulation of up to 36 sites on the head domain and 12 sites on the c - terminal tail of human nfm , which are regulated by at least four potential proline - directed kinases and multiple protein phosphatases . the d1 receptor has been shown to interact with the c - terminal tail of nfm and it will be important for future studies to address the role of phosphorylation of this domain in regulating d1r binding . our demonstrations of nfm involvement in d1r - mediated ltp and d1r - mediated behavior provide evidence that a unique population of nf assemblies is functional in synapses and that the observed effects on synaptic function are not a result of abnormal transport or abnormal effects on nerve conduction or axonal diameter . only deletion of nfm but not other nf subunits markedly exaggerated d1r - mediated response to cocaine , even though deletions of nfl or nfh markedly alter caliber and conduction or conduction , respectively . besides nfm , it might be predicted that -internexin , nfh and nfl also have specific functions in synapses because deletion of all nfs has profound effects on synaptic plasticity and social memory . supporting this view , our further observations also indicate that nfl selectively influences glutamate receptor function ( veeranna / basavarajappa et al . unpublished data ) . moreover , depressed hippocampal ltp induction is also nf subunit - selective : maintenance of ltp is deficient in nfh - null mice while basal neurotransmission and induction of ltp are normal in nfm - null mice ( not shown ) . binding of nfm to d1r has been previously shown in non - neuronal cells of epithelial origin , although both nfm and d1r were over - expressed and foreign to these cultured human embryonic kidney cells . these findings strongly support the view that nf proteins are integral components of synapses , providing insight into the causes and functional significance of nf subunit alterations observed in neuropsychiatric diseases .

synaptic roles for neurofilament proteins have rarely been considered . here , we establish all four neurofilament subunits as integral resident proteins of synapses . compared to the population in axons , neurofilament subunits isolated from synapses have distinctive stoichiometry and phosphorylation state , and respond differently to perturbations in vivo . completely eliminating neurofilament proteins from brain by genetically deleting three subunits ( -internexin , nfh and nfl ) markedly depresses hippocampal ltp induction without detectably altering synapse morphology . deletion of nfm in mice , but not the deletion of any other neurofilament subunit , amplifies dopamine d1-receptor - mediated motor responses to cocaine while redistributing postsynaptic d1-receptors from endosomes to plasma membrane , consistent with a specific modulatory role of nfm in d1-receptor recycling . these results identify a distinct pool of synaptic neurofilament subunits and establish their key role in neurotransmission in vivo , suggesting potential novel influences of neurofilament proteins in psychiatric as well as neurological states .

INTRODUCTION MATERIALS AND METHODS RESULTS Identification of a unique population of NF proteins within synapses Elimination of neurofilaments impairs hippocampal LTP induction NFM specifically influences dopamine D1 receptor mediated behavior DISCUSSION Supplementary Material

unlike the intermediate filaments ( if ) of other cell types , cns neurofilaments are hetero - polymers composed of nfl , nfm , nfh and -internexin . nfm , nfl , and nfh subunits also differ from most other if proteins in being regulated by phosphorylation at many sites by multiple protein kinases . synapses have long been considered to be degradative sites for nf reaching terminals by axonal transport . a functional role for nf proteins within synapses , however , has rarely been considered although scattered observations hint at this possibility . in this study , we used multiple independent approaches to establish that all four nf subunits of the mature cns are integral proteins of synapses , particularly the post - synaptic area . we further observed that eliminating nf proteins from the cns profoundly disrupts synaptic plasticity without altering the structural integrity of synapses . finally , using a large panel of genetic mouse models in which individual nf subunits were selectively deleted , we establish in vivo a specific functional role of post - synaptic nfm subunits in modulating d1 receptor - mediated behavior . our findings indicate that distinctive pools of nf proteins within synapses perform roles in neurotransmission quite different from the conventional role of nf in axons . although nf subunits have diverse ligands , including several structural and receptor proteins enriched in synaptic terminals , it is not known whether or not nf proteins are actually stable constituents of synapses or simply axonal nf contaminants . in analyses of > 100 digitized micrographs using bioquant software , we quantified the densities of gold particles contained within the cytoplasm of pre - synaptic or post - synaptic bouton areas , respectively identified by clear synaptic vesicles or a post - synaptic density , and separately analyzed the dendrites adjacent to synaptic terminals . neurons ( 21 div ) immunostained with mouse mab rmo-44 or anti - nfm rabbit polyclonal antibody displayed concentrations of nfm immunofluorescence that colocalized with the post - synaptic marker , psd95 , identified by the monoclonal antibody from sigma , and were apposed to presynaptic terminals labeled with antibodies to synaptophysin ( monoclonal , sigma ) or synapsin i ( polyclonal , sigma ) ( figure 1g nfm immunofluorescence in the post - synaptic terminal was stronger than in the pre - terminal dendrite . finally , we also confirmed the immuno - gold labeling of nfm was 150kda intact subunit and not the degradation products of this subunit by immunoblot analysis , which showed that full - length nfm is the major nfm - related protein species of synaptosomes ( supplementary figure s1 ) . with each antibody , we analyzed , as negative controls , brain sections from mice lacking all nf proteins through knockout of -internexin , nfh and nfl ( triple ihl - tko mice , described in detail in figure 3 ) to assess the specific binding of antibodies to each nf subunit ( supplementary figure s2c d ) . under the conditions used in our labeling studies , morphometric analyses showed that each nf subunit antibody negligibly labeled ihl - tko striatum ( figure 2c d ) or hippocampus ( not shown ) in contrast to the strong decoration of the abundant nfs in myelinated axons of wt mice , which served as a positive control ( supplementary figure s2a b ) . similar to nfm labeling , more labeling of nfl , nfh and -internexin was seen in post - synaptic areas than in pre - synaptic areas . in further confirmation of the immunoem results , western blot profiles of fractions from 30% optiprep gradients demonstrated that all four nf subunits co - fractionate in significant quantities with vesicular fractions containing the synaptosomal marker psd95 ( figure 2h , fractions 914 ) . moreover , we observed a significant difference in phosphorylation states of the long c - terminal tails of synaptic nfm and nfh compared to their counterparts in the total tissue ( mainly axonal ) nf population , as evidenced by significantly less phosphorylation of nfm ( p<0.01 , n=3 , student s t - test ) ( figure 2j ) and greater phosphorylation of nfh ( p<0.01 , n=3 , student s t - test ) ( figure 2k ) . when nfm was deleted from mice , the proportion of nfh relative to nfl increased 107% in the synaptic pool compared to the total pool ( p = 0.05 , n=3 , student s t test ) while the nfh to -internexin ratio showed a similar though non - significant trend ( p = 0.16 , student s t test , n=3)(figure 2o q ) . moreover , in the absence of nfm , phosphorylation of synaptic nfh increased relative to that of total ( mainly axonal ) nf ( p=0.02 , n=3 , student s t test ) . these results indicate a differential effect of nfm loss on axonal and synaptic nf pools . in the absence of the three subunits from these mice , nfm was also undetectable in the cns ( figure 3a , b ) , including in synaptosomal preparations from hippocampus ( figure 3c ) . we evaluated the effect of genetically deleting the nfm subunit on a classic d1 receptor - mediated behavior -- locomotor activity response to cocaine administration . to achieve sensitization to cocaine , we then injected the mice with saline on day one followed by daily cocaine injections ( 25 mg / kg ) for 3 days . we showed that these enhanced responses to stimulant drugs in mko mice were d1r - mediated by measuring stimulant drug responses in the presence of the specific d1r antagonist sch-23390 . in the absence of sch-23390 , mko mice exhibited a higher degree of sensitization than wild - type controls ; however , in sch-23390-treated mko and wt mice , the sensitization responses were markedly attenuated to the same low level ( figure 4b ) . in contrast to mko mice , these three mouse lines exhibited normal responses to cocaine ( figure 4c ) . replacing half the normal level of nfm in mko ( mko ) mice by cross - breeding them with wild - type mice restored normal cocaine responses ( figure 4c ) , indicating that the effect on d1r - mediated locomotor behavior was related to nfm ( supplementary figure s5 ) and could be achieved at half the normal nfm gene dosage . to investigate this relationship in neurons in vivo in the context of cocaine sensitization , we isolated striata from 12 mko and 12 wt mice injected daily for 7 days with saline or cocaine to induce sensitization and assesses d1r distribution within plasma membrane - enriched and endosome - enriched subcellular fractions . as predicted , cocaine treatment caused more d1r to shift from endosomes ( es ) to plasma membranes ( pm ) in mko mice as demonstrated by sch ligand binding ( p<0.05 , student s t- test ) ( figure 4d ) . to further establish the relationship of d1r - positive endosomes to nfm , we performed immuno - gold double labeling of d1r and nfm , which demonstrated co - localization of d1r and nfm in postsynaptic boutons ( figure 4e h ) . moreover , in synaptosomal fractions , we could show that co - immunoprecipitation of nfm and endosomal markers also pulls down d1r further indicating in vivo interaction between nfm and d1r - containing endosomes ( figure 4i ) . although nf subunits have diverse ligands , including several structural and receptor proteins enriched in synaptic terminals , it is not known whether or not nf proteins are actually stable constituents of synapses or simply axonal nf contaminants . in analyses of > 100 digitized micrographs using bioquant software , we quantified the densities of gold particles contained within the cytoplasm of pre - synaptic or post - synaptic bouton areas , respectively identified by clear synaptic vesicles or a post - synaptic density , and separately analyzed the dendrites adjacent to synaptic terminals . immunogold particle density for nfm in post - synaptic boutons was 8-fold higher ( p<0.0001 , mann - whitney test ) than that associated with mitochondria and 4 fold greater than that in adjacent pre - terminal regions of dendrites ( p<0.0001 , mann - whitney test ) . neurons ( 21 div ) immunostained with mouse mab rmo-44 or anti - nfm rabbit polyclonal antibody displayed concentrations of nfm immunofluorescence that colocalized with the post - synaptic marker , psd95 , identified by the monoclonal antibody from sigma , and were apposed to presynaptic terminals labeled with antibodies to synaptophysin ( monoclonal , sigma ) or synapsin i ( polyclonal , sigma ) ( figure 1g nfm immunofluorescence in the post - synaptic terminal was stronger than in the pre - terminal dendrite . finally , we also confirmed the immuno - gold labeling of nfm was 150kda intact subunit and not the degradation products of this subunit by immunoblot analysis , which showed that full - length nfm is the major nfm - related protein species of synaptosomes ( supplementary figure s1 ) . with each antibody , we analyzed , as negative controls , brain sections from mice lacking all nf proteins through knockout of -internexin , nfh and nfl ( triple ihl - tko mice , described in detail in figure 3 ) to assess the specific binding of antibodies to each nf subunit ( supplementary figure s2c d ) . under the conditions used in our labeling studies , morphometric analyses showed that each nf subunit antibody negligibly labeled ihl - tko striatum ( figure 2c d ) or hippocampus ( not shown ) in contrast to the strong decoration of the abundant nfs in myelinated axons of wt mice , which served as a positive control ( supplementary figure s2a b ) . similar to nfm labeling , more labeling of nfl , nfh and -internexin was seen in post - synaptic areas than in pre - synaptic areas . moreover , we observed a significant difference in phosphorylation states of the long c - terminal tails of synaptic nfm and nfh compared to their counterparts in the total tissue ( mainly axonal ) nf population , as evidenced by significantly less phosphorylation of nfm ( p<0.01 , n=3 , student s t - test ) ( figure 2j ) and greater phosphorylation of nfh ( p<0.01 , n=3 , student s t - test ) ( figure 2k ) . when nfm was deleted from mice , the proportion of nfh relative to nfl increased 107% in the synaptic pool compared to the total pool ( p = 0.05 , n=3 , student s t test ) while the nfh to -internexin ratio showed a similar though non - significant trend ( p = 0.16 , student s t test , n=3)(figure 2o q ) . moreover , in the absence of nfm , phosphorylation of synaptic nfh increased relative to that of total ( mainly axonal ) nf ( p=0.02 , n=3 , student s t test ) . these results indicate a differential effect of nfm loss on axonal and synaptic nf pools . in the absence of the three subunits from these mice , nfm was also undetectable in the cns ( figure 3a , b ) , including in synaptosomal preparations from hippocampus ( figure 3c ) . we evaluated the effect of genetically deleting the nfm subunit on a classic d1 receptor - mediated behavior -- locomotor activity response to cocaine administration . to achieve sensitization to cocaine , we then injected the mice with saline on day one followed by daily cocaine injections ( 25 mg / kg ) for 3 days . we showed that these enhanced responses to stimulant drugs in mko mice were d1r - mediated by measuring stimulant drug responses in the presence of the specific d1r antagonist sch-23390 . in the absence of sch-23390 , mko mice exhibited a higher degree of sensitization than wild - type controls ; however , in sch-23390-treated mko and wt mice , the sensitization responses were markedly attenuated to the same low level ( figure 4b ) . to determine whether or not these enhanced cocaine responses were selective for nfm among the four cns nf subunits , we also analyzed mice lacking either nfh , nfl , or -internexin . in contrast to mko mice , these three mouse lines exhibited normal responses to cocaine ( figure 4c ) . replacing half the normal level of nfm in mko ( mko ) mice by cross - breeding them with wild - type mice restored normal cocaine responses ( figure 4c ) , indicating that the effect on d1r - mediated locomotor behavior was related to nfm ( supplementary figure s5 ) and could be achieved at half the normal nfm gene dosage . hplc - electrochemical detection of dopamine , serotonin , and their metabolites revealed no differences in levels between wt and mko mice ( supplementary figure s7 ) , thus excluding a change in biogenic amine levels as an explanation for the enhancement of cocaine - induced locomotor activity in mko mice . to investigate this relationship in neurons in vivo in the context of cocaine sensitization , we isolated striata from 12 mko and 12 wt mice injected daily for 7 days with saline or cocaine to induce sensitization and assesses d1r distribution within plasma membrane - enriched and endosome - enriched subcellular fractions . as predicted , cocaine treatment caused more d1r to shift from endosomes ( es ) to plasma membranes ( pm ) in mko mice as demonstrated by sch ligand binding ( p<0.05 , student s t- test ) ( figure 4d ) . to further establish the relationship of d1r - positive endosomes to nfm , we performed immuno - gold double labeling of d1r and nfm , which demonstrated co - localization of d1r and nfm in postsynaptic boutons ( figure 4e h ) . moreover , in synaptosomal fractions , we could show that co - immunoprecipitation of nfm and endosomal markers also pulls down d1r further indicating in vivo interaction between nfm and d1r - containing endosomes ( figure 4i ) . although nf proteins have been detected in synaptic fractions and bound to synaptic proteins in vitro , they are usually considered to be axonal nf contaminants and their presence , let alone their possible function , in synapses has been controversial . nf proteins within the synapses not only exhibit different relative subunit ratios than the ones in axons , but they are also differently phosphorylated , indicating that nf proteins in synapses may serve different functions from those in axons . the relatively high proportions of -internexin and lowered phosphorylation state of nfm in synaptic nf assemblies would be expected to confer greater plasticity to the cytoskeleton and allow increased interaction with binding partners . although nf proteins are present in both pre- and postsynaptic areas , 10 nm nf polymers are not abundant in synaptic areas compared to myelinated axons , estimated by conventional electron microscopy . consistent with this interpretation , transport of nfm in non - filamentous , but oligomeric form , has been reported in vivo . our findings that nf proteins within the synapses contain more -internexin and a less phosphorylated form of nfm support the notion that nf proteins within the synaptic cytoskeleton network are in a relatively immature state as in developing neurons , making the cytoskeletal structure more dynamic and susceptible to disassembly . because nfl and nfm monomers are inefficiently transported along axons , nf proteins in synapses are at least oligomeric in form and can be transported as such at least in axons , providing one possible basis for their postsynaptic location . furthermore , bdnf - induced synaptic plasticity from hippocampal slices is blocked by inhibitors of protein synthesis and schaffer - collateral ca1 synapses that were isolated from their pre- and postsynaptic cell bodies still exhibited protein synthesis - dependent plasticity , suggesting a local dendritic source of protein synthesis . in our triple knockout mice lacking -internexin , nfh , and nfl , or a second mouse line lacking -internexin , nfh and nfm ( not shown ) , the content of nfm or nfl proteins in the cns , respectively , is negligible suggesting that single subunits are unlikely to be a functional form of nf proteins in synapses . our gene deletion studies demonstrated for the first time that lack of nfm leads to changes of d1r - mediated ltp and d1r - mediated behavior . our findings are consistent with a model ( figure 5 ) in which nfm , within a synaptic cytoskeletal structure , anchors d1r - containing endosomes within synapses , thereby establishing a reservoir of receptors available for rapid recycling to the synaptic plasma membrane following dopamine agonist stimulation . nfm is one of two neurofilament subunits with exceptionally long carboxyl - terminal tail domains but it seems to play a much more important role than its larger counterpart , nfh , in regulating the structure and function of neurofilaments in axons . the diversity of roles for nfm in neuronal function is consistent with its complex regulation by phosphorylation , which involves second - messenger regulated kinase regulation of up to 36 sites on the head domain and 12 sites on the c - terminal tail of human nfm , which are regulated by at least four potential proline - directed kinases and multiple protein phosphatases . the d1 receptor has been shown to interact with the c - terminal tail of nfm and it will be important for future studies to address the role of phosphorylation of this domain in regulating d1r binding . our demonstrations of nfm involvement in d1r - mediated ltp and d1r - mediated behavior provide evidence that a unique population of nf assemblies is functional in synapses and that the observed effects on synaptic function are not a result of abnormal transport or abnormal effects on nerve conduction or axonal diameter . only deletion of nfm but not other nf subunits markedly exaggerated d1r - mediated response to cocaine , even though deletions of nfl or nfh markedly alter caliber and conduction or conduction , respectively . besides nfm , it might be predicted that -internexin , nfh and nfl also have specific functions in synapses because deletion of all nfs has profound effects on synaptic plasticity and social memory . moreover , depressed hippocampal ltp induction is also nf subunit - selective : maintenance of ltp is deficient in nfh - null mice while basal neurotransmission and induction of ltp are normal in nfm - null mice ( not shown ) . these findings strongly support the view that nf proteins are integral components of synapses , providing insight into the causes and functional significance of nf subunit alterations observed in neuropsychiatric diseases .

animal procedures were conducted in accord with the arvo statement for the use of animals in ophthalmic and vision research and were reviewed and approved by the national eye institute animal care and use committee . the retinoschisin knockout ( rs1-ko ) mouse model was generated as described previously . these mice have been backcrossed for at least 20 generations onto the c57bl/6j line ( jackson laboratory , bar harbor , me , usa ) , and the phenotype and natural history of this animal are well described . four independent groups of mice were reared in low light ( ll ) or moderate light ( ml ) and analyzed by erg and optical coherence tomography ( oct ) at either 1 or 4 months . in a second experiment , two groups of rs1-ko mice were reared in ll or ml and treated at 21 days with an intravitreal injection of scaav8-hrs - irbp vector . an electroretinogram was recorded in both groups at 4 months of age . light conditions were controlled by rearing mice from birth in two different rooms at either 20 lux ( ll ) or 300 lux ( ml ) ( measured outside and in front of the cage ) from an overhead fluorescent light , on either a 12/12 ( ll ) or 14/10 ( ml ) hour light / dark cycle . animals were weaned at 3 weeks of age , and then housed 5 or fewer per cage . rs1-ko mice were genotyped from tail dna to verify that they carried the rs1-ko construct . briefly , mice were dark - adapted overnight and all subsequent procedures were performed in dim red light or darkness . animals were anesthetized by intraperitoneal ketamine ( 100 mg / kg ) and xylazine ( 10 mg / kg ) , pupils were dilated with a mixture of 0.5% tropicamide and 0.5% phenylephrine , and 0.5% tetracaine topical anesthetic drops were applied before placing the recording electrodes . after anesthesia and pupil dilation , mice were placed on a heating pad at 37c , and gold wire electrodes were placed on the center of the cornea with a drop of methylcellulose for corneal hydration . electroretinogram recordings were averaged ( 120 responses ) to 10-ms full - field flashes ( grass photic stimulator ps33 ; astro - med , inc . , west warwick , ri , usa ) over a range of intensities eliciting threshold through maximum isolated rod responses and mixed rod cone responses . the erg signals were amplified 5000 times and bandpass filtered ( 0.11 khz , 3 db / decade ) with a 60-hz line filter . electroretinogram amplitudes in response to the maximum stimulus intensity of 0.6 log cds / m were used to compare mice reared in ml and ll . the a - wave was measured from the 50-ms prestimulus baseline to the maximum value of the negative trough preceding the b - wave . the b - wave amplitude was measured from the a - wave trough to the maximum peak immediately following the a - wave . the a - wave reflects the activation phase of the rod and cone photoresponse ; the b - wave results from the response of bipolar cells that are transynaptically activated by photoreceptors . the b - wave amplitude was divided by the a - wave ( b-/a - wave ratio ) to normalize for the activation of bipolar cells by photoreceptors and better estimate the gain in the bipolar cell response . optical coherence tomography images were obtained as described previously , with the envisu r2200 sd - oct ophthalmic imaging system ( bioptigen , durham , nc , usa ) . animals were anesthetized by intraperitoneal injection of ketamine ( 100 mg / kg ) and xylazine ( 10 mg / kg ) and placed in a custom holder . radial volume scans consisting of 10 b - scans ( 1000 a - scans per b - scan ) were collected at 18 angular intervals and were each an average of five frames . the cavity size was measured from bitmap images created by averaging 10 frames of linear b - scans from the same horizontal plane of the optic nerve ( imagej , http://imagej.nih.gov/ij ; provided in the public domain by the national institutes of health , bethesda , md , usa ) . in each image , the width of the inner nuclear layer plus the outer plexiform layer was measured perpendicularly to the retinal surface at the level of largest cavity on both sides of the optic nerve . this was done in four radial scans through the optic nerve head : nasal to temporal , inferior to superior , nasal inferior quadrant to temporal superior quadrant , and temporal inferior quadrant to nasal superior quadrant . the average of these eight measurements was used as the cavity size for that retina . this method was found to produce results comparable to area measurements of cavities isolated by using intensity threshold segmentation in imagej . retinal cell lysates were prepared in ripa buffer , ph 7.4 ( 50 mm tris - hcl , 1% np-40 , 0.25% sodium deoxycholate , 150 mm nacl , 1 mm egta , and protease inhibitor cocktail ) , and their protein concentrations were determined by bicinchoninic acid ( bca ) reagent kit ( thermo scientific , rockford , il , usa ) . for each light condition and time point , three independent samples were prepared , each being a pool of three retinas from three different mice . cell lysates corresponding to 10 g total protein were loaded on a 10% sds - page gel and transferred to polyvinylidene difluoride membrane ( bio - rad hercules , ca , usa ) for subsequent immunoblotting . the membranes were incubated in odyssey blocking buffer ( li - cor biosciences , lincoln , ne , usa ) for 1 hour and later incubated overnight with one of the indicated primary antibodies diluted in pbs containing 0.05% tween 20 ( pbst ) , ph 7.5 , at 4c : anti inward rectifier k+ ion channel 4.1 ( kir4.1 , 1:400 ; alomone labs , jerusalem , israel ) , anti water channel aquaporin-4 ( aqp4 , 1:1000 ; alomone labs ) , anti glial fibrillary acidic protein ( gfap , 1:5000 ; sigma - aldrich corp . , st . louis , mo , usa ) , and anti-actin ( mouse monoclonal , 1:6000 ; sigma - aldrich ) . after overnight incubation , membranes were rinsed three times in pbst and incubated with one of the corresponding infrared ( ir ) dye - conjugated secondary antibodies : ir dye 800cw conjugated goat ( polyclonal ) anti - rabbit igg ( from li - cor biosciences ) ; or alexa fluor 680conjugated goat anti - mouse igg ( thermo scientific ) . blots were scanned on li - cor odyssey infrared imaging system ( model 9120 , li - cor biosciences ) , and intensity of bands corresponding to aqp4 ( 28 kda ) , kir4.1 ( 30 kda ) , and gfap ( 50 kda ) were analyzed and normalized against an internal control protein -actin ( 43 kda ) , using odyssey software . the final values represent the average of three independent samples each being a pool of three retinas from three different rs1-ko mice . the wild type ( wt ) group contained two independent samples each with a pool of two retinas . recombinant aav - rs1 gene therapy vector was administered into one eye of 55 rs1-ko mice on postnatal day 21 to 26 by intravitreal injection at 2.5 10 vector genomes per eye , as described previously ; the contralateral eye remained untouched . the viral vector was injected into the vitreous body through the sclera on the nasal side of the eye approximately 1 mm posteriorly to the limbus with a 10-l nanofil syringe and a removable 35-gauge needle ( world precision instruments , inc . , after injection , triple antibiotic ophthalmic ointment ( neomycin , polymyxin b , and bacitracin ) was applied to the eye . electroretinogram parameters ( a - wave and b - wave amplitude and b-/a - wave ratio ) and cavity size measured from oct images were compared by 1-way anova between groups of 1- and 4-month - old rs1-ko mice reared in either 20 or 300 lux . two - way anova was used to assess effects of light rearing on the erg parameters of rs1-ko mice after uniocular gene therapy . both treated and light exposure and treatment were independent variables , set as between - subjects and within - subjects variables , respectively . animal procedures were conducted in accord with the arvo statement for the use of animals in ophthalmic and vision research and were reviewed and approved by the national eye institute animal care and use committee . the retinoschisin knockout ( rs1-ko ) mouse model was generated as described previously . these mice have been backcrossed for at least 20 generations onto the c57bl/6j line ( jackson laboratory , bar harbor , me , usa ) , and the phenotype and natural history of this animal are well described . four independent groups of mice were reared in low light ( ll ) or moderate light ( ml ) and analyzed by erg and optical coherence tomography ( oct ) at either 1 or 4 months . in a second experiment , two groups of rs1-ko mice were reared in ll or ml and treated at 21 days with an intravitreal injection of scaav8-hrs - irbp vector . an electroretinogram was recorded in both groups at 4 months of age . light conditions were controlled by rearing mice from birth in two different rooms at either 20 lux ( ll ) or 300 lux ( ml ) ( measured outside and in front of the cage ) from an overhead fluorescent light , on either a 12/12 ( ll ) or 14/10 ( ml ) hour light / dark cycle . animals were weaned at 3 weeks of age , and then housed 5 or fewer per cage . rs1-ko mice were genotyped from tail dna to verify that they carried the rs1-ko construct . briefly , mice were dark - adapted overnight and all subsequent procedures were performed in dim red light or darkness . animals were anesthetized by intraperitoneal ketamine ( 100 mg / kg ) and xylazine ( 10 mg / kg ) , pupils were dilated with a mixture of 0.5% tropicamide and 0.5% phenylephrine , and 0.5% tetracaine topical anesthetic drops were applied before placing the recording electrodes . after anesthesia and pupil dilation , mice were placed on a heating pad at 37c , and gold wire electrodes were placed on the center of the cornea with a drop of methylcellulose for corneal hydration . electroretinogram recordings were averaged ( 120 responses ) to 10-ms full - field flashes ( grass photic stimulator ps33 ; astro - med , inc . , west warwick , ri , usa ) over a range of intensities eliciting threshold through maximum isolated rod responses and mixed rod cone responses . the erg signals were amplified 5000 times and bandpass filtered ( 0.11 khz , 3 db / decade ) with a 60-hz line filter . electroretinogram amplitudes in response to the maximum stimulus intensity of 0.6 log cds / m were used to compare mice reared in ml and ll . the a - wave was measured from the 50-ms prestimulus baseline to the maximum value of the negative trough preceding the b - wave . the b - wave amplitude was measured from the a - wave trough to the maximum peak immediately following the a - wave . the a - wave reflects the activation phase of the rod and cone photoresponse ; the b - wave results from the response of bipolar cells that are transynaptically activated by photoreceptors . the b - wave amplitude was divided by the a - wave ( b-/a - wave ratio ) to normalize for the activation of bipolar cells by photoreceptors and better estimate the gain in the bipolar cell response . optical coherence tomography images were obtained as described previously , with the envisu r2200 sd - oct ophthalmic imaging system ( bioptigen , durham , nc , usa ) . animals were anesthetized by intraperitoneal injection of ketamine ( 100 mg / kg ) and xylazine ( 10 mg / kg ) and placed in a custom holder . radial volume scans consisting of 10 b - scans ( 1000 a - scans per b - scan ) were collected at 18 angular intervals and were each an average of five frames . the cavity size was measured from bitmap images created by averaging 10 frames of linear b - scans from the same horizontal plane of the optic nerve ( imagej , http://imagej.nih.gov/ij ; provided in the public domain by the national institutes of health , bethesda , md , usa ) . in each image , the width of the inner nuclear layer plus the outer plexiform layer was measured perpendicularly to the retinal surface at the level of largest cavity on both sides of the optic nerve . this was done in four radial scans through the optic nerve head : nasal to temporal , inferior to superior , nasal inferior quadrant to temporal superior quadrant , and temporal inferior quadrant to nasal superior quadrant . the average of these eight measurements was used as the cavity size for that retina . this method was found to produce results comparable to area measurements of cavities isolated by using intensity threshold segmentation in imagej . retinal cell lysates were prepared in ripa buffer , ph 7.4 ( 50 mm tris - hcl , 1% np-40 , 0.25% sodium deoxycholate , 150 mm nacl , 1 mm egta , and protease inhibitor cocktail ) , and their protein concentrations were determined by bicinchoninic acid ( bca ) reagent kit ( thermo scientific , rockford , il , usa ) . for each light condition and time point , three independent samples were prepared , each being a pool of three retinas from three different mice . cell lysates corresponding to 10 g total protein were loaded on a 10% sds - page gel and transferred to polyvinylidene difluoride membrane ( bio - rad hercules , ca , usa ) for subsequent immunoblotting . the membranes were incubated in odyssey blocking buffer ( li - cor biosciences , lincoln , ne , usa ) for 1 hour and later incubated overnight with one of the indicated primary antibodies diluted in pbs containing 0.05% tween 20 ( pbst ) , ph 7.5 , at 4c : anti inward rectifier k+ ion channel 4.1 ( kir4.1 , 1:400 ; alomone labs , jerusalem , israel ) , anti water channel aquaporin-4 ( aqp4 , 1:1000 ; alomone labs ) , anti glial fibrillary acidic protein ( gfap , 1:5000 ; sigma - aldrich corp . , st . louis , mo , usa ) , and anti-actin ( mouse monoclonal , 1:6000 ; sigma - aldrich ) . after overnight incubation , membranes were rinsed three times in pbst and incubated with one of the corresponding infrared ( ir ) dye - conjugated secondary antibodies : ir dye 800cw conjugated goat ( polyclonal ) anti - rabbit igg ( from li - cor biosciences ) ; or alexa fluor 680conjugated goat anti - mouse igg ( thermo scientific ) . blots were scanned on li - cor odyssey infrared imaging system ( model 9120 , li - cor biosciences ) , and intensity of bands corresponding to aqp4 ( 28 kda ) , kir4.1 ( 30 kda ) , and gfap ( 50 kda ) were analyzed and normalized against an internal control protein -actin ( 43 kda ) , using odyssey software . the final values represent the average of three independent samples each being a pool of three retinas from three different rs1-ko mice . the wild type ( wt ) group contained two independent samples each with a pool of two retinas . recombinant aav - rs1 gene therapy vector was administered into one eye of 55 rs1-ko mice on postnatal day 21 to 26 by intravitreal injection at 2.5 10 vector genomes per eye , as described previously ; the contralateral eye remained untouched . the viral vector was injected into the vitreous body through the sclera on the nasal side of the eye approximately 1 mm posteriorly to the limbus with a 10-l nanofil syringe and a removable 35-gauge needle ( world precision instruments , inc . , after injection , triple antibiotic ophthalmic ointment ( neomycin , polymyxin b , and bacitracin ) was applied to the eye . electroretinogram parameters ( a - wave and b - wave amplitude and b-/a - wave ratio ) and cavity size measured from oct images were compared by 1-way anova between groups of 1- and 4-month - old rs1-ko mice reared in either 20 or 300 lux . two - way anova was used to assess effects of light rearing on the erg parameters of rs1-ko mice after uniocular gene therapy . both treated and light exposure and treatment were independent variables , set as between - subjects and within - subjects variables , respectively . rs1-ko mice reared in different light intensities for 4 months showed substantial differences in retinal structure and function ( fig . had increased retinal thickness , compared to rs1-ko mice reared at 20 lux , owing to extensive schisis cavities , which spanned the outer plexiform and inner nuclear layers . rs1-ko mice reared in 20 lux had fewer cavities and the inner retina appeared structurally more organized . rs1-ko mice reared in 300 lux had a smaller erg b - wave and b / a ratio indicating greater impairment of inner retinal function . the a - wave was similar for the two lighting conditions . retinal function and morphology in two representative 4-month - old rs1-ko mice reared in low light ( ll ) and moderate light ( ml ) conditions . at 4 months , rs1-ko mice reared in ml showed substantial differences in both retinal morphology and function , compared to rs1-ko mice reared in ll . ml - reared rs1-ko mice showed large schisis cavities in the outer plexiform and inner nuclear layers , which were less numerous and smaller in ll - reared rs1-ko mice ( asterisks ) . consistent with a severe structural impairment of the inner retina , the erg b - wave amplitude in the ml rs1-ko mouse was considerably reduced , resulting in a typical electronegative erg . compared with the ml - reared rs1-ko mouse , ll - reared rs1-ko mouse had a much larger b-/a - wave ratio ( 1.9 vs. 1.2 ) , indicating a better post - photoreceptor function . calipers indicate the thickness of the inner nuclear layer plus the outer plexiform layer at the level of the largest cavity on each side of the retinal section . inl , inner nuclear layer ; opl , outer plexiform layer . the changes in retinal function and structure over time were systematically explored by comparing the erg and the cavity size in rs1-ko mice reared in 20 and 300 lux at 1 and 4 months of age ( fig . electroretinogram findings and cavity size in 1- and 4-month - old rs1-ko mice reared at either 20 lux ( ll ) or 300 lux ( ml ) light intensity . ( a ) a - wave amplitude did not change significantly between 1 and 4 months for both light intensities and no significant difference was found between ll and ml mice at any time point . b - wave amplitude ( b ) declined significantly in ml and ll rs1-ko mice by 4 months and at 4 months was significantly smaller in ml condition than ll . when we normalized the b - wave by the a - wave , b-/a - wave amplitude ratio ( c ) decreased significantly only in the ml group and was unchanged in ll . by 4 months of age , cavity size ( d ) increased significantly in the ml - reared rs1-ko mice , whereas it remained unchanged respect to 1 month in ll - reared animals . in each graph , dot is the mean value and error bars indicate standard errors . * p < 0.05 , * * p < 0.001 . , no effect of rearing light intensity was identified on either retinal function or structure . the erg a - wave , b - wave , and b / a ratio of ll- and ml - reared mice were not significantly different ( p = 0.98 , p = 0.637 , and p = 0.12 , respectively ) . the oct showed mild schisis cavities in the inner retina of both ll- and ml - reared mice but did not show any difference in size ( p = 0.68 ) . at 4 months of age , no significant change in a - wave amplitude was observed for either ll- or ml - reared mice with respect to 1 month ( table 1 ) , suggesting no effect of aging and light exposure on photoreceptor function . as expected by the natural progressive decline of post - photoreceptor function in rs1-ko mice , the b - wave declined significantly from 1 to 4 months in both ll and ml groups ( by 23% , p = 0.015 and by 40% , p = 0.002 , respectively ) . when the two light conditions were compared , however , ml - reared mice had significantly smaller b - waves than rs1-ko mice reared in ll ( p = 0.012 ) , indicating a faster decline in post - photoreceptor function in mice reared in 300 lux . the effect of a brighter light on inner retinal function was even more evident in the b-/a - wave ratio , which estimates the gain in signaling between photoreceptors and bipolar cell . between 1 and 4 months , the b-/a - wave ratio remained unchanged in the ll - reared rs1-ko mice ( p = 0.09 ) , whereas it was reduced by 30% in the ml - reared animals ( p = 0.000 ) . erg and oct parameters in rs1-ko mice reared in either ll or ml consistent with these functional findings , oct analysis ( fig . 2 ; table 1 ) showed that the cavity size was essentially unaltered between 1 and 4 months in ll - reared mice ( p = 0.50 ) , whereas cavities increased in size by 88% in mice reared in ml ( p = 0.000 ) . fluid homeostasis in the inner retina and outer plexiform layer is regulated by mller glial cells . we investigated whether effects of light rearing on cavity formation might be mediated by changes in expression of ion and water channels such as kir4.1 and aqp4 on mller cells . aqp4 channel expression remained unchanged between 1 and 4 months in both ll and ml rs1-ko mice , and no difference was identified between the two light intensities . compared to ll - reared rs1-ko mice in which kir4.1 expression remained unchanged between 1 and 4 months , ml rearing caused kir4.1 levels to increase 7-fold by 4 months ( p < 0.05 ) ( fig . glial fibrillary acidic protein levels , an indicator of mller cell activation , were not significantly different between ll- and ml - reared rs1-ko mice at either 1 or 4 months , though there was a trend toward an increase of gfap between 1 and 4 months in both groups , and especially for ml - reared mice . aqp4 , kir4.1 , and gfap levels were unchanged in wt mice irrespective of light conditions and age , but their levels were overall higher in rs1-ko mice than in wt animals , likely as a result of mller cell response to the presence of intraretinal cavities . effect of light rearing on aqp-4 , kir4.1 , and gfap levels in 1- and 4-month - old rs1-ko and wt mice . expression levels of aqp4 , kir4.1 , and gfap normalized to internal control protein actin are presented as mean standard error for 1- and 4-month - old rs1-ko and wt mice reared in either ll or ml . rs1-ko mice showed no significant difference in aqp4 and gfap levels between ll and ml conditions at 1 and 4 months ( a , c ) . kir4.1 levels were unchanged between ll and ml rs1-ko mice at 1 month , but at 4 months they were significantly increased by 7-fold in ml - reared rs1-ko mice compared to those reared in ll ( b ) . aqp4 , kir4.1 , and gfap levels were unchanged in wt mice irrespective of light conditions and age ( d f ) but their levels were overall higher in rs1-ko mice than in wt animals , likely as a result of mller cell response to the presence of intraretinal cavities . * p < 0.05 . two groups of rs1-ko mice were reared at 20 ( n = 26 ) or 300 lux ( n = 29 ) and were treated at 21 days with an intravitreal injection of scaav8-hrs - irbp vector as we have done previously . as we found in the previous set of rs1-ko mice , functional erg recordings at 4 months showed that untreated eyes of rs1-ko mice reared in ll had significantly larger b - wave amplitudes and b-/a - wave ratios than untreated eyes of mice reared in ml ( p = 0.012 and p = 0.000 , respectively ) . no significant difference in a - wave amplitude was observed between the two groups ( fig . effect of light rearing on the functional outcome after aav8-mediated rs1 gene replacement in rs1-ko mice . average erg a - wave ( a ) , b - wave ( b ) , and b-/a - wave ratio ( c ) in two groups of 4-month - old rs1-ko mice reared in low light ( ll ) ( n = 26 ) and moderate light ( ml ) ( n = 29 ) and treated with aav8-rs1 at 21 days . although ll mice showed significantly larger b - wave amplitude and b-/a - wave ratio than ml mice in the aav8-rs1treated eye , 2-way anova analysis did not show significant interaction between treatment and rearing light exposure , indicating that aav8-mediated rs1 expression improved inner retinal function in ll and ml reared mice by the same extent . untreated eyes in ll - reared rs1-ko mice had significantly larger b - wave and b-/a - wave than ml mice . this indicates that gene therapy and dark rearing have an additive beneficial effect . in each graph , gene replacement significantly increased the b - wave amplitude and the b-/a - wave ratio in the rs1-ko treated eyes for mice reared in ml ( by 65% , p < 0.001 and 50% , p < 0.001 , respectively ) and ll ( by 77% , p < 0.001 and 35% , p < 0.001 , respectively ) ( table 2 ) . low light reared mice had significantly larger b - wave amplitudes and b-/a - wave ratios than ml - reared mice , though there was no significant interaction between light rearing and treatment on the erg parameters ( 2-way anova , p > 0.05 for both ) . this indicates that replacing rs1 protein improved inner retinal function in both ll- and ml - reared mice independently of effects from the rearing light intensity . rs1-ko mice reared in different light intensities for 4 months showed substantial differences in retinal structure and function ( fig . had increased retinal thickness , compared to rs1-ko mice reared at 20 lux , owing to extensive schisis cavities , which spanned the outer plexiform and inner nuclear layers . rs1-ko mice reared in 20 lux had fewer cavities and the inner retina appeared structurally more organized . rs1-ko mice reared in 300 lux had a smaller erg b - wave and b / a ratio indicating greater impairment of inner retinal function . the a - wave was similar for the two lighting conditions . retinal function and morphology in two representative 4-month - old rs1-ko mice reared in low light ( ll ) and moderate light ( ml ) conditions . at 4 months , rs1-ko mice reared in ml showed substantial differences in both retinal morphology and function , compared to rs1-ko mice reared in ll . ml - reared rs1-ko mice showed large schisis cavities in the outer plexiform and inner nuclear layers , which were less numerous and smaller in ll - reared rs1-ko mice ( asterisks ) . consistent with a severe structural impairment of the inner retina , the erg b - wave amplitude in the ml rs1-ko mouse was considerably reduced , resulting in a typical electronegative erg . compared with the ml - reared rs1-ko mouse , ll - reared rs1-ko mouse had a much larger b-/a - wave ratio ( 1.9 vs. 1.2 ) , indicating a better post - photoreceptor function . calipers indicate the thickness of the inner nuclear layer plus the outer plexiform layer at the level of the largest cavity on each side of the retinal section . inl , inner nuclear layer ; opl , outer plexiform layer . the changes in retinal function and structure over time were systematically explored by comparing the erg and the cavity size in rs1-ko mice reared in 20 and 300 lux at 1 and 4 months of age ( fig . electroretinogram findings and cavity size in 1- and 4-month - old rs1-ko mice reared at either 20 lux ( ll ) or 300 lux ( ml ) light intensity . ( a ) a - wave amplitude did not change significantly between 1 and 4 months for both light intensities and no significant difference was found between ll and ml mice at any time point . b - wave amplitude ( b ) declined significantly in ml and ll rs1-ko mice by 4 months and at 4 months was significantly smaller in ml condition than ll . when we normalized the b - wave by the a - wave , b-/a - wave amplitude ratio ( c ) decreased significantly only in the ml group and was unchanged in ll . by 4 months of age , cavity size ( d ) increased significantly in the ml - reared rs1-ko mice , whereas it remained unchanged respect to 1 month in ll - reared animals . in each graph , dot is the mean value and error bars indicate standard errors . * p < 0.05 , * * p < 0.001 . , no effect of rearing light intensity was identified on either retinal function or structure . the erg a - wave , b - wave , and b / a ratio of ll- and ml - reared mice were not significantly different ( p = 0.98 , p = 0.637 , and p = 0.12 , respectively ) . the oct showed mild schisis cavities in the inner retina of both ll- and ml - reared mice but did not show any difference in size ( p = 0.68 ) . at 4 months of age , no significant change in a - wave amplitude was observed for either ll- or ml - reared mice with respect to 1 month ( table 1 ) , suggesting no effect of aging and light exposure on photoreceptor function . as expected by the natural progressive decline of post - photoreceptor function in rs1-ko mice , the b - wave declined significantly from 1 to 4 months in both ll and ml groups ( by 23% , p = 0.015 and by 40% , p = 0.002 , respectively ) . when the two light conditions were compared , however , ml - reared mice had significantly smaller b - waves than rs1-ko mice reared in ll ( p = 0.012 ) , indicating a faster decline in post - photoreceptor function in mice reared in 300 lux . the effect of a brighter light on inner retinal function was even more evident in the b-/a - wave ratio , which estimates the gain in signaling between photoreceptors and bipolar cell . between 1 and 4 months , the b-/a - wave ratio remained unchanged in the ll - reared rs1-ko mice ( p = 0.09 ) , whereas it was reduced by 30% in the ml - reared animals ( p = 0.000 ) . erg and oct parameters in rs1-ko mice reared in either ll or ml consistent with these functional findings , oct analysis ( fig . 2 ; table 1 ) showed that the cavity size was essentially unaltered between 1 and 4 months in ll - reared mice ( p = 0.50 ) , whereas cavities increased in size by 88% in mice reared in ml ( p = 0.000 ) . fluid homeostasis in the inner retina and outer plexiform layer is regulated by mller glial cells . we investigated whether effects of light rearing on cavity formation might be mediated by changes in expression of ion and water channels such as kir4.1 and aqp4 on mller cells . aqp4 channel expression remained unchanged between 1 and 4 months in both ll and ml rs1-ko mice , and no difference was identified between the two light intensities . compared to ll - reared rs1-ko mice in which kir4.1 expression remained unchanged between 1 and 4 months , ml rearing caused kir4.1 levels to increase 7-fold by 4 months ( p < 0.05 ) ( fig . glial fibrillary acidic protein levels , an indicator of mller cell activation , were not significantly different between ll- and ml - reared rs1-ko mice at either 1 or 4 months , though there was a trend toward an increase of gfap between 1 and 4 months in both groups , and especially for ml - reared mice . aqp4 , kir4.1 , and gfap levels were unchanged in wt mice irrespective of light conditions and age , but their levels were overall higher in rs1-ko mice than in wt animals , likely as a result of mller cell response to the presence of intraretinal cavities . effect of light rearing on aqp-4 , kir4.1 , and gfap levels in 1- and 4-month - old rs1-ko and wt mice . expression levels of aqp4 , kir4.1 , and gfap normalized to internal control protein actin are presented as mean standard error for 1- and 4-month - old rs1-ko and wt mice reared in either ll or ml . rs1-ko mice showed no significant difference in aqp4 and gfap levels between ll and ml conditions at 1 and 4 months ( a , c ) . kir4.1 levels were unchanged between ll and ml rs1-ko mice at 1 month , but at 4 months they were significantly increased by 7-fold in ml - reared rs1-ko mice compared to those reared in ll ( b ) . aqp4 , kir4.1 , and gfap levels were unchanged in wt mice irrespective of light conditions and age ( d f ) but their levels were overall higher in rs1-ko mice than in wt animals , likely as a result of mller cell response to the presence of intraretinal cavities . two groups of rs1-ko mice were reared at 20 ( n = 26 ) or 300 lux ( n = 29 ) and were treated at 21 days with an intravitreal injection of scaav8-hrs - irbp vector as we have done previously . as we found in the previous set of rs1-ko mice , functional erg recordings at 4 months showed that untreated eyes of rs1-ko mice reared in ll had significantly larger b - wave amplitudes and b-/a - wave ratios than untreated eyes of mice reared in ml ( p = 0.012 and p = 0.000 , respectively ) . no significant difference in a - wave amplitude was observed between the two groups ( fig . effect of light rearing on the functional outcome after aav8-mediated rs1 gene replacement in rs1-ko mice . average erg a - wave ( a ) , b - wave ( b ) , and b-/a - wave ratio ( c ) in two groups of 4-month - old rs1-ko mice reared in low light ( ll ) ( n = 26 ) and moderate light ( ml ) ( n = 29 ) and treated with aav8-rs1 at 21 days . although ll mice showed significantly larger b - wave amplitude and b-/a - wave ratio than ml mice in the aav8-rs1treated eye , 2-way anova analysis did not show significant interaction between treatment and rearing light exposure , indicating that aav8-mediated rs1 expression improved inner retinal function in ll and ml reared mice by the same extent . untreated eyes in ll - reared rs1-ko mice had significantly larger b - wave and b-/a - wave than ml mice . this indicates that gene therapy and dark rearing have an additive beneficial effect . in each graph , * p < 0.05 , * * p < 0.001 . n.s . , not significant . gene replacement significantly increased the b - wave amplitude and the b-/a - wave ratio in the rs1-ko treated eyes for mice reared in ml ( by 65% , p < 0.001 and 50% , p < 0.001 , respectively ) and ll ( by 77% , p < 0.001 and 35% , p < 0.001 , respectively ) ( table 2 ) . reared mice had significantly larger b - wave amplitudes and b-/a - wave ratios than ml - reared mice , though there was no significant interaction between light rearing and treatment on the erg parameters ( 2-way anova , p > 0.05 for both ) . this indicates that replacing rs1 protein improved inner retinal function in both ll- and ml - reared mice independently of effects from the rearing light intensity . this study demonstrated that the light intensity in which rs1-ko mice are reared exerts a significant effect on the inner retinal phenotype of these animals . rearing them in ll retards the progression of the xlrs disease as judged by cavity size and post - photoreceptor erg function . our previous natural history studies in rs1-ko mice have shown schisis cavities as early as 3 weeks and then a significant increase in their size between 1 and 4 months . cavity formation in the inner retina is accompanied by considerable impairment in post - photoreceptor erg b - wave function with little effect on the a - wave , causing the prototypical electronegative erg . however , in ll - reared mice , both cavity size and inner retinal function , ( i.e. , b-/a - wave ratio ) remained unchanged between 1 and 4 months , indicating that light restriction was beneficial for evolution of inner retinal pathology in rs1-ko mice . in other animal models of retinal degeneration , exposure to bright light can promote photoreceptor degeneration . however , the light intensities have been usually much higher ( e.g. , 5000 lux for 8 hours with dilated pupils ) than those used in this study in which the ml condition was standard colony room lighting . so , it is not surprising that ml rearing did not increase the rate of photoreceptor loss in rs1-ko mice . however , ml rearing exacerbated the inner retinal phenotype of cavity formation and decreased erg responses . this effect of relatively modest lighting on post - photoreceptor structure and function is surprising , since visible light is absorbed by photoreceptors and retinal pigmented epithelium , and no photosensitive elements are known to be present at the outer plexiform layer where schisis cavities specifically develop . there is some evidence that light environment during rearing can influence the inner retinal structure and/or function , in some cases without detectable effects on the outer retina . for example , vistamehr and tian have shown that dark rearing suppresses erg oscillatory potentials , which represent the activity of rod bipolar , amacrine , and retinal ganglion cells , in mature animals , without suppressing the erg a- or b - waves . we do not have a clear picture of how light influences inner retinal pathology in rs1-ko mice , but one hypothesis is that a brighter light environment may alter inner retinal metabolism , ion gradients , and extracellular fluid movement , exerting more stress on retinal structure than in an ll environment . in this context the extracellular adhesion properties of retinoschisin may play a role in maintaining structural integrity under these conditions and its lack may result in cavity formation . photoreceptors and rpe primarily control outer retinal homeostasis , while mller cells regulate ion and water homeostasis of the inner retina through specific membrane channels . specifically , aqp4 channels control water flux across the mller cell membrane , and kir4.1 buffers k ions released into the extracellular space by synaptic activity during retinal illumination . these two channels colocalize on the mller cell membrane and are thought to couple water transport with potassium currents . we found that by 4 months , kir4.1 was strongly upregulated in rs1-ko mice reared in ml , indicating dysregulation of k balance in the inner retina . this upregulation may also contribute to cavity formation in rs1-ko mice by accumulation of ions and fluid in the extracellular space . however , as kir4.1 channels mainly mediate the k efflux from mller cells into the vitreous , blood vessels , and subretinal space , increased kir4.1 levels may represent compensatory activity of mller cells to augment k clearance from the intraretinal extracellular space in conjunction with removal of fluid from the schisis cavities . despite aqp4 and kir4.1 channel activity being coupled to extracellular fluid reabsorption , we did not observe an increase of aqp4 expression along with kir4.1 in ml 4-month - old rs1-ko mice . in fact , aqp4 levels were not affected by either age or light condition , though rs1-ko mice had higher levels of aqp4 expression than wt mice in both ll and ml conditions at 1 month . at this age , rs1-ko mice already show retinal cavities , and it may be that the increase of aqp4 channels on mller cells as a compensatory mechanism for fluid accumulation is already saturated in 1-month - old mice . consequently , further increase in intraretinal fluid in ml rearing can not be compensated for , causing cavities to increase . a recent study in aqp4-null mice has shown that cellular k reuptake from the extracellular space is impaired when aqp4 channels are not expressed . likewise , saturation of aqp4 expression in rs1-ko mice may increase the extracellular k and result in the compensatory increase in kir4.1 channel expression in 4-month - old rs1-ko mice . effects of ml exposure on inner retinal pathology were not evident in 1-month - old rs1-ko mice , as both ll- and ml - reared animals showed comparable cavity size and similar degree of inner retinal dysfunction . it could be that , as the ml intensity differed by approximately only 1 log from the ll intensity , the light - induced changes were not immediate but took longer to develop . we have previously observed that photoreceptors in rs1-ko mouse are not fully matured by 1 month of age , and their outer segments require additional weeks to reach normal length . photoreceptors were more mature by 4 months , and outer segments reached normal length , which would enhance the synaptic signaling to the post - photoreceptor pathway . we postulate that this may exacerbate the effects of a brighter light environment on the inner retina , leading to cavity formation . we have shown that delivering a normal rs1 gene by using an adeno - associated viral vector significantly improves retinal structure and function in rs1-ko mice with reduction of the size and number of cavities and an increase in the b - wave , and b-/a - wave ratio . in the present study , we found that the rearing light intensity does not interfere with the functional improvement of rs1 gene therapy . both ll- and ml - reared rs1-ko mice showed comparable improvements in b - wave amplitude and b-/a - wave ratio at 4 months of age relative to fellow untreated eyes . gene therapy and ll rearing effects were independent and had additive beneficial effect on inner retinal function of rs1-ko mice . thus , after gene therapy both b - wave amplitude and b-/a - wave ratio were larger in ll- than ml - reared animals . figure 5 compares the natural history of ll and ml rs1-ko mice with respect to those which were treated with rs1 gene therapy . although after treatment the inner retinal function increased by the same amount in ll- and ml - reared rs1-ko mice , replacing rs1 further improved the inner retinal function in ll - reared mice , whereas it preserved function in ml - reared mice with respect to levels at 1 month of age . at 4 months , ll - reared animals showed an increase in b-/a - wave ratio after treatment , whereas the latter was unchanged in rs1-ko mice reared in ml . effects of rs1 gene therapy on low light ( ll)- and moderate light ( ml)-reared rs1-ko mice natural history . rs1 gene replacement increased the inner retinal function ( b-/a - wave ratio ) by the same extent in 4-month - old ll- and ml - reared rs1-ko mice . when compared to 1-month - old rs1-ko mice , gene therapy improved inner retinal function in 4-month - old ll - reared animals but only preserved inner retinal function in ml - reared animals . since ll - reared rs1-ko mice do not show any change in cavity size between 1 and 4 months , rs1 gene transfer improves inner retinal function independently from an effect on retinal cavities , most likely by restoring synapse integrity between photoreceptors and bipolar cells . the fact that treating ll - reared rs1-ko mice produces a functional improvement , although no change in cavity size occurs naturally at this light intensity between 1 and 4 months , indicates that rs1 gene replacement can improve the inner retinal function independently from its beneficial effect on cavities . this is consistent with our previous finding that retinoschisin acts as a synaptic stabilizer element . when rs1-ko mice are reared in ml , despite rs1 gene transfer producing the same degree of functional improvement , light exposure itself or the development of cavities may irreversibly impact the inner retina and prevent them from reaching the same level of retinal function as in ll conditions . in conclusion , this study showed that rearing light intensities contribute to the severity of the rs1-ko phenotype and that even a moderate light intensity significantly impacts the pathology of the inner retina . we do not know whether the ambient illumination has a similar effect on human xlrs . if it does , environmental exposure to different light conditions may contribute to heterogeneity of the human xlrs phenotype , and light restriction could be considered as a potential prophylactic intervention . effects of ml exposure on inner retinal pathology were not evident in 1-month - old rs1-ko mice , as both ll- and ml - reared animals showed comparable cavity size and similar degree of inner retinal dysfunction . it could be that , as the ml intensity differed by approximately only 1 log from the ll intensity , the light - induced changes were not immediate but took longer to develop . we have previously observed that photoreceptors in rs1-ko mouse are not fully matured by 1 month of age , and their outer segments require additional weeks to reach normal length . photoreceptors were more mature by 4 months , and outer segments reached normal length , which would enhance the synaptic signaling to the post - photoreceptor pathway . we postulate that this may exacerbate the effects of a brighter light environment on the inner retina , leading to cavity formation . we have shown that delivering a normal rs1 gene by using an adeno - associated viral vector significantly improves retinal structure and function in rs1-ko mice with reduction of the size and number of cavities and an increase in the b - wave , and b-/a - wave ratio . in the present study , we found that the rearing light intensity does not interfere with the functional improvement of rs1 gene therapy . both ll- and ml - reared rs1-ko mice showed comparable improvements in b - wave amplitude and b-/a - wave ratio at 4 months of age relative to fellow untreated eyes . gene therapy and ll rearing effects were independent and had additive beneficial effect on inner retinal function of rs1-ko mice . thus , after gene therapy both b - wave amplitude and b-/a - wave ratio were larger in ll- than ml - reared animals . figure 5 compares the natural history of ll and ml rs1-ko mice with respect to those which were treated with rs1 gene therapy . although after treatment the inner retinal function increased by the same amount in ll- and ml - reared rs1-ko mice , replacing rs1 further improved the inner retinal function in ll - reared mice , whereas it preserved function in ml - reared mice with respect to levels at 1 month of age . at 4 months , ll - reared animals showed an increase in b-/a - wave ratio after treatment , whereas the latter was unchanged in rs1-ko mice reared in ml . effects of rs1 gene therapy on low light ( ll)- and moderate light ( ml)-reared rs1-ko mice natural history . rs1 gene replacement increased the inner retinal function ( b-/a - wave ratio ) by the same extent in 4-month - old ll- and ml - reared rs1-ko mice . when compared to 1-month - old rs1-ko mice , gene therapy improved inner retinal function in 4-month - old ll - reared animals but only preserved inner retinal function in ml - reared animals . since ll - reared rs1-ko mice do not show any change in cavity size between 1 and 4 months , rs1 gene transfer improves inner retinal function independently from an effect on retinal cavities , most likely by restoring synapse integrity between photoreceptors and bipolar cells . the fact that treating ll - reared rs1-ko mice produces a functional improvement , although no change in cavity size occurs naturally at this light intensity between 1 and 4 months , indicates that rs1 gene replacement can improve the inner retinal function independently from its beneficial effect on cavities . this is consistent with our previous finding that retinoschisin acts as a synaptic stabilizer element . when rs1-ko mice are reared in ml , despite rs1 gene transfer producing the same degree of functional improvement , light exposure itself or the development of cavities may irreversibly impact the inner retina and prevent them from reaching the same level of retinal function as in ll conditions . in conclusion , this study showed that rearing light intensities contribute to the severity of the rs1-ko phenotype and that even a moderate light intensity significantly impacts the pathology of the inner retina . we do not know whether the ambient illumination has a similar effect on human xlrs . if it does , environmental exposure to different light conditions may contribute to heterogeneity of the human xlrs phenotype , and light restriction could be considered as a potential prophylactic intervention .

purposeto test the effects of rearing light intensity on retinal function and morphology in the retinoschisis knockout ( rs1-ko ) mouse model of x - linked retinoschisis , and whether it affects functional outcome of rs1 gene replacement.methodsseventy-six rs1-ko mice were reared in either cyclic low light ( ll , 20 lux ) or moderate light ( ml , 300 lux ) and analyzed at 1 and 4 months . retinal function was assessed by electroretinogram and cavity size by optical coherence tomography . expression of inward - rectifier k+ channel ( kir4.1 ) , water channel aquaporin-4 ( aqp4 ) , and glial fibrillary acidic protein ( gfap ) were analyzed by western blotting . in a separate study , rs1-ko mice reared in ll ( n = 29 ) or ml ( n = 27 ) received a unilateral intravitreal injection of scaav8-hrs - irbp at 21 days , and functional outcome was evaluated at 4 months by electroretinogram.resultsat 1 month , no functional or structural differences were found between ll- or ml - reared rs1-ko mice . at 4 months , ml - reared rs1-ko mice showed significant reduction of b - wave amplitude and b-/a - wave ratio with no changes in a - wave , and a significant increase in cavity size , compared to ll - reared animals . moderate light rearing increased kir4.1 expression in rs1-ko mice by 4 months , but not aqp4 and gfap levels . administration of scaav8-hrs1-irbp to rs1-ko mice showed similar improvement of inner retinal erg function independent of ll or ml rearing.conclusionsrearing light conditions affect the development of retinal cavities and post - photoreceptor function in rs1-ko mice . however , the effect of rearing light intensity does not interact with the efficacy of rs1 gene replacement in rs1-ko mice .

Materials and Methods Animal Handling and Light Conditions Electroretinogram Optical Coherence Tomography Western Blotting Intravitreal Injection of scAAV8-hRS1-IRBP Statistical Analysis Results Low-Light Rearing Preserves Inner Retinal Structure and Function in Kir4.1 Channels but Not Aquaporin-4 Channels Are Upregulated in Rearing Light Intensity Does Not Interact With Discussion Photoreceptor Maturation Gene Therapy Versus Light Rearing Effect

four independent groups of mice were reared in low light ( ll ) or moderate light ( ml ) and analyzed by erg and optical coherence tomography ( oct ) at either 1 or 4 months . in a second experiment , two groups of rs1-ko mice were reared in ll or ml and treated at 21 days with an intravitreal injection of scaav8-hrs - irbp vector . the membranes were incubated in odyssey blocking buffer ( li - cor biosciences , lincoln , ne , usa ) for 1 hour and later incubated overnight with one of the indicated primary antibodies diluted in pbs containing 0.05% tween 20 ( pbst ) , ph 7.5 , at 4c : anti inward rectifier k+ ion channel 4.1 ( kir4.1 , 1:400 ; alomone labs , jerusalem , israel ) , anti water channel aquaporin-4 ( aqp4 , 1:1000 ; alomone labs ) , anti glial fibrillary acidic protein ( gfap , 1:5000 ; sigma - aldrich corp . electroretinogram parameters ( a - wave and b - wave amplitude and b-/a - wave ratio ) and cavity size measured from oct images were compared by 1-way anova between groups of 1- and 4-month - old rs1-ko mice reared in either 20 or 300 lux . four independent groups of mice were reared in low light ( ll ) or moderate light ( ml ) and analyzed by erg and optical coherence tomography ( oct ) at either 1 or 4 months . in a second experiment , two groups of rs1-ko mice were reared in ll or ml and treated at 21 days with an intravitreal injection of scaav8-hrs - irbp vector . the membranes were incubated in odyssey blocking buffer ( li - cor biosciences , lincoln , ne , usa ) for 1 hour and later incubated overnight with one of the indicated primary antibodies diluted in pbs containing 0.05% tween 20 ( pbst ) , ph 7.5 , at 4c : anti inward rectifier k+ ion channel 4.1 ( kir4.1 , 1:400 ; alomone labs , jerusalem , israel ) , anti water channel aquaporin-4 ( aqp4 , 1:1000 ; alomone labs ) , anti glial fibrillary acidic protein ( gfap , 1:5000 ; sigma - aldrich corp . electroretinogram parameters ( a - wave and b - wave amplitude and b-/a - wave ratio ) and cavity size measured from oct images were compared by 1-way anova between groups of 1- and 4-month - old rs1-ko mice reared in either 20 or 300 lux . rs1-ko mice reared in 300 lux had a smaller erg b - wave and b / a ratio indicating greater impairment of inner retinal function . retinal function and morphology in two representative 4-month - old rs1-ko mice reared in low light ( ll ) and moderate light ( ml ) conditions . at 4 months , rs1-ko mice reared in ml showed substantial differences in both retinal morphology and function , compared to rs1-ko mice reared in ll . compared with the ml - reared rs1-ko mouse , ll - reared rs1-ko mouse had a much larger b-/a - wave ratio ( 1.9 vs. 1.2 ) , indicating a better post - photoreceptor function . the changes in retinal function and structure over time were systematically explored by comparing the erg and the cavity size in rs1-ko mice reared in 20 and 300 lux at 1 and 4 months of age ( fig . electroretinogram findings and cavity size in 1- and 4-month - old rs1-ko mice reared at either 20 lux ( ll ) or 300 lux ( ml ) light intensity . by 4 months of age , cavity size ( d ) increased significantly in the ml - reared rs1-ko mice , whereas it remained unchanged respect to 1 month in ll - reared animals . at 4 months of age , no significant change in a - wave amplitude was observed for either ll- or ml - reared mice with respect to 1 month ( table 1 ) , suggesting no effect of aging and light exposure on photoreceptor function . as expected by the natural progressive decline of post - photoreceptor function in rs1-ko mice , the b - wave declined significantly from 1 to 4 months in both ll and ml groups ( by 23% , p = 0.015 and by 40% , p = 0.002 , respectively ) . when the two light conditions were compared , however , ml - reared mice had significantly smaller b - waves than rs1-ko mice reared in ll ( p = 0.012 ) , indicating a faster decline in post - photoreceptor function in mice reared in 300 lux . the effect of a brighter light on inner retinal function was even more evident in the b-/a - wave ratio , which estimates the gain in signaling between photoreceptors and bipolar cell . between 1 and 4 months , the b-/a - wave ratio remained unchanged in the ll - reared rs1-ko mice ( p = 0.09 ) , whereas it was reduced by 30% in the ml - reared animals ( p = 0.000 ) . 2 ; table 1 ) showed that the cavity size was essentially unaltered between 1 and 4 months in ll - reared mice ( p = 0.50 ) , whereas cavities increased in size by 88% in mice reared in ml ( p = 0.000 ) . compared to ll - reared rs1-ko mice in which kir4.1 expression remained unchanged between 1 and 4 months , ml rearing caused kir4.1 levels to increase 7-fold by 4 months ( p < 0.05 ) ( fig . glial fibrillary acidic protein levels , an indicator of mller cell activation , were not significantly different between ll- and ml - reared rs1-ko mice at either 1 or 4 months , though there was a trend toward an increase of gfap between 1 and 4 months in both groups , and especially for ml - reared mice . rs1-ko mice showed no significant difference in aqp4 and gfap levels between ll and ml conditions at 1 and 4 months ( a , c ) . kir4.1 levels were unchanged between ll and ml rs1-ko mice at 1 month , but at 4 months they were significantly increased by 7-fold in ml - reared rs1-ko mice compared to those reared in ll ( b ) . two groups of rs1-ko mice were reared at 20 ( n = 26 ) or 300 lux ( n = 29 ) and were treated at 21 days with an intravitreal injection of scaav8-hrs - irbp vector as we have done previously . as we found in the previous set of rs1-ko mice , functional erg recordings at 4 months showed that untreated eyes of rs1-ko mice reared in ll had significantly larger b - wave amplitudes and b-/a - wave ratios than untreated eyes of mice reared in ml ( p = 0.012 and p = 0.000 , respectively ) . average erg a - wave ( a ) , b - wave ( b ) , and b-/a - wave ratio ( c ) in two groups of 4-month - old rs1-ko mice reared in low light ( ll ) ( n = 26 ) and moderate light ( ml ) ( n = 29 ) and treated with aav8-rs1 at 21 days . although ll mice showed significantly larger b - wave amplitude and b-/a - wave ratio than ml mice in the aav8-rs1treated eye , 2-way anova analysis did not show significant interaction between treatment and rearing light exposure , indicating that aav8-mediated rs1 expression improved inner retinal function in ll and ml reared mice by the same extent . untreated eyes in ll - reared rs1-ko mice had significantly larger b - wave and b-/a - wave than ml mice . in each graph , gene replacement significantly increased the b - wave amplitude and the b-/a - wave ratio in the rs1-ko treated eyes for mice reared in ml ( by 65% , p < 0.001 and 50% , p < 0.001 , respectively ) and ll ( by 77% , p < 0.001 and 35% , p < 0.001 , respectively ) ( table 2 ) . rs1-ko mice reared in 300 lux had a smaller erg b - wave and b / a ratio indicating greater impairment of inner retinal function . retinal function and morphology in two representative 4-month - old rs1-ko mice reared in low light ( ll ) and moderate light ( ml ) conditions . at 4 months , rs1-ko mice reared in ml showed substantial differences in both retinal morphology and function , compared to rs1-ko mice reared in ll . compared with the ml - reared rs1-ko mouse , ll - reared rs1-ko mouse had a much larger b-/a - wave ratio ( 1.9 vs. 1.2 ) , indicating a better post - photoreceptor function . the changes in retinal function and structure over time were systematically explored by comparing the erg and the cavity size in rs1-ko mice reared in 20 and 300 lux at 1 and 4 months of age ( fig . electroretinogram findings and cavity size in 1- and 4-month - old rs1-ko mice reared at either 20 lux ( ll ) or 300 lux ( ml ) light intensity . by 4 months of age , cavity size ( d ) increased significantly in the ml - reared rs1-ko mice , whereas it remained unchanged respect to 1 month in ll - reared animals . at 4 months of age , no significant change in a - wave amplitude was observed for either ll- or ml - reared mice with respect to 1 month ( table 1 ) , suggesting no effect of aging and light exposure on photoreceptor function . as expected by the natural progressive decline of post - photoreceptor function in rs1-ko mice , the b - wave declined significantly from 1 to 4 months in both ll and ml groups ( by 23% , p = 0.015 and by 40% , p = 0.002 , respectively ) . when the two light conditions were compared , however , ml - reared mice had significantly smaller b - waves than rs1-ko mice reared in ll ( p = 0.012 ) , indicating a faster decline in post - photoreceptor function in mice reared in 300 lux . the effect of a brighter light on inner retinal function was even more evident in the b-/a - wave ratio , which estimates the gain in signaling between photoreceptors and bipolar cell . between 1 and 4 months , the b-/a - wave ratio remained unchanged in the ll - reared rs1-ko mice ( p = 0.09 ) , whereas it was reduced by 30% in the ml - reared animals ( p = 0.000 ) . 2 ; table 1 ) showed that the cavity size was essentially unaltered between 1 and 4 months in ll - reared mice ( p = 0.50 ) , whereas cavities increased in size by 88% in mice reared in ml ( p = 0.000 ) . compared to ll - reared rs1-ko mice in which kir4.1 expression remained unchanged between 1 and 4 months , ml rearing caused kir4.1 levels to increase 7-fold by 4 months ( p < 0.05 ) ( fig . glial fibrillary acidic protein levels , an indicator of mller cell activation , were not significantly different between ll- and ml - reared rs1-ko mice at either 1 or 4 months , though there was a trend toward an increase of gfap between 1 and 4 months in both groups , and especially for ml - reared mice . kir4.1 levels were unchanged between ll and ml rs1-ko mice at 1 month , but at 4 months they were significantly increased by 7-fold in ml - reared rs1-ko mice compared to those reared in ll ( b ) . two groups of rs1-ko mice were reared at 20 ( n = 26 ) or 300 lux ( n = 29 ) and were treated at 21 days with an intravitreal injection of scaav8-hrs - irbp vector as we have done previously . as we found in the previous set of rs1-ko mice , functional erg recordings at 4 months showed that untreated eyes of rs1-ko mice reared in ll had significantly larger b - wave amplitudes and b-/a - wave ratios than untreated eyes of mice reared in ml ( p = 0.012 and p = 0.000 , respectively ) . average erg a - wave ( a ) , b - wave ( b ) , and b-/a - wave ratio ( c ) in two groups of 4-month - old rs1-ko mice reared in low light ( ll ) ( n = 26 ) and moderate light ( ml ) ( n = 29 ) and treated with aav8-rs1 at 21 days . although ll mice showed significantly larger b - wave amplitude and b-/a - wave ratio than ml mice in the aav8-rs1treated eye , 2-way anova analysis did not show significant interaction between treatment and rearing light exposure , indicating that aav8-mediated rs1 expression improved inner retinal function in ll and ml reared mice by the same extent . gene replacement significantly increased the b - wave amplitude and the b-/a - wave ratio in the rs1-ko treated eyes for mice reared in ml ( by 65% , p < 0.001 and 50% , p < 0.001 , respectively ) and ll ( by 77% , p < 0.001 and 35% , p < 0.001 , respectively ) ( table 2 ) . , b-/a - wave ratio ) remained unchanged between 1 and 4 months , indicating that light restriction was beneficial for evolution of inner retinal pathology in rs1-ko mice . we found that by 4 months , kir4.1 was strongly upregulated in rs1-ko mice reared in ml , indicating dysregulation of k balance in the inner retina . we have shown that delivering a normal rs1 gene by using an adeno - associated viral vector significantly improves retinal structure and function in rs1-ko mice with reduction of the size and number of cavities and an increase in the b - wave , and b-/a - wave ratio . in the present study , we found that the rearing light intensity does not interfere with the functional improvement of rs1 gene therapy . both ll- and ml - reared rs1-ko mice showed comparable improvements in b - wave amplitude and b-/a - wave ratio at 4 months of age relative to fellow untreated eyes . thus , after gene therapy both b - wave amplitude and b-/a - wave ratio were larger in ll- than ml - reared animals . although after treatment the inner retinal function increased by the same amount in ll- and ml - reared rs1-ko mice , replacing rs1 further improved the inner retinal function in ll - reared mice , whereas it preserved function in ml - reared mice with respect to levels at 1 month of age . at 4 months , ll - reared animals showed an increase in b-/a - wave ratio after treatment , whereas the latter was unchanged in rs1-ko mice reared in ml . rs1 gene replacement increased the inner retinal function ( b-/a - wave ratio ) by the same extent in 4-month - old ll- and ml - reared rs1-ko mice . when compared to 1-month - old rs1-ko mice , gene therapy improved inner retinal function in 4-month - old ll - reared animals but only preserved inner retinal function in ml - reared animals . since ll - reared rs1-ko mice do not show any change in cavity size between 1 and 4 months , rs1 gene transfer improves inner retinal function independently from an effect on retinal cavities , most likely by restoring synapse integrity between photoreceptors and bipolar cells . the fact that treating ll - reared rs1-ko mice produces a functional improvement , although no change in cavity size occurs naturally at this light intensity between 1 and 4 months , indicates that rs1 gene replacement can improve the inner retinal function independently from its beneficial effect on cavities . when rs1-ko mice are reared in ml , despite rs1 gene transfer producing the same degree of functional improvement , light exposure itself or the development of cavities may irreversibly impact the inner retina and prevent them from reaching the same level of retinal function as in ll conditions . we have shown that delivering a normal rs1 gene by using an adeno - associated viral vector significantly improves retinal structure and function in rs1-ko mice with reduction of the size and number of cavities and an increase in the b - wave , and b-/a - wave ratio . in the present study , we found that the rearing light intensity does not interfere with the functional improvement of rs1 gene therapy . both ll- and ml - reared rs1-ko mice showed comparable improvements in b - wave amplitude and b-/a - wave ratio at 4 months of age relative to fellow untreated eyes . thus , after gene therapy both b - wave amplitude and b-/a - wave ratio were larger in ll- than ml - reared animals . although after treatment the inner retinal function increased by the same amount in ll- and ml - reared rs1-ko mice , replacing rs1 further improved the inner retinal function in ll - reared mice , whereas it preserved function in ml - reared mice with respect to levels at 1 month of age . at 4 months , ll - reared animals showed an increase in b-/a - wave ratio after treatment , whereas the latter was unchanged in rs1-ko mice reared in ml . rs1 gene replacement increased the inner retinal function ( b-/a - wave ratio ) by the same extent in 4-month - old ll- and ml - reared rs1-ko mice . when compared to 1-month - old rs1-ko mice , gene therapy improved inner retinal function in 4-month - old ll - reared animals but only preserved inner retinal function in ml - reared animals . since ll - reared rs1-ko mice do not show any change in cavity size between 1 and 4 months , rs1 gene transfer improves inner retinal function independently from an effect on retinal cavities , most likely by restoring synapse integrity between photoreceptors and bipolar cells . the fact that treating ll - reared rs1-ko mice produces a functional improvement , although no change in cavity size occurs naturally at this light intensity between 1 and 4 months , indicates that rs1 gene replacement can improve the inner retinal function independently from its beneficial effect on cavities . when rs1-ko mice are reared in ml , despite rs1 gene transfer producing the same degree of functional improvement , light exposure itself or the development of cavities may irreversibly impact the inner retina and prevent them from reaching the same level of retinal function as in ll conditions .

the amount that reaches the skin depends on the solubility of the vitamins and their transepidermal diffusion . permeability of vitamins can be increased by use of a transdermal carrier . in recent decades , the most explored macromolecular vehicles have been polyamidoamine ( pamam ) dendrimers.1 these monodispersed dendritic molecules have a strictly defined shape and size and are able to encapsulate drug molecules , resulting in formation of host guest complexes.210 on the other hand , full - generation pamam dendrimers have surface amine groups ( 32 amine groups in the third - generation pamam dendrimer , g3 ) , offering an easy route for formation of conjugates with drugs.1121 due to the relatively low toxicity of pamam dendrimers,2225 they are frequently used as transdermal diffusion promoters.26 in previous studies , we have used pamam dendrimers as carriers for 8-methoxypsoralen , the photosensitizer used in psoralen and ultraviolet light a therapy ( puva),4 riboflavin,5 and vitamin c.27 in all these cases , the pamam dendrimers formed host guest complexes with 8-methoxypsoralen , vitamin b2 and vitamin c , thereby modifying the transdermal flux of the guest molecule . another approach to influencing the rate of transdermal diffusion is based on covalent binding of a prodrug and using such bioconjugates as a platform for introducing the drug into skin tissue . in terms of potential dermatological application , bioconjugates of folate,11 biotin,12 riboflavin,13 cholic acid,14 and phosphorylcholine15 have been synthesized and tested as membrane permeability enhancers , increasing the cellular uptake of the drug . here we report on bioconjugates obtained from third - generation pamam dendrimers , retinal ( [ ret ] vitamin a ) , pyridoxal ( [ pyr ] vitamin b6 ) , or pyridoxal phosphate ( [ plp ] metabolically active coenzyme of vitamin b6 ) . the g3 dendrimer was chosen in accordance with our previously reported results for optimization of dendrimer size as a transdermal carrier for vitamins b2 and c. the important issue concerning application of bioconjugates carrying biologically active molecules is their bioavailability , which was tested here in vitro . the common solvents and reagents used for synthesis of pamam dendrimers or pamam bioconjugates , ie , ethylenediamine , methyl acrylate , 13-cis - retinal and all- trans - retinal , pyr , and plp were purchased from sigma- aldrich ( st louis , mo ) . glutamic pyruvic transaminase from porcine heart ( ec 2.6.1.2 , activity 75 units / mg of protein ; sigma - aldrich ) was used for catalytic tests . h and c nuclear magnetic resonance ( nmr ) spectra were recorded using a bruker 300 mhz instrument . permeation of g3 and g3 conjugates was studied using a franz diffusion assembly ( model dc 600 ; copley scientific ltd , nottingham , uk ) equipped with 6 cm acceptor compartments . a polyvinylidene difluoride ( pvdf ) model membrane ( 0.125 mm thickness ) the bioconjugates were released from emulsion , which was prepared using cetearyl alcohol ( 1.5 g ) , brij 72 ( 1.2 g ) , and brij 58 ( 0.3 g ) as emulsifiers , vaseline ( 5.0 g ) , stearin ( 0.5 g ) , glycerine ( 1.5 g ) ( all from sigma - aldrich ) , and water ( 40.0 g ) . samples for the permeability studies were prepared by dissolving 90 mg of g3 or g3 in 1 g of emulsion . samples weighing approximately 250 mg were mounted over a commercial pvdf membrane for assessment of permeability . the receiving medium was 0.067 m phosphate buffer ( ph 7.4 ) in ethanol 7:3 v / v . the progress of diffusion was monitored spectrophotometrically at 355 nm for g3 or 275 nm for g3 using extinction coefficients calculated for the conjugate solutions in receiving medium . next , 10 ml aliquots of receptor solution were taken at 0.5 hours or longer time intervals and the receiver compartment was filled with a fresh 6 ml of receptor solution . the experiments were conducted until at least 1.5% of the initial amount of vitamin was received in the receptor solution . the results are presented as the mean ( standard deviation , shown as bars ) of six sets of measurements . the active area of the membrane determined by size of the ring in the franz cell was 0.176 cm . the cumulative amounts of g3 and g3 received as a function of diffusion time were crucial to determine the diffusion properties of the bioconjugates . for comparison of diffusion efficiency , the slope of the linear part of the plot showing the cumulative percentage of conjugate versus time was used as the quantitative parameter . the slope was obtained using a linear regression procedure with origin 8.6 packet software ( gambit cod , krakow , poland ) . a third - generation pamam dendrimer with an ethylenediamine core ( g3 ) was synthesized according to a previously published method28 and purified using dialysis in a water- water system through a cellulose membrane ( zellutrans / roth 3.5 with a 2 kda cutoff ) , as described elsewhere.29 the purity of g3 was confirmed by h and c nmr spectra in deuterium oxide and in methanol - d4 . standard one - dimensional and two - dimensional correlation spectroscopy , nuclear overhauser enhancement spectroscopy , heteronuclear single - quantum correlation spectroscopy , and heteronuclear multiple bond correlation measurements were performed to obtain the spectral assignments of g3 and its bioconjugates . synthesis of g3 and g3 was performed at a 300 mg scale of g3 ( 0.42 mmol ) in methanol as follows : 1.138 g 13-cis- or 13-trans - retinal ( 1.68 mmol ) in 20 ml of methanol was added dropwise to 30 ml of g3 solution with vigorous stirring in a nitrogen atmosphere in the dark . then the mixture was then left for 30 minutes , followed by evaporation of the methanol under a stream of nitrogen . the progress of the reaction and final products ( see figure 1 for schematic formula of g3 ) was characterized by h nmr spectroscopy . the bioconjugates were purified by extensive dialysis in a methanol - methanol system through a cellulose membrane ( zellutrans / roth 3.5 with a 2 kda cutoff ) . performed using 40 mg of g3 , a stock solution of 13-trans - retinal was added in portions to obtain g3 bioconjugates ( where n = 1 , 2 , 3 , 4 , 6 , 8 , 10 , 12 , and 16 ) as a methanolic solution , and ultraviolet - visible spectra were recorded in methanol ( figure 2 ) . h nmr ( g3 in cd3od , for atom numbering see figure 1 ) showed 8.48 ppm ( 1h , d , h-15 ) , 7.08 ( 1h , dd , h-11 ) , 6.50 ( 1h , d , h-12 ) , 6.383.23 ( 3h , three overlapped doublets , h-7 , h-8 , h-10 ) , 6.21 ( 1h , d , h-14 ) , 3.70 ( 2h , m , h-20 ) , 3.51 ( 2h , m , h-21 ) , 2.23 ( 3h , s , ch319 ) , 2.11 ( 2h , h-4 ) , 2.08 ( 3h , s , ch318 ) , 1.78 ( 3h , s , ch317 ) , 1.72 ( 2h , h-3 ) , 1.57 ( 2h , h-2 ) , and 1.27 ( 6h , s , ch316 , 16 ) . ultraviolet - visible ( g3 in ch3oh ) : 355 nm ( = 1.5 10 mol dm cm ) . condensation of pyr with amine group - terminated g3 in methanol or plp with g3 was performed in methanol and water , respectively , by stepwise addition of a 0.01 m solution of pyr in methanol or plp in water into an approximately 0.001 m solution of g3 in methanol or water , respectively ( 15 cm containing about 105 mg of g3 ) . the stoichiometry of g3 and g3 bioconjugates ( n = number of pyr or plp molecules covalently attached to terminal nitrogen atoms via an aldimine bond , figure 3 ) was monitored by h nmr spectroscopy . finally , the g3 and g3 were purified by dialysis , as mentioned earlier , isolated , and characterized by h and p nmr spectra . in a separate smaller experiment , stepwise addition of plp was continued across a broader range of plp : g3 molar ratios in order to characterize the g3 bioconjugates by ultraviolet - visible spectra ( figure 4 ) . h nmr ( g3 in cd3od ; see figure 3 for atom numbering ) : 8.89 ppm ( 1h , h-7 ) , 7.57 ( 1h , h6 ) , 4.79 ( 2h , h-8 ) , 3.86 ( 2h , h-9 ) , 3.55 ( 2h , h-10 ) , 2.58 ( 3h , ch32 ) . ultraviolet- visible ( g3 in ch3oh ) : 288 nm ( = 2.5 10 mol dm cm ) , 332 ( = 9.4 10 mol dm cm ) . h nmr ( g3 in cdod ) : 8.93 ppm ( 1h , h-7 ) , 7.84 ( 1h , h6 ) , 4.76 ( 2h , h-8 ) , 3.84 ( 2h , h-9 ) , 3.58 ( 2h , h-10 ) 2.58 ( 3h , ch32 ) . in vitro studies of transfer of an amine group from l - alanine to -ketoglutarate a solution of l - alanine and -ketoglutarate ( both at a concentration of 20 mm ) and 2 mm pyr or plp were adjusted to ph 7.4 with sodium deuteroxide solution in d2o , and the h nmr spectrum was recorded . to this sample , 10 l of stock solution of transaminase ( 5 mg / ml ) was injected and nmr spectra were then recorded at 5-minute intervals . the same conditions were used to study the bioconjugates , except that pyr and plp were replaced with g3 and g3 at 0.5 mm concentrations . the overlay spectra are presented in figure 5 and in the supplementary materials as figures s1 and s2 . a third - generation pamam dendrimer with an ethylenediamine core ( g3 ) was synthesized according to a previously published method28 and purified using dialysis in a water- water system through a cellulose membrane ( zellutrans / roth 3.5 with a 2 kda cutoff ) , as described elsewhere.29 the purity of g3 was confirmed by h and c nmr spectra in deuterium oxide and in methanol - d4 . standard one - dimensional and two - dimensional correlation spectroscopy , nuclear overhauser enhancement spectroscopy , heteronuclear single - quantum correlation spectroscopy , and heteronuclear multiple bond correlation measurements were performed to obtain the spectral assignments of g3 and its bioconjugates . synthesis of g3 and g3 was performed at a 300 mg scale of g3 ( 0.42 mmol ) in methanol as follows : 1.138 g 13-cis- or 13-trans - retinal ( 1.68 mmol ) in 20 ml of methanol was added dropwise to 30 ml of g3 solution with vigorous stirring in a nitrogen atmosphere in the dark . then the mixture was then left for 30 minutes , followed by evaporation of the methanol under a stream of nitrogen . the progress of the reaction and final products ( see figure 1 for schematic formula of g3 ) was characterized by h nmr spectroscopy . the bioconjugates were purified by extensive dialysis in a methanol - methanol system through a cellulose membrane ( zellutrans / roth 3.5 with a 2 kda cutoff ) . performed using 40 mg of g3 , a stock solution of 13-trans - retinal was added in portions to obtain g3 bioconjugates ( where n = 1 , 2 , 3 , 4 , 6 , 8 , 10 , 12 , and 16 ) as a methanolic solution , and ultraviolet - visible spectra were recorded in methanol ( figure 2 ) . h nmr ( g3 in cd3od , for atom numbering see figure 1 ) showed 8.48 ppm ( 1h , d , h-15 ) , 7.08 ( 1h , dd , h-11 ) , 6.50 ( 1h , d , h-12 ) , 6.383.23 ( 3h , three overlapped doublets , h-7 , h-8 , h-10 ) , 6.21 ( 1h , d , h-14 ) , 3.70 ( 2h , m , h-20 ) , 3.51 ( 2h , m , h-21 ) , 2.23 ( 3h , s , ch319 ) , 2.11 ( 2h , h-4 ) , 2.08 ( 3h , s , ch318 ) , 1.78 ( 3h , s , ch317 ) , 1.72 ( 2h , h-3 ) , 1.57 ( 2h , h-2 ) , and 1.27 ( 6h , s , ch316 , 16 ) . ultraviolet - visible ( g3 in ch3oh ) : 355 nm ( = 1.5 10 mol dm cm ) . condensation of pyr with amine group - terminated g3 in methanol or plp with g3 was performed in methanol and water , respectively , by stepwise addition of a 0.01 m solution of pyr in methanol or plp in water into an approximately 0.001 m solution of g3 in methanol or water , respectively ( 15 cm containing about 105 mg of g3 ) . the stoichiometry of g3 and g3 bioconjugates ( n = number of pyr or plp molecules covalently attached to terminal nitrogen atoms via an aldimine bond , figure 3 ) was monitored by h nmr spectroscopy . finally , the g3 and g3 were purified by dialysis , as mentioned earlier , isolated , and characterized by h and p nmr spectra . in a separate smaller experiment , stepwise addition of plp was continued across a broader range of plp : g3 molar ratios in order to characterize the g3 bioconjugates by ultraviolet - visible spectra ( figure 4 ) . h nmr ( g3 in cd3od ; see figure 3 for atom numbering ) : 8.89 ppm ( 1h , h-7 ) , 7.57 ( 1h , h6 ) , 4.79 ( 2h , h-8 ) , 3.86 ( 2h , h-9 ) , 3.55 ( 2h , h-10 ) , 2.58 ( 3h , ch32 ) . ultraviolet- visible ( g3 in ch3oh ) : 288 nm ( = 2.5 10 mol dm cm ) , 332 ( = 9.4 10 mol dm cm ) . h nmr ( g3 in cdod ) : 8.93 ppm ( 1h , h-7 ) , 7.84 ( 1h , h6 ) , 4.76 ( 2h , h-8 ) , 3.84 ( 2h , h-9 ) , 3.58 ( 2h , h-10 ) 2.58 ( 3h , ch32 ) . in vitro studies of transfer of an amine group from l - alanine to -ketoglutarate were performed on an nmr sample scale . a solution of l - alanine and -ketoglutarate ( both at a concentration of 20 mm ) and 2 mm pyr or plp were adjusted to ph 7.4 with sodium deuteroxide solution in d2o , and the h nmr spectrum was recorded . to this sample , 10 l of stock solution of transaminase ( 5 mg / ml ) was injected and nmr spectra were then recorded at 5-minute intervals . the same conditions were used to study the bioconjugates , except that pyr and plp were replaced with g3 and g3 at 0.5 mm concentrations . the overlay spectra are presented in figure 5 and in the supplementary materials as figures s1 and s2 . the amine groups of the pamam dendrimers are readily convertible into schiff bases via condensation with aldehydes . 29 we used a simple protocol for this reaction to obtain conjugates of g3 pamam dendrimer containing 32 terminal amine groups with biologically important vitamins , ie , ret ( 13-cis and all - trans ) and pyr or plp . stepwise addition of 13-cis - retinal or all - trans - retinal into g3 in methanol resulted in formation of g3 bioconjugates at variable substitution steps ( n = 116 ) , accompanied by marked changes in the ultraviolet - visible spectra ( figure 2 ) . initially the absorption band attributed to aldimine n * transition was centered at 363 nm ( for n = 1 ) , which gradually shifted to a higher energy level ( 328 nm ) upon increasing n to 16 , ie , to the point where 50% of the amine groups in g3 were converted into schiff base . presumably the hypsochromic shift observed was due to interaction between the unsaturated hydrocarbon chains of all - trans - retinal within the bioconjugate . the sigmoidal plot of max versus n ( figure 5 ) showed the inflection point to be at about n = 6 , corresponding to the statistical situation whereby every retinal substituent had at least one other retinal molecule attached to the proximal amine group of g3 . both 13-cis - retinal and 13-trans - retinal were used to obtain g34 and g34 bioconjugates . the h nmr spectra of these compounds were recorded in methanol - d4 ( figure 6 ) . the most characteristic resonance of the bioconjugates was that for the aldimine proton ( h-15 ) which was diagnostic for schiff base formation . the signal shifted from 10.15 ppm ( 13-cis - retinal ) or 10.03 ppm ( 13-trans - retinal ) to 8.59 ppm for g34 or 8.48 ppm for g34 . the g34 bioconjugate was stable in solution when the sample was protected from air and light ( figure 6 , lower spectrum ) , while g34 underwent spontaneous conversion within 12 hours under the same conditions into a mixture of g34 species ( where x = cis or trans , figure 6 , upper spectrum ) . the g34 bioconjugate actually corresponds to a mixture of species with predominantly n = 4 . the high - intensity resonances within the 2.33.4 ppm region belong to the pamam dendrimer , while those below 2.25 ppm correspond to the methyl and methylene protons of the retinal substituent . the most characteristic resonances of pamam in g34 and the other bioconjugates obtained here are those of two methylene group protons , h-20 and h-21 ( for atom numbering see figure 1 ) , seen at 3.70 and 3.51 ppm , respectively . the broad resonance at 8.2 ppm ( lower spectrum ) belongs to the internal n - h protons of the pamam dendrimer , which slowly undergoes substitution with the deuterium solvent and eventually disappears , and hence is not present in the upper spectrum for the bioconjugate mixture which was recorded after 12 hours of incubation of initial g34 in methanol - d4 . the 13-cis - trans conversion in the g3 conjugates is not surprising in view of previous studies of retinal conformation in schiff bases . ab initio quantum chemical and free energy simulation30 and dynamic simulation31 show that lowest energy configuration is all - trans , which is about 2.1 kcal / mol lower than that of 13-cis . moreover , the flexibility induced by the polyene chain of retinal in the bacteriorhodopsin - retinal schiff base might be responsible for enabling the cis - trans transition and modifying the pka of the retinal - protonated schiff base in bacteriorhodopsin.31 stepwise addition of pyridoxal hydrochloride solution into g3 ( both in methanol ) resulted in formation of g3 bioconjugates , which were characterized by h nmr spectra . the characteristic resonances of this bioconjugate are those of the aldimine proton ( h-7 ) at 8.93 ppm , the aromatic proton ( h-6 ) at 7.84 ppm , and the 5-ch2oh singlet at 4.76 ppm . the solubility of the g3 analog in methanol is lower than that of g3 , nevertheless the h nmr spectrum was recorded in methanol - d4 for comparative purposes . in this solvent , the chemical shift of the h-6 and h-7 protons was similar to those for g3 ( 7.57 and 8.89 ppm , respectively ) , while the doublet of 5-ch2opo3 protons ( h-8 ) is shifted downfield to 4.79 ppm . stepwise addition of plp in water into an aqueous solution of g3 enabled recording of the ultraviolet - visible spectra for g3 bioconjugates ( figure 4 ) , which differed considerably from that for plp itself , in that the * transition band centered at 295 nm for the plp aldehyde split into three bands centered at 340 nm , 275 nm , and 255 nm ( shoulder ) of almost equal intensity upon formation of the schiff base . the averaged extinction coefficient per one adjacent plp remains constant ( 5.0 10 mol dm cm ) and is slightly lower than that for plp itself ( 6.2 10 mol dm cm ) , suggesting that no strong interaction between adjacent plp substituents is present in g3 until n = 16 . therefore , both types of pyr bioconjugate containing four equivalents of vitamin , ie , g3 and g3 , were used for subsequent biochemical and pharmacokinetic tests in vitro . the presence of hemiaminals and their back conversion into free aldehyde has been demonstrated using model systems,29,32 including schiff bases obtained from pamam dendrimers and aldehydes . because plp is a crucial cofactor for transaminases,33 we have tested g3 and also g3 as potential cofactor donors . in order to test the bioavailability of plp incorporated into a g3 bioconjugate , we used glutamic pyruvic transaminase from porcine heart tissue ( ec 2.6.1.2 ) and monitored amine transfer from the l - alanine donor to -ketoglutarate under h nmr spectral control in h2o . amine transfer and formation of l - glutamate was triggered by addition of plp ( final concentration 0.002 m , ie , 2 mol in 1 ml of an nmr sample ) into the solution containing 0.02 m l - alanine and -ketoglutarate and glutamic pyruvic transaminase ( approximately 0.05 mg corresponding to three units ) in a 1 cm sample at ph 7.6 . fifty percent conversion of -ketoglutarate to l - glutamate was achieved in 45 minutes ( see figure s1 for h nmr spectra of the reaction mixture ) . an almost analogous spectral image was obtained when g3 was added as the cofactor source ( figure 7 ) . in this case , the transamination reaction was triggered by addition of l - alanine into a solution already containing -ketoglutarate , glutamic pyruvic transaminase , and g3 ( 0.5 mol , all other concentrations and numbers of mol were the same as in the control experiment ) . the h nmr spectral assignments were done by two - dimensional correlation spectroscopy . for the starting solution , from which l - alanine was absent , the 3-ch2 and 4-ch2 triplet resonances are seen at 5.35 ppm and 3.50 ppm , respectively , while all other broad resonances belonging to the pamam protons and plp residues are shown in the full - range spectrum at 8.73 ( n = ch-7 ) and 7.50 ( 6-h ) . four minutes after addition of l - alanine , the resonances of l - glutamate appear as multiplets from two magnetically nonequivalent 4-ch2 centered at 1.96 ppm and 3-ch2 centered at 2.24 ppm . no 2-ch ( ala ) resonance was observed due to involvement of the deuterium oxide solvent . in the course of the reaction , the resonances from l - alanine disappear ( 2-ch ) or broaden slightly due to formation of a schiff base between l - alanine and plp , which in fact is the species actively involved in transamination . again , 50% conversion of -ketoglutarate l - glutamate was achieved within approximately 45 minutes , while the methyl group resonance of l - alanine was no longer detected in the h nmr spectrum of the reaction mixture . thus , the puzzling issue remains concerning the fate of the amine groups not involved in transamination with -ketoglutarate . the most surprising result was obtained in the last transamination experiment , in which g3 was used as a potential cofactor source for transaminase . no reaction was observed when pyr was used instead of g3 in the control experiment . the cofactor activity of g3 in the amine transfer from l - alanine to -ketoglutarate suggests that activation of pyr as a cofactor for transaminase occurs via covalent bonding of pyr to pamam g3 . amine group transfer from pyridoxamine covalently incorporated into the core of pamam to phenylpyruvic and pyruvic acid,34,35 as well as racemization of alanine by pyr covalently incorporated into the core of polyethyleneimine dendrimers has been reported.36 in order to characterize the roles of the pamam carrier and the pamam - plp and pamam - pyr bioconjugates further , we performed additional control experiments . amine transfer from pamam to -ketoglutarate did not occur in the absence or presence of glutamic pyruvic transaminase when l - alanine was absent . g3 and g3 in the absence of glutamic pyruvic transaminase did not catalyze amine transfer from l - alanine to -ketoglutarate , demonstrating that the bioconjugates did not play a catalytic role . finally , transamination did not occur when g3 was added to the system containing l - alanine , -ketoglutarate , and glutamic pyruvic transaminase , indicating that g3 was not an activator of glutamic pyruvic transaminase . permeation of g3 and g3 conjugates through pvdf was tested using a buffered water : ethanol receiving solution . the conjugates were soluble in this solvent to a concentration of 10 mol dm , so we could obtain the ultraviolet - visible spectra . we measured the extinction at max for g3 and g3 conjugates at 355 nm and 275 nm , respectively , and took the full spectra in order to confirm the identity of the conjugates by comparing them with the original spectra of these species . the flux , measured under reproducible conditions for both the test systems , changed in a typical manner , being high during the first half hour of the experiment and becoming stable ( in a stationary condition ) thereafter . the diffusion profiles were plotted as the cumulative amount of conjugate versus time or percentage of diffused species versus time . the conjugates showed different fluxes in stationary conditions , ie , the flux of g3 was 1.5 mol h cm , while that of g3 was as low as 0.4 mol h cm . the cumulative percentage plots for the diffused species versus time are shown in figure 8 . the linear fit for these experimental profiles was applied in the region of stationary conditions , and the slopes of the fitted lines ( expressed in % h ) can be interpreted in terms of the diffusion ability of the conjugates . thus , the slopes of the fitted line for g3 ( 0.44% 0.03% h ) and for g3 ( 1.36% 0.06% h ) demonstrate clearly an approximately two - fold lower permeation ability for the g3 conjugate than for the g3 . for comparison , control experiments were performed on release of trans - retinal and plp from the same emulsion ( figure 8) . comparing the diffusion rates for the g3 conjugate with those of ret alone , one can see that the bioconjugate acts as a vitamin permeation promoter , while comparison of the diffusion profiles for g3 and plp indicate that , in this case , the bioconjugate slows diffusion of plp . generally , by introducing enhancement factors ( ef ) as the ratio of the diffusion rate of bioconjugates ( or host guest complexes g3-x , where x = ascorbic acid , riboflavin , or 8-methoxypsoralen ) to the diffusion rate of the free species x , thus , ef values of > 1 demonstrate the ability of the g3 pamam dendrimer to promote transdermal diffusion , while ef values < 1 indicate that the dendrimer retards diffusion ( table 1 ) . permeation efficiency ( diffusion rate of drug or prodrug , including bioconjugates ) is influenced by two main factors , ie , size and flexibility of the species and its water solubility . we have determined previously the rate of diffusion of g3 itself ( g3 labeled with one fluorescein substituent [ g3]),5 which gave us a reference value of 2.18% 0.23% h. for bioconjugates bearing four substituents of ret , pyr , or plp , the diffusion rate is lower , which can be attributed to the larger size of the bioconjugate than g3 itself ( 2.2 nm in diameter ) . nevertheless , the bioconjugates do modify the diffusion of free ret or plp . in the case of ret , which is insoluble in water ( but soluble in oil phase emulsion ) the bioconjugate diffuses faster than ret due to better solubility of the g3 bioconjugate than ret in water . in the case of plp , the effect is opposite , in that plp diffuses faster than the macromolecular g3 bioconjugate ( both water - soluble ) due to a steric effect . for host guest complexes with pamam as the host , the pamam carrier diminishes the rate of diffusion of water - soluble vitamin c by four times through porcine ear skin and by 1.5 times through pvdf.27 on the other hand , g3 acts as a solubilizer for water - insoluble riboflavin5 and 8-methoxypsoralene,4 and promotes their diffusion . the enhancement factor was 4 through pvdf and 3 through porcine ear skin for riboflavin5 and 3 for 8-methoxypsoralen as demonstrated in vitro using pvdf and porcine ear skin models , as well as in vivo studies using rat skin with confocal microscopy monitoring.37 stepwise addition of 13-cis - retinal or all - trans - retinal into g3 in methanol resulted in formation of g3 bioconjugates at variable substitution steps ( n = 116 ) , accompanied by marked changes in the ultraviolet - visible spectra ( figure 2 ) . initially the absorption band attributed to aldimine n * transition was centered at 363 nm ( for n = 1 ) , which gradually shifted to a higher energy level ( 328 nm ) upon increasing n to 16 , ie , to the point where 50% of the amine groups in g3 were converted into schiff base . presumably the hypsochromic shift observed was due to interaction between the unsaturated hydrocarbon chains of all - trans - retinal within the bioconjugate . the sigmoidal plot of max versus n ( figure 5 ) showed the inflection point to be at about n = 6 , corresponding to the statistical situation whereby every retinal substituent had at least one other retinal molecule attached to the proximal amine group of g3 . both 13-cis - retinal and 13-trans - retinal were used to obtain g34 and g34 bioconjugates . the h nmr spectra of these compounds were recorded in methanol - d4 ( figure 6 ) . the most characteristic resonance of the bioconjugates was that for the aldimine proton ( h-15 ) which was diagnostic for schiff base formation . the signal shifted from 10.15 ppm ( 13-cis - retinal ) or 10.03 ppm ( 13-trans - retinal ) to 8.59 ppm for g34 or 8.48 ppm for g34 . the g34 bioconjugate was stable in solution when the sample was protected from air and light ( figure 6 , lower spectrum ) , while g34 underwent spontaneous conversion within 12 hours under the same conditions into a mixture of g34 species ( where x = cis or trans , figure 6 , upper spectrum ) . the g34 bioconjugate actually corresponds to a mixture of species with predominantly n = 4 . the high - intensity resonances within the 2.33.4 ppm region belong to the pamam dendrimer , while those below 2.25 ppm correspond to the methyl and methylene protons of the retinal substituent . the most characteristic resonances of pamam in g34 and the other bioconjugates obtained here are those of two methylene group protons , h-20 and h-21 ( for atom numbering see figure 1 ) , seen at 3.70 and 3.51 ppm , respectively . the broad resonance at 8.2 ppm ( lower spectrum ) belongs to the internal n - h protons of the pamam dendrimer , which slowly undergoes substitution with the deuterium solvent and eventually disappears , and hence is not present in the upper spectrum for the bioconjugate mixture which was recorded after 12 hours of incubation of initial g34 in methanol - d4 . the 13-cis - trans conversion in the g3 conjugates is not surprising in view of previous studies of retinal conformation in schiff bases . ab initio quantum chemical and free energy simulation30 and dynamic simulation31 show that lowest energy configuration is all - trans , which is about 2.1 kcal / mol lower than that of 13-cis . moreover , the flexibility induced by the polyene chain of retinal in the bacteriorhodopsin - retinal schiff base might be responsible for enabling the cis - trans transition and modifying the pka of the retinal - protonated schiff base in bacteriorhodopsin.31 stepwise addition of pyridoxal hydrochloride solution into g3 ( both in methanol ) resulted in formation of g3 bioconjugates , which were characterized by h nmr spectra . the characteristic resonances of this bioconjugate are those of the aldimine proton ( h-7 ) at 8.93 ppm , the aromatic proton ( h-6 ) at 7.84 ppm , and the 5-ch2oh singlet at 4.76 ppm . the solubility of the g3 analog in methanol is lower than that of g3 , nevertheless the h nmr spectrum was recorded in methanol - d4 for comparative purposes . in this solvent , the chemical shift of the h-6 and h-7 protons was similar to those for g3 ( 7.57 and 8.89 ppm , respectively ) , while the doublet of 5-ch2opo3 protons ( h-8 ) is shifted downfield to 4.79 ppm . stepwise addition of plp in water into an aqueous solution of g3 enabled recording of the ultraviolet - visible spectra for g3 bioconjugates ( figure 4 ) , which differed considerably from that for plp itself , in that the * transition band centered at 295 nm for the plp aldehyde split into three bands centered at 340 nm , 275 nm , and 255 nm ( shoulder ) of almost equal intensity upon formation of the schiff base . the averaged extinction coefficient per one adjacent plp remains constant ( 5.0 10 mol dm cm ) and is slightly lower than that for plp itself ( 6.2 10 mol dm cm ) , suggesting that no strong interaction between adjacent plp substituents is present in g3 until n = 16 . therefore , both types of pyr bioconjugate containing four equivalents of vitamin , ie , g3 and g3 , were used for subsequent biochemical and pharmacokinetic tests in vitro . the presence of hemiaminals and their back conversion into free aldehyde has been demonstrated using model systems,29,32 including schiff bases obtained from pamam dendrimers and aldehydes . because plp is a crucial cofactor for transaminases,33 we have tested g3 and also g3 as potential cofactor donors . in order to test the bioavailability of plp incorporated into a g3 bioconjugate , we used glutamic pyruvic transaminase from porcine heart tissue ( ec 2.6.1.2 ) and monitored amine transfer from the l - alanine donor to -ketoglutarate under h nmr spectral control in h2o . in the control experiment , amine transfer and formation of l - glutamate was triggered by addition of plp ( final concentration 0.002 m , ie , 2 mol in 1 ml of an nmr sample ) into the solution containing 0.02 m l - alanine and -ketoglutarate and glutamic pyruvic transaminase ( approximately 0.05 mg corresponding to three units ) in a 1 cm sample at ph 7.6 . fifty percent conversion of -ketoglutarate to l - glutamate was achieved in 45 minutes ( see figure s1 for h nmr spectra of the reaction mixture ) . an almost analogous spectral image was obtained when g3 was added as the cofactor source ( figure 7 ) . in this case , the transamination reaction was triggered by addition of l - alanine into a solution already containing -ketoglutarate , glutamic pyruvic transaminase , and g3 ( 0.5 mol , all other concentrations and numbers of mol were the same as in the control experiment ) . for the starting solution , from which l - alanine was absent , the 3-ch2 and 4-ch2 triplet resonances are seen at 5.35 ppm and 3.50 ppm , respectively , while all other broad resonances belonging to the pamam protons and plp residues are shown in the full - range spectrum at 8.73 ( n = ch-7 ) and 7.50 ( 6-h ) . four minutes after addition of l - alanine , the resonances of l - glutamate appear as multiplets from two magnetically nonequivalent 4-ch2 centered at 1.96 ppm and 3-ch2 centered at 2.24 ppm . no 2-ch ( ala ) resonance was observed due to involvement of the deuterium oxide solvent . in the course of the reaction , the resonances from l - alanine disappear ( 2-ch ) or broaden slightly due to formation of a schiff base between l - alanine and plp , which in fact is the species actively involved in transamination . again , 50% conversion of -ketoglutarate l - glutamate was achieved within approximately 45 minutes , while the methyl group resonance of l - alanine was no longer detected in the h nmr spectrum of the reaction mixture . thus , the puzzling issue remains concerning the fate of the amine groups not involved in transamination with -ketoglutarate . the most surprising result was obtained in the last transamination experiment , in which g3 was used as a potential cofactor source for transaminase . no reaction was observed when pyr was used instead of g3 in the control experiment . the cofactor activity of g3 in the amine transfer from l - alanine to -ketoglutarate suggests that activation of pyr as a cofactor for transaminase occurs via covalent bonding of pyr to pamam g3 . amine group transfer from pyridoxamine covalently incorporated into the core of pamam to phenylpyruvic and pyruvic acid,34,35 as well as racemization of alanine by pyr covalently incorporated into the core of polyethyleneimine dendrimers has been reported.36 in order to characterize the roles of the pamam carrier and the pamam - plp and pamam - pyr bioconjugates further , we performed additional control experiments . amine transfer from pamam to -ketoglutarate did not occur in the absence or presence of glutamic pyruvic transaminase when l - alanine was absent . g3 and g3 in the absence of glutamic pyruvic transaminase did not catalyze amine transfer from l - alanine to -ketoglutarate , demonstrating that the bioconjugates did not play a catalytic role . finally , transamination did not occur when g3 was added to the system containing l - alanine , -ketoglutarate , and glutamic pyruvic transaminase , indicating that g3 was not an activator of glutamic pyruvic transaminase . permeation of g3 and g3 conjugates through pvdf was tested using a buffered water : ethanol receiving solution . the conjugates were soluble in this solvent to a concentration of 10 mol dm , so we could obtain the ultraviolet - visible spectra . we measured the extinction at max for g3 and g3 conjugates at 355 nm and 275 nm , respectively , and took the full spectra in order to confirm the identity of the conjugates by comparing them with the original spectra of these species . the flux , measured under reproducible conditions for both the test systems , changed in a typical manner , being high during the first half hour of the experiment and becoming stable ( in a stationary condition ) thereafter . the diffusion profiles were plotted as the cumulative amount of conjugate versus time or percentage of diffused species versus time . the conjugates showed different fluxes in stationary conditions , ie , the flux of g3 was 1.5 mol h cm , while that of g3 was as low as 0.4 mol h cm . the cumulative percentage plots for the diffused species versus time are shown in figure 8 . the linear fit for these experimental profiles was applied in the region of stationary conditions , and the slopes of the fitted lines ( expressed in % h ) can be interpreted in terms of the diffusion ability of the conjugates . thus , the slopes of the fitted line for g3 ( 0.44% 0.03% h ) and for g3 ( 1.36% 0.06% h ) demonstrate clearly an approximately two - fold lower permeation ability for the g3 conjugate than for the g3 . for comparison , control experiments were performed on release of trans - retinal and plp from the same emulsion ( figure 8) . comparing the diffusion rates for the g3 conjugate with those of ret alone , one can see that the bioconjugate acts as a vitamin permeation promoter , while comparison of the diffusion profiles for g3 and plp indicate that , in this case , the bioconjugate slows diffusion of plp . generally , by introducing enhancement factors ( ef ) as the ratio of the diffusion rate of bioconjugates ( or host guest complexes g3-x , where x = ascorbic acid , riboflavin , or 8-methoxypsoralen ) to the diffusion rate of the free species x , the ability of the g3 pamam dendrimer to influence permeation can be estimated quantitatively . thus , ef values of > 1 demonstrate the ability of the g3 pamam dendrimer to promote transdermal diffusion , while ef values < 1 indicate that the dendrimer retards diffusion ( table 1 ) . permeation efficiency ( diffusion rate of drug or prodrug , including bioconjugates ) is influenced by two main factors , ie , size and flexibility of the species and its water solubility . we have determined previously the rate of diffusion of g3 itself ( g3 labeled with one fluorescein substituent [ g3]),5 which gave us a reference value of 2.18% 0.23% h. for bioconjugates bearing four substituents of ret , pyr , or plp , the diffusion rate is lower , which can be attributed to the larger size of the bioconjugate than g3 itself ( 2.2 nm in diameter ) . nevertheless , the bioconjugates do modify the diffusion of free ret or plp . in the case of ret , which is insoluble in water ( but soluble in oil phase emulsion ) the bioconjugate diffuses faster than ret due to better solubility of the g3 bioconjugate than ret in water . in the case of plp , the effect is opposite , in that plp diffuses faster than the macromolecular g3 bioconjugate ( both water - soluble ) due to a steric effect . for host guest complexes with pamam as the host , the pamam carrier diminishes the rate of diffusion of water - soluble vitamin c by four times through porcine ear skin and by 1.5 times through pvdf.27 on the other hand , g3 acts as a solubilizer for water - insoluble riboflavin5 and 8-methoxypsoralene,4 and promotes their diffusion . the enhancement factor was 4 through pvdf and 3 through porcine ear skin for riboflavin5 and 3 for 8-methoxypsoralen as demonstrated in vitro using pvdf and porcine ear skin models , as well as in vivo studies using rat skin with confocal microscopy monitoring.37 vitamin b6 and vitamin a readily form conjugates with third - generation pamam dendrimers as schiff bases . condensation of 13-cis and 13-trans- retinal with g3 resulted in formation of conjugates with variable degrees of pamam substitution . the attached molecules of 13-cis - retinal undergo 13-cis 13-trans conversion within the schiff base . plp is readily available as a cofactor for transaminase when released from a g3 bioconjugate , which was demonstrated in vitro by amine transfer from l - alanine to -ketoglutarate , as confirmed by h nmr spectroscopy . the pyr bioconjugated with g3 behaves the same as g3 and in contrary to free pyridoxal , which is not cofactor for transaminase . bioconjugates of plp and ret with a g3 pamam dendrimer diffuse through a pvdf skin model membrane slightly slower than g3 labeled with single fluorescein . finally , bioconjugates of g3 with vitamin a and vitamin b6 aldehydes can be used as transdermal drug delivery vehicles . in the case of bioconjugates with pyr and plp , the vitamins become available as transaminase cofactors , which is crucial when using these conjugates to feed skin . the h nmr spectra of reaction mixture containing pyridoxal phosphate ( 2 mm ) , l - alanine and -ketoglutarate ( both 20 mmm ) and transaminase ( 3 units ) in d2o , ph = 7.4 . the relevant fragments of h nmr spectra of reaction mixture containing g3 ( 2 mm ) , l - alanine and -ketoglutarate ( both 20 mmm ) and transaminase ( 3 units ) in d2o , ph = 7.4 , recorded after ( from bottom to top ) : 2 , 6 , 10 , 20 , and 45 minutes , respectively . abbreviation : nmr , nuclear magnetic resonance .

backgroundbioconjugates of a polyamidoamine ( pamam ) g3 dendrimer and an aldehyde were synthesized as carriers for vitamins a and b6 , and the bioavailability of these vitamins for skin nutrition was investigated.methodsnuclear magnetic resonance ( nmr ) and ultraviolet - visible methods were used to characterize the structure of the bioconjugates and for monitoring release of pyridoxal ( pyr ) and pyridoxal phosphate ( plp ) from these bioconjugates in vitro . a skin model permeation of bioconjugates was also studied in a franz chamber.resultsa transdermal g3 pamam dendrimer was used to synthesize bioconjugates with trans - retinal ( ret ) , pyridoxal ( pyr ) , or plp . these nanomolecules , containing up to four covalently linked ret , pyr , or plp ( g34ret , g34pyr , and g34plp ) , were able to permeate the skin , as demonstrated in vitro using a model skin membrane . plp and pyr bound to a macromolecular vehicle were active cofactors for glutamic pyruvic transaminase , as shown by 1h nmr spectral monitoring of the progress of the l - alanine + -ketoglutarate glutamic acid + pyruvic acid reaction.conclusionpamam-plp , pamam - pyr , and pamam - ret bioconjugates are able to permeate the skin . plp and pyr are available as cofactors for glutamic pyruvic transaminase .

Introduction Materials and methods Synthesis of PAMAM dendrimers Synthesis of PAMAM G3-Ret bioconjugates Synthesis of PAMAM G3-Pyr and G3-PLP bioconjugates Catalytic tests Results and discussion G3 G3 Release of Pyr and PLP from bioconjugates and transamination Permeation through PVDF membrane Conclusion Supplementary figures

in recent decades , the most explored macromolecular vehicles have been polyamidoamine ( pamam ) dendrimers.1 these monodispersed dendritic molecules have a strictly defined shape and size and are able to encapsulate drug molecules , resulting in formation of host guest complexes.210 on the other hand , full - generation pamam dendrimers have surface amine groups ( 32 amine groups in the third - generation pamam dendrimer , g3 ) , offering an easy route for formation of conjugates with drugs.1121 due to the relatively low toxicity of pamam dendrimers,2225 they are frequently used as transdermal diffusion promoters.26 in previous studies , we have used pamam dendrimers as carriers for 8-methoxypsoralen , the photosensitizer used in psoralen and ultraviolet light a therapy ( puva),4 riboflavin,5 and vitamin c.27 in all these cases , the pamam dendrimers formed host guest complexes with 8-methoxypsoralen , vitamin b2 and vitamin c , thereby modifying the transdermal flux of the guest molecule . here we report on bioconjugates obtained from third - generation pamam dendrimers , retinal ( [ ret ] vitamin a ) , pyridoxal ( [ pyr ] vitamin b6 ) , or pyridoxal phosphate ( [ plp ] metabolically active coenzyme of vitamin b6 ) . the g3 dendrimer was chosen in accordance with our previously reported results for optimization of dendrimer size as a transdermal carrier for vitamins b2 and c. the important issue concerning application of bioconjugates carrying biologically active molecules is their bioavailability , which was tested here in vitro . the common solvents and reagents used for synthesis of pamam dendrimers or pamam bioconjugates , ie , ethylenediamine , methyl acrylate , 13-cis - retinal and all- trans - retinal , pyr , and plp were purchased from sigma- aldrich ( st louis , mo ) . h and c nuclear magnetic resonance ( nmr ) spectra were recorded using a bruker 300 mhz instrument . permeation of g3 and g3 conjugates was studied using a franz diffusion assembly ( model dc 600 ; copley scientific ltd , nottingham , uk ) equipped with 6 cm acceptor compartments . a third - generation pamam dendrimer with an ethylenediamine core ( g3 ) was synthesized according to a previously published method28 and purified using dialysis in a water- water system through a cellulose membrane ( zellutrans / roth 3.5 with a 2 kda cutoff ) , as described elsewhere.29 the purity of g3 was confirmed by h and c nmr spectra in deuterium oxide and in methanol - d4 . performed using 40 mg of g3 , a stock solution of 13-trans - retinal was added in portions to obtain g3 bioconjugates ( where n = 1 , 2 , 3 , 4 , 6 , 8 , 10 , 12 , and 16 ) as a methanolic solution , and ultraviolet - visible spectra were recorded in methanol ( figure 2 ) . in a separate smaller experiment , stepwise addition of plp was continued across a broader range of plp : g3 molar ratios in order to characterize the g3 bioconjugates by ultraviolet - visible spectra ( figure 4 ) . in vitro studies of transfer of an amine group from l - alanine to -ketoglutarate a solution of l - alanine and -ketoglutarate ( both at a concentration of 20 mm ) and 2 mm pyr or plp were adjusted to ph 7.4 with sodium deuteroxide solution in d2o , and the h nmr spectrum was recorded . a third - generation pamam dendrimer with an ethylenediamine core ( g3 ) was synthesized according to a previously published method28 and purified using dialysis in a water- water system through a cellulose membrane ( zellutrans / roth 3.5 with a 2 kda cutoff ) , as described elsewhere.29 the purity of g3 was confirmed by h and c nmr spectra in deuterium oxide and in methanol - d4 . performed using 40 mg of g3 , a stock solution of 13-trans - retinal was added in portions to obtain g3 bioconjugates ( where n = 1 , 2 , 3 , 4 , 6 , 8 , 10 , 12 , and 16 ) as a methanolic solution , and ultraviolet - visible spectra were recorded in methanol ( figure 2 ) . in a separate smaller experiment , stepwise addition of plp was continued across a broader range of plp : g3 molar ratios in order to characterize the g3 bioconjugates by ultraviolet - visible spectra ( figure 4 ) . a solution of l - alanine and -ketoglutarate ( both at a concentration of 20 mm ) and 2 mm pyr or plp were adjusted to ph 7.4 with sodium deuteroxide solution in d2o , and the h nmr spectrum was recorded . 29 we used a simple protocol for this reaction to obtain conjugates of g3 pamam dendrimer containing 32 terminal amine groups with biologically important vitamins , ie , ret ( 13-cis and all - trans ) and pyr or plp . stepwise addition of 13-cis - retinal or all - trans - retinal into g3 in methanol resulted in formation of g3 bioconjugates at variable substitution steps ( n = 116 ) , accompanied by marked changes in the ultraviolet - visible spectra ( figure 2 ) . initially the absorption band attributed to aldimine n * transition was centered at 363 nm ( for n = 1 ) , which gradually shifted to a higher energy level ( 328 nm ) upon increasing n to 16 , ie , to the point where 50% of the amine groups in g3 were converted into schiff base . the broad resonance at 8.2 ppm ( lower spectrum ) belongs to the internal n - h protons of the pamam dendrimer , which slowly undergoes substitution with the deuterium solvent and eventually disappears , and hence is not present in the upper spectrum for the bioconjugate mixture which was recorded after 12 hours of incubation of initial g34 in methanol - d4 . stepwise addition of plp in water into an aqueous solution of g3 enabled recording of the ultraviolet - visible spectra for g3 bioconjugates ( figure 4 ) , which differed considerably from that for plp itself , in that the * transition band centered at 295 nm for the plp aldehyde split into three bands centered at 340 nm , 275 nm , and 255 nm ( shoulder ) of almost equal intensity upon formation of the schiff base . in order to test the bioavailability of plp incorporated into a g3 bioconjugate , we used glutamic pyruvic transaminase from porcine heart tissue ( ec 2.6.1.2 ) and monitored amine transfer from the l - alanine donor to -ketoglutarate under h nmr spectral control in h2o . amine transfer and formation of l - glutamate was triggered by addition of plp ( final concentration 0.002 m , ie , 2 mol in 1 ml of an nmr sample ) into the solution containing 0.02 m l - alanine and -ketoglutarate and glutamic pyruvic transaminase ( approximately 0.05 mg corresponding to three units ) in a 1 cm sample at ph 7.6 . in this case , the transamination reaction was triggered by addition of l - alanine into a solution already containing -ketoglutarate , glutamic pyruvic transaminase , and g3 ( 0.5 mol , all other concentrations and numbers of mol were the same as in the control experiment ) . for the starting solution , from which l - alanine was absent , the 3-ch2 and 4-ch2 triplet resonances are seen at 5.35 ppm and 3.50 ppm , respectively , while all other broad resonances belonging to the pamam protons and plp residues are shown in the full - range spectrum at 8.73 ( n = ch-7 ) and 7.50 ( 6-h ) . in the course of the reaction , the resonances from l - alanine disappear ( 2-ch ) or broaden slightly due to formation of a schiff base between l - alanine and plp , which in fact is the species actively involved in transamination . amine group transfer from pyridoxamine covalently incorporated into the core of pamam to phenylpyruvic and pyruvic acid,34,35 as well as racemization of alanine by pyr covalently incorporated into the core of polyethyleneimine dendrimers has been reported.36 in order to characterize the roles of the pamam carrier and the pamam - plp and pamam - pyr bioconjugates further , we performed additional control experiments . amine transfer from pamam to -ketoglutarate did not occur in the absence or presence of glutamic pyruvic transaminase when l - alanine was absent . g3 and g3 in the absence of glutamic pyruvic transaminase did not catalyze amine transfer from l - alanine to -ketoglutarate , demonstrating that the bioconjugates did not play a catalytic role . finally , transamination did not occur when g3 was added to the system containing l - alanine , -ketoglutarate , and glutamic pyruvic transaminase , indicating that g3 was not an activator of glutamic pyruvic transaminase . we measured the extinction at max for g3 and g3 conjugates at 355 nm and 275 nm , respectively , and took the full spectra in order to confirm the identity of the conjugates by comparing them with the original spectra of these species . generally , by introducing enhancement factors ( ef ) as the ratio of the diffusion rate of bioconjugates ( or host guest complexes g3-x , where x = ascorbic acid , riboflavin , or 8-methoxypsoralen ) to the diffusion rate of the free species x , thus , ef values of > 1 demonstrate the ability of the g3 pamam dendrimer to promote transdermal diffusion , while ef values < 1 indicate that the dendrimer retards diffusion ( table 1 ) . we have determined previously the rate of diffusion of g3 itself ( g3 labeled with one fluorescein substituent [ g3]),5 which gave us a reference value of 2.18% 0.23% h. for bioconjugates bearing four substituents of ret , pyr , or plp , the diffusion rate is lower , which can be attributed to the larger size of the bioconjugate than g3 itself ( 2.2 nm in diameter ) . the enhancement factor was 4 through pvdf and 3 through porcine ear skin for riboflavin5 and 3 for 8-methoxypsoralen as demonstrated in vitro using pvdf and porcine ear skin models , as well as in vivo studies using rat skin with confocal microscopy monitoring.37 stepwise addition of 13-cis - retinal or all - trans - retinal into g3 in methanol resulted in formation of g3 bioconjugates at variable substitution steps ( n = 116 ) , accompanied by marked changes in the ultraviolet - visible spectra ( figure 2 ) . both 13-cis - retinal and 13-trans - retinal were used to obtain g34 and g34 bioconjugates . the most characteristic resonance of the bioconjugates was that for the aldimine proton ( h-15 ) which was diagnostic for schiff base formation . the broad resonance at 8.2 ppm ( lower spectrum ) belongs to the internal n - h protons of the pamam dendrimer , which slowly undergoes substitution with the deuterium solvent and eventually disappears , and hence is not present in the upper spectrum for the bioconjugate mixture which was recorded after 12 hours of incubation of initial g34 in methanol - d4 . moreover , the flexibility induced by the polyene chain of retinal in the bacteriorhodopsin - retinal schiff base might be responsible for enabling the cis - trans transition and modifying the pka of the retinal - protonated schiff base in bacteriorhodopsin.31 stepwise addition of pyridoxal hydrochloride solution into g3 ( both in methanol ) resulted in formation of g3 bioconjugates , which were characterized by h nmr spectra . stepwise addition of plp in water into an aqueous solution of g3 enabled recording of the ultraviolet - visible spectra for g3 bioconjugates ( figure 4 ) , which differed considerably from that for plp itself , in that the * transition band centered at 295 nm for the plp aldehyde split into three bands centered at 340 nm , 275 nm , and 255 nm ( shoulder ) of almost equal intensity upon formation of the schiff base . in order to test the bioavailability of plp incorporated into a g3 bioconjugate , we used glutamic pyruvic transaminase from porcine heart tissue ( ec 2.6.1.2 ) and monitored amine transfer from the l - alanine donor to -ketoglutarate under h nmr spectral control in h2o . in the control experiment , amine transfer and formation of l - glutamate was triggered by addition of plp ( final concentration 0.002 m , ie , 2 mol in 1 ml of an nmr sample ) into the solution containing 0.02 m l - alanine and -ketoglutarate and glutamic pyruvic transaminase ( approximately 0.05 mg corresponding to three units ) in a 1 cm sample at ph 7.6 . in this case , the transamination reaction was triggered by addition of l - alanine into a solution already containing -ketoglutarate , glutamic pyruvic transaminase , and g3 ( 0.5 mol , all other concentrations and numbers of mol were the same as in the control experiment ) . in the course of the reaction , the resonances from l - alanine disappear ( 2-ch ) or broaden slightly due to formation of a schiff base between l - alanine and plp , which in fact is the species actively involved in transamination . again , 50% conversion of -ketoglutarate l - glutamate was achieved within approximately 45 minutes , while the methyl group resonance of l - alanine was no longer detected in the h nmr spectrum of the reaction mixture . amine group transfer from pyridoxamine covalently incorporated into the core of pamam to phenylpyruvic and pyruvic acid,34,35 as well as racemization of alanine by pyr covalently incorporated into the core of polyethyleneimine dendrimers has been reported.36 in order to characterize the roles of the pamam carrier and the pamam - plp and pamam - pyr bioconjugates further , we performed additional control experiments . amine transfer from pamam to -ketoglutarate did not occur in the absence or presence of glutamic pyruvic transaminase when l - alanine was absent . g3 and g3 in the absence of glutamic pyruvic transaminase did not catalyze amine transfer from l - alanine to -ketoglutarate , demonstrating that the bioconjugates did not play a catalytic role . finally , transamination did not occur when g3 was added to the system containing l - alanine , -ketoglutarate , and glutamic pyruvic transaminase , indicating that g3 was not an activator of glutamic pyruvic transaminase . the linear fit for these experimental profiles was applied in the region of stationary conditions , and the slopes of the fitted lines ( expressed in % h ) can be interpreted in terms of the diffusion ability of the conjugates . for comparison , control experiments were performed on release of trans - retinal and plp from the same emulsion ( figure 8) . generally , by introducing enhancement factors ( ef ) as the ratio of the diffusion rate of bioconjugates ( or host guest complexes g3-x , where x = ascorbic acid , riboflavin , or 8-methoxypsoralen ) to the diffusion rate of the free species x , the ability of the g3 pamam dendrimer to influence permeation can be estimated quantitatively . we have determined previously the rate of diffusion of g3 itself ( g3 labeled with one fluorescein substituent [ g3]),5 which gave us a reference value of 2.18% 0.23% h. for bioconjugates bearing four substituents of ret , pyr , or plp , the diffusion rate is lower , which can be attributed to the larger size of the bioconjugate than g3 itself ( 2.2 nm in diameter ) . the enhancement factor was 4 through pvdf and 3 through porcine ear skin for riboflavin5 and 3 for 8-methoxypsoralen as demonstrated in vitro using pvdf and porcine ear skin models , as well as in vivo studies using rat skin with confocal microscopy monitoring.37 vitamin b6 and vitamin a readily form conjugates with third - generation pamam dendrimers as schiff bases . plp is readily available as a cofactor for transaminase when released from a g3 bioconjugate , which was demonstrated in vitro by amine transfer from l - alanine to -ketoglutarate , as confirmed by h nmr spectroscopy . bioconjugates of plp and ret with a g3 pamam dendrimer diffuse through a pvdf skin model membrane slightly slower than g3 labeled with single fluorescein . in the case of bioconjugates with pyr and plp , the vitamins become available as transaminase cofactors , which is crucial when using these conjugates to feed skin . the h nmr spectra of reaction mixture containing pyridoxal phosphate ( 2 mm ) , l - alanine and -ketoglutarate ( both 20 mmm ) and transaminase ( 3 units ) in d2o , ph = 7.4 . the relevant fragments of h nmr spectra of reaction mixture containing g3 ( 2 mm ) , l - alanine and -ketoglutarate ( both 20 mmm ) and transaminase ( 3 units ) in d2o , ph = 7.4 , recorded after ( from bottom to top ) : 2 , 6 , 10 , 20 , and 45 minutes , respectively .

human rad52 belongs to a ubiquitous class of proteins that helps to overcome the thermodynamic barrier required to anneal complementary dna strands under biological conditions ( iyer et al . , 2002 , sugiman - marangos et al . , 2016 ) . in mammalian cells , rad52 is important for repair of dna double - strand breaks ( dsbs ) by the mutagenic rad51-independent single - strand annealing pathway ( ssa ) ( bhargava et al . , 2016 , morales et al . , 2015 , stark et al . , 2004 ) . however , unlike its yeast ortholog , vertebrate rad52 does not seem to be crucial for recombinational repair of dsbs via rad51 ( rijkers et al . the reason for this is that the rad52 function to facilitate the loading of rad51 on replication protein a ( rpa)-coated single - stranded dna ( ssdna ) appears to have been taken over by breast cancer susceptibility protein 2 ( brca2 ) ( see commentary by liu and heyer , 2011 and references therein ) . however , the finding that rad52 deficiencies are synthetically lethal with brca2 deficiencies suggests a functional redundancy between rad52 and brca2 ( feng et al . , 2011 , these reports , together with the recent discovery that rad52 is implicated in promoting dna synthesis after replication stress ( bhowmick et al . , 2016 , 2016 ) and in modulating antibody class - switch recombination ( zan et al . , 2017 ) , are fueling a regain of interest in studying the function of rad52 for both fundamental and therapeutic purposes ( hanamshet et al . , 2016 ) . rad52 forms ring - shaped structures in vitro ( shinohara et al . , 1998 ) . in solution , full - length rad52 forms stable heptameric rings with a large central channel , a structural organization reminiscent of hexameric dna helicases ( stasiak et al . , 2000 ) . however , unlike hexameric dna helicases , there is no evidence indicating that dna passes through the channel of the rad52 ring . in contrast , it has been proposed that ssdna wraps around the outer surface of the rad52 ring , interacting with an exposed positively charged groove ( singleton et al . , 2002 ) . annealing of complementary ssdna might then involve ssdna wrapping and dynamic interactions between multiple rad52 rings ( grimme et al . , we directly visualize and quantify the interaction of fluorescently labeled human rad52 with individual ssdna and double - stranded dna ( dsdna ) molecules using a single - molecule approach that combines optical trapping with microfluidics and fluorescence microscopy . we report intrinsic properties of rad52-dna interactions , including binding stoichiometry , diffusivity , and effect on dna mechanics , and discuss the implications of our findings on the biological roles of rad52 . after expression in bacteria , human rad52 tagged at the n terminus with egfp ( gfp - rad52 ) was purified ( figure s1a ) and tested in strand - annealing kinetic assays ( details of experimental procedures can be found in supplemental experimental procedures ) . we found no appreciable differences in activity compared to the untagged variant ( figures s1b and s1c ) , in agreement with previous studies where gfp - rad52 fully rescued the synthetic lethality of brca2 rad52 double - deficient cells ( feng et al . to study the interaction of gfp - rad52 with individual dna molecules , we used a combination of optical trapping , fluorescence microscopy , and microfluidics ( brouwer et al . , 2016 , heller et al . , 2014a , heller et al . , 2014b ) using two independent optical traps , individual dna molecules could be manipulated while simultaneously detecting the tension on the dna . dsdna molecules were biotinylated on the 3 ends of both top and bottom dna strands ( candelli et al . , 2013 ) and were tethered to optically trapped streptavidin - coated polystyrene microspheres ( figure s1d ) . single ssdna tethers ( figure s1e ) were generated by biotinylation of both the 5 and 3 ends of the top strand of a dsdna molecule and subsequent detachment of the bottom strand by force - induced melting ( candelli et al . , 2013 ) . after incubation of the dsdna and ssdna constructs in a gfp - rad52 solution , the dna tethers were brought into a buffer channel where dna - bound proteins could be visualized in the absence of fluid flow and fluorescent proteins in solution ( figures s1f and s1 g ) . to facilitate in situ generation of ssdna templates and dna - protein complexes , our approach included laminar flow microfluidics , which allows fast exchange between buffers containing microspheres , dna , protein - free , and protein solutions ( figures s1h and s1i ) . the affinity of rad52 for ssdna has been reported to be much higher than for dsdna ( van dyck et al . , 1998 ) . to quantitatively assess the affinity for ssdna , single ssdna molecules were ( unless otherwise indicated ) incubated at a tension of 10 pn in a buffer containing 1 nm of gfp - rad52 ; 20 mm tris - hcl ( ph 7.6 ) ; 100 mm kcl ; and either 1 mm ca , 1 mm mg , or no divalent cations . protein complexes formation on the dna was assessed from the fluorescence intensity ( under constant continuous excitation with 500 ms exposure time / frame ) immediately after transfer of the construct to a protein - free environment . we observed discrete fluorescent patches along the ssdna constructs , with each patch corresponding to an oligomeric dna - bound gfp - rad52 complex . in the presence of ca , loading of gfp - rad52 is remarkably fast : after 5 s incubation in 100 pm gfp - rad52 , significant amounts of fluorescent protein were bound to the ssdna ( figure 1a , left panel ) . under these conditions , the observed rate of patch formation was 35 3 oligomers s nm ( n = 43 ) . in the presence of mg , a much lower rate of ( 5 1)10 oligomers s nm ( n = 11 ) was observed and loading of similar amounts of protein on ssdna required a much longer incubation ( 100 s ) at 10-fold higher gfp - rad52 concentration ( figure 1a , middle panel ) . when no divalent cation was present , binding of gfp - rad52 was even less efficient and the patch formation rate was ( 1.2 0.5)10 oligomers s nm ( n = 25 ; figure 1a , right panel ) . interactions appeared independent of dna sequence ( figure s2a ) ; most patches detected on ssdna appeared static ( see analysis of protein diffusion below ) ; and within the ( limited ) observation time of our experiments ( about 2 min ) , we did not observe dissociation of the gfp - rad52 patches from the dna , which implies a dissociation rate smaller than 0.008 s. we thus show that gfp - rad52 interacts efficiently and statically with ssdna in a cation - dependent manner . next , we determined the stoichiometry of the dna - bound complexes by quantifying the fluorescence intensity of the complexes and normalizing to that of an individual gfp ( figure s3 ) . stoichiometry distributions were typically very broad , ranging from one to several tens of gfp - rad52 monomers per complex ( figure 1b ) . we found no evidence for the strict heptameric structure reported previously ( blue dotted lines in figure 1b ; stasiak et al . , 2000 ) . heptamers , the dominant species in solution , may thus rearrange into different oligomeric complexes when the protein interacts with ssdna . nevertheless , further experiments are needed to directly observe this putative rearrangement and assess to what extent the deviation from the 7n distribution expected for heptamers and multiples of heptamers is caused by quenching of the egfp fluorescence by homo - fret or because of a dark , non - fluorescent fraction in the gfp - rad52 preparations . in the presence of ca , the average patch size was significantly larger than in the presence of mg or without divalent cations ( figure 1c ) . applying tension to the ssdna substrate did not have a significant effect on the average number of patches ( figure s2b ) and their size ( figure s2c ) . to further address how rad52 interacts with ssdna , we performed an experiment where a ssdna construct was repeatedly incubated in the gfp - rad52 channel , each incubation lasting 2 s. the position of the fluorescent patches was recorded and photobleached afterward . subsequently , the recorded positions of the successive incubations were compared ( figure s2d ; representative example out of eight experiments ) . the probability of detecting a fluorescent patch at the same position during successive incubations is in the order of 14% 4% ( sem ) , indicating that the rates of initial patch formation and of patch growth are of the same order , unlike rad51 , which exhibits rate - limiting nucleation and a fast polymerization rate ( candelli et al . we also studied the impact of gfp - rad52 binding on the mechanical properties of ssdna . to this end , we incubated ssdna constructs in a buffer containing gfp - rad52 at very low tension , allowing different segments of the ssdna to interact with each other . next , the constructs were brought into a protein - free buffer channel , where force - extension and force - relaxation curves were measured . in these curves , two clear effects were observed . in the extension curves ( figure 1d ) , rupture events were observed , where a large , abrupt increase in dna extension was observed without an increase in force ( orange arrows ) . the average increase in contour length for a single rupture event was 0.35 0.02 m ( mean sem ; figure s2e ) . the relaxation curves , on the other hand , showed no such ruptures and appeared smooth , but the ssdna was significantly shorter than naked ssdna . the rupture events could be attributed to tension - induced rupture of protein - protein bridges , as can be observed in the corresponding fluorescence kymographs ( figure 1e ) or to the rupture of short stretches of dsdna that were formed through rad52-mediated annealing of partially complementary dna segments . in addition , the shortening of the gfp - rad52-ssdna construct with respect to naked ssdna can be attributed either to very strong ( and thus unbroken ) protein - protein bridges or to ssdna that is wrapped around the protein complexes . hence , this behavior is consistent with the proposal that ssdna wrapping and ring - ring contacts might be involved in rad52-promoted strand annealing ( grimme et al . , 2010 , rothenberg et al . , 2008 ) . although biochemical studies have mostly focused on the binding of rad52 to ssdna , there is also evidence for rad52 interacting with dsdna ( hengel et al . , 2016 , van dyck et al . , 1998 ) , despite the biological relevance of such interaction remaining subject of debate . to detect dsdna binding , we worked at 20 nm gfp - rad52 concentration ; as reference , the rad52 concentration in yeast is 1 nm ( ghaemmaghami et al . , 2003 ) . interestingly , rad52 has a secondary dna binding site that is important for dsdna binding , likely regulated by phosphorylation , and required to introduce positive supercoiling in dsdna upon rad52 binding ( honda et al . , 2011 , , we used our single - molecule approach to directly observe the binding of gfp - rad52 to dsdna . as for the interaction with ssdna , binding was dependent on the divalent cation present : gfp - rad52 binds to dsdna more readily in the presence of ca ( average patch formation rate [ 26 4]10 oligomers s nm [ n = 66 ] ) , than in the presence of mg ( [ 3.9 0.3]10 oligomers s nm [ n = 101 ] ) or in the absence of divalent cations ( [ 2.9 0.5]10 oligomers s nm [ n = 38 ] ; figure 2a ) . from these numbers , it is clear that , in the presence of divalent ions , the affinity for dsdna is one to three orders of magnitude lower compared to the affinity for ssdna . again , the interaction of gfp - rad52 with dsdna appeared not to depend on dna sequence ( figure s2a ) , and dissociation was slower than photobleaching , as found for the interaction with ssdna . unlike gfp - rad52 binding to ssdna , which predominantly involved static complexes , binding to dsdna involved both static and diffusive complexes ( figure 2a ) , depending on the cation and applied tension . also , when measured in ca and at 50 pn , the average patch size on dsdna did not depend on incubation time ( figure s4a ) , indicating that patch growth is not cooperative . stoichiometry distributions of complexes bound to dsdna were even broader than for ssdna ( figure 2b ) , and also for dsdna , we did not discern a clear 7n distribution reminiscent of heptameric complexes . again , a dependence of the stoichiometry distributions on the divalent cation present was observed , as well as a significant dependence on the tension applied to the construct ( figure 2c ) . at lower dna tensions , this observation is in contrast to the interaction with ssdna , which did not show dramatic tension dependence . most likely , this is due to structural changes of bare dsdna upon application of tension , causing disruption of base pairing resulting in force - induced melting or other structural states that are more ssdna - like where rad52 would preferentially bind with a stoichiometry reminiscent of that observed for ssdna ( king et al . , 2013 ) . for ssdna the size of these complexes depends on the presence of ca and mg . whereas the difference in the presence or absence of divalent cation can be explained by the shielding effect of their positive charges on the negatively charged phosphodiester backbone of dna , the difference between ca and mg can not be directly explained . nevertheless , it is interesting to note here that ca greatly favors association kinetics of gfp - rad52 with dna . in light of previous studies that showed an important role of ca in the control of homologous recombination in human by affecting the atpase activity of rad51 ( bugreev and mazin , 2004 , mazina and mazin , 2004 ) , our findings suggest that ca could have a much wider impact on dna recombination transactions in human cells not only by stimulating rad51-mediated strand exchange but also rad52-mediated strand annealing . next , we investigated the effect of gfp - rad52 binding on the mechanical properties of dsdna using dsdna constructs with a high coverage of gfp - rad52 . we observe smooth force - extension and relaxation curves ( figures 2d , s4b , and s4c ) that did not show evidence for protein - protein bridges or dna wrapping , in contrast to our observation for ssdna . individual force - extension curves show substantial variations ( figure s4b ) , likely due to dna - bound protein complexes sticking nonspecifically to the microspheres . relaxation curves , however , were reproducible and smooth ( figure s4c ) . for forces below 30 pn , these curves can be accurately described by the extensible - worm - like - chain ( ewlc ) model ( broekmans et al . , 2016 ; figure 2d ) . from the fit parameters in the presence and absence of gfp - rad52 ( table 1 ) , we deduce that gfp - rad52 binding results in a ( 9 1)-fold decrease of the persistence length and a ( 1.21 0.03)-fold increase in contour length , whereas the stretch modulus is not affected . these properties suggest a possible binding mechanism of rad52 to dsdna through intercalation and opening of the double helix , which might be crucial for atp - independent homology recognition and strand exchange by rad52 ( bi et al . , 2004 , reddy et al . , 1997 ) . in the buffer conditions used , at forces above 30 pn , extension - relaxation cycles of bare dsdna typically show a saw - tooth - like overstretching transition with a large hysteresis between the extension and relaxation curve ( figure s4d ) , signature of the force - induced melting of the dna strands ( gross et al . , 2011 ) . for dsdna coated with gfp - rad52 , the behavior is different : the curves remain smooth and the hysteresis between extension and relaxation curves is much smaller , indicating that force - induced melting of the dna strands no longer occurs . the formation of ssdna is thus prevented by gfp - rad52 , providing evidence for strand annealing and clamping activity for rad52 reminiscent of the activity proposed for the bacteriophage red ortholog , which is thought to clamp dna strands together to secure homology recognition ( ander et al . , 2015 ) . rad52 dna - strand clamping might be an important property during second - end capture , for holding together annealed dna repeats to allow processing of ssdna flaps and dna repair synthesis during the various types of homologous recombination after d - loop formation . moreover , as was discovered in yeast , rad52 could be part of a complex that tightly tethers the two ends of broken chromosomes , allowing them to withstand the pulling forces of the mitotic spindle ( lisby and rothstein , 2004 , lobachev et al . , 2004 ) . when examining the binding of gfp - rad52 to dsdna ( figure 2a ) , we observed that , depending on the buffer and the tension applied on the construct , a fraction of the fluorescent patches moved along the dna in a diffusive manner . to quantify this diffusive motion , we used custom - written tracking software to determine the trajectory of each individual fluorescent patch over time and used mean squared displacement ( msd ) analysis to determine the one - dimensional diffusion coefficient of each patch ( figures s5a s5d ) . then , we applied a threshold of 583 nm / s ( the minimal detectable diffusion coefficient in our experimental conditions ; see supplemental experimental procedures ) to determine whether the complex was static or diffusive . for each tension and buffer condition studied , we generated the distribution of above - threshold diffusion coefficients ( figure 3a ) and used two parameters to quantify the diffusive behavior : the average diffusion coefficient ( calculated based on only the diffusive particles ) and the diffusive fraction ( the fraction of particles that diffused ) . diffusion was most prominent at forces below 15 pn , where almost 100% of the particles were mobile . both the average diffusion coefficient ( figure 3b ) and the diffusive fraction ( figure 3c ) decreased with increasing tension . the transition between static and diffusive behavior is reversible : when the force is increased , particles switch from a diffusive to a static state , and when the force is decreased again , particles may switch back to the diffusive mode ( figure 3d ) . yet , no dependence of the diffusion coefficient on the nature of the divalent cation was observed ( figure 3e ) , and the diffusive fraction also did not change accordingly ( figure s5e ) . finally , our data show no strong correlation between the diffusion coefficient and the size of the fluorescent patch ( figure 3f ) , indicating that diffusion is not limited by the drag force acting on the protein complex . a quantitative analysis revealed that rad52 complexes also diffuse on ssdna , albeit with smaller diffusion coefficients and diffusive fractions than on dsdna and in a manner independent of the tension applied on the ssdna construct ( figures s5f and s5 g ) . in total , 25% 3% of the complexes bound to ssdna showed diffusive behavior and the average diffusion coefficient was roughly 50-fold smaller for particles diffusing on ssdna than on dsdna ( figure 3e ) . from these data , we conclude that gfp - rad52 can interact with dna in either static or diffusive binding modes . the diffusive binding mode is predominantly observed on dsdna and is slowed down when tension is applied on the dsdna , which favors immobilization of rad52 complexes by intercalation in the double helix ( figure 3 g , top panel ) . this diffusive binding mechanism suggests a role for rad52 in a diffusive search mechanism for localizing dna structural intermediates , such as ssdna - dsdna interfaces . given the involvement of rad52 in second - end capture during homologous recombination ( mcilwraith and west , 2008 , nimonkar et al . , 2009 , shi et al . , 2009 ) , we explored its ability to capture dna in trans . gfp - rad52 was first bound to dsdna or to ssdna , and the constructs were subsequently exposed to a solution of 60-mer ssdna oligonucleotides fluorescently end labeled with atto647n . gfp - rad52 bound to dsdna exhibits a remarkably efficient ability to capture the ssdna oligonucleotide in trans ( figure 4a ) . all out of 27 individual gfp - rad52 fluorescent patches ( green ) observed on six independent dsdna molecules held at 50 pn captured at least one ssdna oligo ( red ) . under the same conditions , no atto647n signal was detected on constructs that were not incubated with gfp - rad52 ( figure 4b ) . next , we performed similar experiments on ssdna constructs held at 5 pn ( figure 4c ) . again , analysis of six independent molecules pre - incubated with gfp - rad52 showed detection of atto647n signal co - localizing with the gfp - rad52 patches . control experiments with ssdna constructs not pre - incubated with gfp - rad52 revealed that the oligonucleotides bind to naked ssdna to a much lesser extent than in the presence of gfp - rad52 ( figure 4d ) . we conclude that dna - bound gfp - rad52 is efficient at capturing ssdna from solution , reminiscent of its role in second - end capture . we have provided a quantitative assessment of the interaction of human rad52 with dna , suggesting properties important for its physiological roles as summarized in figure 3 g . while interacting tightly with ssdna through a combination of wrapping and bridging , rad52 complexes bound to dsdna profoundly affect dsdna mechanics and the substantial decrease in persistence length and slight increase in contour length observed upon rad52 binding indicate that rad52 binding increases dsdna flexibility probably by destabilizing and intercalating into duplex dna . our findings suggest that the way by which rad52 promotes strand exchange in vitro is not by a strand - invasion mechanism like the rad51 or reca nucleoprotein filament but rather results from the ability of rad52 to change dsdna structure , intercalating in the helix to make the bases available for pairing . consistent with our model , it was previously observed that increasing the fractional at content of dna increases the yields of in vitro strand exchange reactions by rad52 , likely because it would be easier for rad52 to intercalate in at regions ( bi et al . , 2004 , also , the overstretching behavior of dsdna is profoundly altered in the presence of rad52 , suggesting that rad52 prevents force - induced melting and thus providing evidence for strand - clamping activity . the methodology and findings reported here can now be used in future experiments to extend this analysis by studying how rad52 interacts with rpa , a pivotal ssdna - binding protein . indeed , rad52 will need to deal with rpa - coated ssdna in the physiological context , and its direct physical and functional interaction with rpa appears to be essential for homologous recombination in yeast and mammalian cells , especially when long ssdna substrates need to be processed ( jackson et al . , 2002 , park et al . , 1996 , sugiyama et al . , 1998 ) . further , the activities of brca2 could be similarly analyzed and directly compared to the ones of rad52 to explore possible functional redundancies between the two proteins suggested by studies of the ustilago maydis orthologs ( mazloum et al . , 2007 ) . expanded methods and details about proteins and dna substrates and annealing and ssdna oligo capture assays are provided in supplemental experimental procedures . single - molecule experiments have been executed using a custom - build instrument ( gross et al . , 2010 ) integrating optical trapping , wide - field fluorescence microscopy , and microfluidics . dna melting for generation of ssdna templates was performed in 20 mm tris - hcl ( ph 7.6 ) . protein , imaging , and ssdna oligo buffer consisted of 20 mm tris - hcl ( ph 7.6 ) ; 100 mm kcl ; and either 1 mm mgcl2 , 1 mm cacl2 , or no divalent cations . the stoichiometry of the dna - bound gfp - rad52 complexes was inferred from the number of gfp molecules in each fluorescent patch , calculated dividing the initial fluorescence intensity of the patch by the average intensity of a single gfp . we used a step - fitting algorithm ( kerssemakers et al . , 2006 ) to extract the intensity of single gfps from photo - bleaching traces ( figure s3a ) of individual patches . the average gfp intensity was obtained from a lorentzian fit to the histogram of step intensities ( figure s3b ) . diffusion was analyzed tracking gfp - rad52 complexes for a large number of frames ( on average 29 2 s ) and quantified using 1d msd analysis ( heller et al . , 2014b ) . diffusion coefficients were calculated by linear fit to the first five points of the msd curves ( figure s5d ) . data are presented as mean sem , and histograms show statistical errors in the number of counts .

summaryhuman rad52 promotes annealing of complementary single - stranded dna ( ssdna ) . in - depth knowledge of rad52-dna interaction is required to understand how its activity is integrated in dna repair processes . here , we visualize individual fluorescent rad52 complexes interacting with single dna molecules . the interaction with ssdna is rapid , static , and tight , where ssdna appears to wrap around rad52 complexes that promote intra - molecular bridging . with double - stranded dna ( dsdna ) , interaction is slower , weaker , and often diffusive . interestingly , force spectroscopy experiments show that rad52 alters the mechanics dsdna by enhancing dna flexibility and increasing dna contour length , suggesting intercalation . rad52 binding changes the nature of the overstretching transition of dsdna and prevents dna melting , which is advantageous for strand clamping during or after annealing . dna - bound rad52 is efficient at capturing ssdna in trans . together , these effects may help key steps in dna repair , such as second - end capture during homologous recombination or strand annealing during rad51-independent recombination reactions .

Introduction Results and Discussion Experimental Procedures Author Contributions

human rad52 belongs to a ubiquitous class of proteins that helps to overcome the thermodynamic barrier required to anneal complementary dna strands under biological conditions ( iyer et al . in mammalian cells , rad52 is important for repair of dna double - strand breaks ( dsbs ) by the mutagenic rad51-independent single - strand annealing pathway ( ssa ) ( bhargava et al . the reason for this is that the rad52 function to facilitate the loading of rad51 on replication protein a ( rpa)-coated single - stranded dna ( ssdna ) appears to have been taken over by breast cancer susceptibility protein 2 ( brca2 ) ( see commentary by liu and heyer , 2011 and references therein ) . however , the finding that rad52 deficiencies are synthetically lethal with brca2 deficiencies suggests a functional redundancy between rad52 and brca2 ( feng et al . , 2011 , these reports , together with the recent discovery that rad52 is implicated in promoting dna synthesis after replication stress ( bhowmick et al . , 2017 ) , are fueling a regain of interest in studying the function of rad52 for both fundamental and therapeutic purposes ( hanamshet et al . however , unlike hexameric dna helicases , there is no evidence indicating that dna passes through the channel of the rad52 ring . in contrast , it has been proposed that ssdna wraps around the outer surface of the rad52 ring , interacting with an exposed positively charged groove ( singleton et al . annealing of complementary ssdna might then involve ssdna wrapping and dynamic interactions between multiple rad52 rings ( grimme et al . , we directly visualize and quantify the interaction of fluorescently labeled human rad52 with individual ssdna and double - stranded dna ( dsdna ) molecules using a single - molecule approach that combines optical trapping with microfluidics and fluorescence microscopy . we report intrinsic properties of rad52-dna interactions , including binding stoichiometry , diffusivity , and effect on dna mechanics , and discuss the implications of our findings on the biological roles of rad52 . we found no appreciable differences in activity compared to the untagged variant ( figures s1b and s1c ) , in agreement with previous studies where gfp - rad52 fully rescued the synthetic lethality of brca2 rad52 double - deficient cells ( feng et al . to study the interaction of gfp - rad52 with individual dna molecules , we used a combination of optical trapping , fluorescence microscopy , and microfluidics ( brouwer et al . , 2014b ) using two independent optical traps , individual dna molecules could be manipulated while simultaneously detecting the tension on the dna . after incubation of the dsdna and ssdna constructs in a gfp - rad52 solution , the dna tethers were brought into a buffer channel where dna - bound proteins could be visualized in the absence of fluid flow and fluorescent proteins in solution ( figures s1f and s1 g ) . to facilitate in situ generation of ssdna templates and dna - protein complexes , our approach included laminar flow microfluidics , which allows fast exchange between buffers containing microspheres , dna , protein - free , and protein solutions ( figures s1h and s1i ) . protein complexes formation on the dna was assessed from the fluorescence intensity ( under constant continuous excitation with 500 ms exposure time / frame ) immediately after transfer of the construct to a protein - free environment . we observed discrete fluorescent patches along the ssdna constructs , with each patch corresponding to an oligomeric dna - bound gfp - rad52 complex . in the presence of ca , loading of gfp - rad52 is remarkably fast : after 5 s incubation in 100 pm gfp - rad52 , significant amounts of fluorescent protein were bound to the ssdna ( figure 1a , left panel ) . interactions appeared independent of dna sequence ( figure s2a ) ; most patches detected on ssdna appeared static ( see analysis of protein diffusion below ) ; and within the ( limited ) observation time of our experiments ( about 2 min ) , we did not observe dissociation of the gfp - rad52 patches from the dna , which implies a dissociation rate smaller than 0.008 s. we thus show that gfp - rad52 interacts efficiently and statically with ssdna in a cation - dependent manner . next , we determined the stoichiometry of the dna - bound complexes by quantifying the fluorescence intensity of the complexes and normalizing to that of an individual gfp ( figure s3 ) . heptamers , the dominant species in solution , may thus rearrange into different oligomeric complexes when the protein interacts with ssdna . nevertheless , further experiments are needed to directly observe this putative rearrangement and assess to what extent the deviation from the 7n distribution expected for heptamers and multiples of heptamers is caused by quenching of the egfp fluorescence by homo - fret or because of a dark , non - fluorescent fraction in the gfp - rad52 preparations . to further address how rad52 interacts with ssdna , we performed an experiment where a ssdna construct was repeatedly incubated in the gfp - rad52 channel , each incubation lasting 2 s. the position of the fluorescent patches was recorded and photobleached afterward . subsequently , the recorded positions of the successive incubations were compared ( figure s2d ; representative example out of eight experiments ) . the probability of detecting a fluorescent patch at the same position during successive incubations is in the order of 14% 4% ( sem ) , indicating that the rates of initial patch formation and of patch growth are of the same order , unlike rad51 , which exhibits rate - limiting nucleation and a fast polymerization rate ( candelli et al . we also studied the impact of gfp - rad52 binding on the mechanical properties of ssdna . to this end , we incubated ssdna constructs in a buffer containing gfp - rad52 at very low tension , allowing different segments of the ssdna to interact with each other . next , the constructs were brought into a protein - free buffer channel , where force - extension and force - relaxation curves were measured . in the extension curves ( figure 1d ) , rupture events were observed , where a large , abrupt increase in dna extension was observed without an increase in force ( orange arrows ) . the average increase in contour length for a single rupture event was 0.35 0.02 m ( mean sem ; figure s2e ) . the rupture events could be attributed to tension - induced rupture of protein - protein bridges , as can be observed in the corresponding fluorescence kymographs ( figure 1e ) or to the rupture of short stretches of dsdna that were formed through rad52-mediated annealing of partially complementary dna segments . in addition , the shortening of the gfp - rad52-ssdna construct with respect to naked ssdna can be attributed either to very strong ( and thus unbroken ) protein - protein bridges or to ssdna that is wrapped around the protein complexes . hence , this behavior is consistent with the proposal that ssdna wrapping and ring - ring contacts might be involved in rad52-promoted strand annealing ( grimme et al . although biochemical studies have mostly focused on the binding of rad52 to ssdna , there is also evidence for rad52 interacting with dsdna ( hengel et al . , 1998 ) , despite the biological relevance of such interaction remaining subject of debate . to detect dsdna binding , we worked at 20 nm gfp - rad52 concentration ; as reference , the rad52 concentration in yeast is 1 nm ( ghaemmaghami et al . interestingly , rad52 has a secondary dna binding site that is important for dsdna binding , likely regulated by phosphorylation , and required to introduce positive supercoiling in dsdna upon rad52 binding ( honda et al . , 2011 , , we used our single - molecule approach to directly observe the binding of gfp - rad52 to dsdna . as for the interaction with ssdna , binding was dependent on the divalent cation present : gfp - rad52 binds to dsdna more readily in the presence of ca ( average patch formation rate [ 26 4]10 oligomers s nm [ n = 66 ] ) , than in the presence of mg ( [ 3.9 0.3]10 oligomers s nm [ n = 101 ] ) or in the absence of divalent cations ( [ 2.9 0.5]10 oligomers s nm [ n = 38 ] ; figure 2a ) . again , the interaction of gfp - rad52 with dsdna appeared not to depend on dna sequence ( figure s2a ) , and dissociation was slower than photobleaching , as found for the interaction with ssdna . unlike gfp - rad52 binding to ssdna , which predominantly involved static complexes , binding to dsdna involved both static and diffusive complexes ( figure 2a ) , depending on the cation and applied tension . also , when measured in ca and at 50 pn , the average patch size on dsdna did not depend on incubation time ( figure s4a ) , indicating that patch growth is not cooperative . stoichiometry distributions of complexes bound to dsdna were even broader than for ssdna ( figure 2b ) , and also for dsdna , we did not discern a clear 7n distribution reminiscent of heptameric complexes . again , a dependence of the stoichiometry distributions on the divalent cation present was observed , as well as a significant dependence on the tension applied to the construct ( figure 2c ) . at lower dna tensions , this observation is in contrast to the interaction with ssdna , which did not show dramatic tension dependence . in light of previous studies that showed an important role of ca in the control of homologous recombination in human by affecting the atpase activity of rad51 ( bugreev and mazin , 2004 , mazina and mazin , 2004 ) , our findings suggest that ca could have a much wider impact on dna recombination transactions in human cells not only by stimulating rad51-mediated strand exchange but also rad52-mediated strand annealing . next , we investigated the effect of gfp - rad52 binding on the mechanical properties of dsdna using dsdna constructs with a high coverage of gfp - rad52 . individual force - extension curves show substantial variations ( figure s4b ) , likely due to dna - bound protein complexes sticking nonspecifically to the microspheres . for forces below 30 pn , these curves can be accurately described by the extensible - worm - like - chain ( ewlc ) model ( broekmans et al . from the fit parameters in the presence and absence of gfp - rad52 ( table 1 ) , we deduce that gfp - rad52 binding results in a ( 9 1)-fold decrease of the persistence length and a ( 1.21 0.03)-fold increase in contour length , whereas the stretch modulus is not affected . these properties suggest a possible binding mechanism of rad52 to dsdna through intercalation and opening of the double helix , which might be crucial for atp - independent homology recognition and strand exchange by rad52 ( bi et al . in the buffer conditions used , at forces above 30 pn , extension - relaxation cycles of bare dsdna typically show a saw - tooth - like overstretching transition with a large hysteresis between the extension and relaxation curve ( figure s4d ) , signature of the force - induced melting of the dna strands ( gross et al . for dsdna coated with gfp - rad52 , the behavior is different : the curves remain smooth and the hysteresis between extension and relaxation curves is much smaller , indicating that force - induced melting of the dna strands no longer occurs . the formation of ssdna is thus prevented by gfp - rad52 , providing evidence for strand annealing and clamping activity for rad52 reminiscent of the activity proposed for the bacteriophage red ortholog , which is thought to clamp dna strands together to secure homology recognition ( ander et al . rad52 dna - strand clamping might be an important property during second - end capture , for holding together annealed dna repeats to allow processing of ssdna flaps and dna repair synthesis during the various types of homologous recombination after d - loop formation . moreover , as was discovered in yeast , rad52 could be part of a complex that tightly tethers the two ends of broken chromosomes , allowing them to withstand the pulling forces of the mitotic spindle ( lisby and rothstein , 2004 , lobachev et al . when examining the binding of gfp - rad52 to dsdna ( figure 2a ) , we observed that , depending on the buffer and the tension applied on the construct , a fraction of the fluorescent patches moved along the dna in a diffusive manner . to quantify this diffusive motion , we used custom - written tracking software to determine the trajectory of each individual fluorescent patch over time and used mean squared displacement ( msd ) analysis to determine the one - dimensional diffusion coefficient of each patch ( figures s5a s5d ) . then , we applied a threshold of 583 nm / s ( the minimal detectable diffusion coefficient in our experimental conditions ; see supplemental experimental procedures ) to determine whether the complex was static or diffusive . for each tension and buffer condition studied , we generated the distribution of above - threshold diffusion coefficients ( figure 3a ) and used two parameters to quantify the diffusive behavior : the average diffusion coefficient ( calculated based on only the diffusive particles ) and the diffusive fraction ( the fraction of particles that diffused ) . diffusion was most prominent at forces below 15 pn , where almost 100% of the particles were mobile . the transition between static and diffusive behavior is reversible : when the force is increased , particles switch from a diffusive to a static state , and when the force is decreased again , particles may switch back to the diffusive mode ( figure 3d ) . yet , no dependence of the diffusion coefficient on the nature of the divalent cation was observed ( figure 3e ) , and the diffusive fraction also did not change accordingly ( figure s5e ) . finally , our data show no strong correlation between the diffusion coefficient and the size of the fluorescent patch ( figure 3f ) , indicating that diffusion is not limited by the drag force acting on the protein complex . a quantitative analysis revealed that rad52 complexes also diffuse on ssdna , albeit with smaller diffusion coefficients and diffusive fractions than on dsdna and in a manner independent of the tension applied on the ssdna construct ( figures s5f and s5 g ) . in total , 25% 3% of the complexes bound to ssdna showed diffusive behavior and the average diffusion coefficient was roughly 50-fold smaller for particles diffusing on ssdna than on dsdna ( figure 3e ) . from these data , we conclude that gfp - rad52 can interact with dna in either static or diffusive binding modes . the diffusive binding mode is predominantly observed on dsdna and is slowed down when tension is applied on the dsdna , which favors immobilization of rad52 complexes by intercalation in the double helix ( figure 3 g , top panel ) . this diffusive binding mechanism suggests a role for rad52 in a diffusive search mechanism for localizing dna structural intermediates , such as ssdna - dsdna interfaces . given the involvement of rad52 in second - end capture during homologous recombination ( mcilwraith and west , 2008 , nimonkar et al . , 2009 ) , we explored its ability to capture dna in trans . gfp - rad52 was first bound to dsdna or to ssdna , and the constructs were subsequently exposed to a solution of 60-mer ssdna oligonucleotides fluorescently end labeled with atto647n . gfp - rad52 bound to dsdna exhibits a remarkably efficient ability to capture the ssdna oligonucleotide in trans ( figure 4a ) . next , we performed similar experiments on ssdna constructs held at 5 pn ( figure 4c ) . we conclude that dna - bound gfp - rad52 is efficient at capturing ssdna from solution , reminiscent of its role in second - end capture . we have provided a quantitative assessment of the interaction of human rad52 with dna , suggesting properties important for its physiological roles as summarized in figure 3 g . while interacting tightly with ssdna through a combination of wrapping and bridging , rad52 complexes bound to dsdna profoundly affect dsdna mechanics and the substantial decrease in persistence length and slight increase in contour length observed upon rad52 binding indicate that rad52 binding increases dsdna flexibility probably by destabilizing and intercalating into duplex dna . our findings suggest that the way by which rad52 promotes strand exchange in vitro is not by a strand - invasion mechanism like the rad51 or reca nucleoprotein filament but rather results from the ability of rad52 to change dsdna structure , intercalating in the helix to make the bases available for pairing . , 2004 , also , the overstretching behavior of dsdna is profoundly altered in the presence of rad52 , suggesting that rad52 prevents force - induced melting and thus providing evidence for strand - clamping activity . indeed , rad52 will need to deal with rpa - coated ssdna in the physiological context , and its direct physical and functional interaction with rpa appears to be essential for homologous recombination in yeast and mammalian cells , especially when long ssdna substrates need to be processed ( jackson et al . further , the activities of brca2 could be similarly analyzed and directly compared to the ones of rad52 to explore possible functional redundancies between the two proteins suggested by studies of the ustilago maydis orthologs ( mazloum et al . single - molecule experiments have been executed using a custom - build instrument ( gross et al . , 2010 ) integrating optical trapping , wide - field fluorescence microscopy , and microfluidics . dna melting for generation of ssdna templates was performed in 20 mm tris - hcl ( ph 7.6 ) . protein , imaging , and ssdna oligo buffer consisted of 20 mm tris - hcl ( ph 7.6 ) ; 100 mm kcl ; and either 1 mm mgcl2 , 1 mm cacl2 , or no divalent cations . the stoichiometry of the dna - bound gfp - rad52 complexes was inferred from the number of gfp molecules in each fluorescent patch , calculated dividing the initial fluorescence intensity of the patch by the average intensity of a single gfp . diffusion was analyzed tracking gfp - rad52 complexes for a large number of frames ( on average 29 2 s ) and quantified using 1d msd analysis ( heller et al . diffusion coefficients were calculated by linear fit to the first five points of the msd curves ( figure s5d ) .

death by suicide is a leading cause of death,1 and initiatives to advance suicide prevention programmes are a high priority in many countries.2 harmful alcohol use has been recognised as a strong risk factor for suicide death.2 in psychological autopsy studies , alcohol use disorder was strongly associated with suicide death ( or=5.2 , 95% ci 3.3 to 8.3),3 and heavy alcohol use was also strongly linked with suicide death ( or=6.2 , 95% ci 5.6 to 6.9 in men).4 in a recent systematic review of the prospective studies , however , the magnitude of the association between alcohol use disorder and suicide death was reported to be moderate ( pooled risk ratio=1.74 , 95% ci 1.26 to 2.21 ) . the strong associations reported in psychological autopsy studies and case control studies may have resulted , at least in part , from several biases such as surrogate interview bias and recall bias . furthermore , when usual alcohol consumption rather than alcohol use disorder is considered in the general population , evidence of the impact of usual alcohol consumption on suicide death is less than convincing . control studies based on a prospective cohort , have indicated that alcohol consumption was not associated with a higher risk of suicide death , compared to no alcohol consumption , even in the group with the highest consumption in each study,511 with a few exceptions.12 13 overall , the association between usual alcohol consumption and suicide death is unclear . since the majority of adults drink alcohol at some point in their life , a better understanding of usual alcohol consumption and suicide death should inform decision - making for better suicide prevention strategies in the general population . the purpose of this study was to prospectively examine the association of usual alcohol intake - related variables including alcohol intake frequency , average alcohol consumption and quantity of alcohol consumed on a drinking day with suicide death in a community dwelling elderly population in korea . we tried to elucidate questions such as whether greater usual alcohol consumption is associated with a greater risk of suicide death ( dose response relationship ) , and whether moderate alcohol consumption ( below 5 drinks / week , or 4 or fewer drinking days a month ) has a protective effect against suicide death compared to abstaining completely . the kangwha cohort study was established in march 1985.14 15 among 9378 residents of kangwha county in korea who were 55 years or older as of february 1985 , 6372 persons ( 67.9% ) participated in the survey . participants who were lost to follow - up after the initial survey ( n=39 ) were excluded , as were those with missing information about drinking status ( n=2 ) , their body mass index ( bmi ; n=143 ) , or other covariates ( n=25 ) . in the end , a total of 6163 elderly ( 2635 men ; 3528 women ) were included as a study population . the institutional review board of yonsei university ( approval no . 4 - 2007 - 0182 ) no to the question , do you drink alcohol? the drinking frequency was reported as daily , almost daily , 23 times a week , 14 times a month or 412 times a year . the question concerning the type of alcoholic beverage and the amount of alcohol consumption was , how much ( in doe ( 1800 ml ) , hop ( 180 ml ) , bottles or glasses ) do you drink of a type of alcoholic beverage? alcohol consumption ( alcohol in grams ) per week was estimated by multiplying the amount of alcohol consumption per drinking day ( ml ) by the alcohol content in the particular type of alcoholic beverage , the drinking days per week , and the specific gravity of alcohol ( 0.8 ) . a different score for drinking days per week was assigned to each drinking frequency ( daily : 7.0 ; almost daily : 5.5 ; 23 times a week : 2.5 ; 14 times a month : 0.625 ; 412 times a year : 0.163 ; non - drinking : 0 ) . participants were asked to fill in up to two types of alcoholic beverage they usually consume on one drinking day . the amount of alcohol consumption was calculated for each type and summed . among drinkers , the alcohol content and the bottle volume of each type of alcoholic beverage were determined on the basis of data from the year 1985 . soju is a distilled alcoholic beverage , a type of liquor , and makkoli is an unfiltered alcoholic beverage made from rice , both native to korea . soju had 25% alcohol and makkoli 6% alcohol at the time of the survey for the kangwha cohort study in 1985 . the amount of ethanol was 10 g in a glass of soju and 9.6 g in a glass of makkoli . the alcohol frequency was merged into four groups in further analysis : daily , 26 days a week ( almost daily and 23 times a week combined ) , 4 or fewer days a month ( 14 times a month and 412 times a year combined ) , and non - drinkers . the alcohol intake amount ( grams of alcohol ) per week was classified into four groups ( grams of alcohol per week ( drinks per week , with 1 drink as 14 g of alcohol16 ; non - drinkers , < 70 ( < 5 drinks ) ; 70209 ( 514 ) , 210419 ( 1529 ) , and 420 ( 30 ) ) . the amount consumed ( grams of alcohol ) per drinking day was classified into four groups ( grams of alcohol per drinking day ( drinks per occasion ) ; non - drinkers , < 70 ( < 5 drinks ) ; 70139 ( 59 ) , and 140 ( 10 ) ) . one standard drink was considered to be around 14 g.16 the alcohol intake frequency ( number of days ) per week , alcohol intake volume per week , and alcohol intake amount per drinking day were also analysed as continuous variables . trained investigators interviewed each participant using a structured questionnaire for sociodemographic factors and health - related factors including alcohol intake - related variables , smoking status and marital status . bmi was calculated as the weight in kilograms divided by the square of the height in metres ( kg / m ) . more details on the data collection have been described elsewhere.14 15 data on deaths from suicide from 1 january 1992 to 31 december 2008 were confirmed by national death records from the national statistical office of korea . data on those who died from march 1985 to 31 december 1991 were collected either through calls or visits of trained surveyors twice a year , or from records of burial and death certificates at local administrative branch offices in each study region . follow - up was performed through record linkage at the national level and was complete , except for the case of emigrants . the test and analysis of variance were performed to compare individual characteristics by drinking status . cox proportional hazards models were used to calculate hrs associated with alcohol intake - related variables . the covariables included in the cox model were age at enrolment ( continuous variable ) , hypertension ( hypertension , no hypertension ; an indicator for cardiovascular health6 ) , smoking ( never smoker , past smoker , or current smoker ; participants were asked to answer i quit smoking to the question , do you smoke cigarettes? ) , bmi ( kg / m ; < 18.5 , 18.524.9 ( reference ) , or 25.0 ) , self - rated health ( good or fair , or poor ) , marital status ( living with a spouse , not living with a spouse ) , education ( ever , never ) and occupation ( non - agriculture , agriculture ) . since the number of suicide deaths is small , there is a possibility of overfitting when all those potential confounders are included in the cox model ; thus , additional analysis was performed , including only those variables with the model p value < 0.2 for the wald test in the univariate cox analysis : sex , smoking status , self - rated health compared to the same age group , marital status and occupation . additionally , analysis according to the follow - up period ( 19851996 vs 19972008 ) was performed to examine whether alcohol - related suicide death may differ by period after alcohol intake assessment . hypertension , marital status and a dummy variable for bmi ( 25 kg / m ) showed some evidence of a violation of the assumption ( p<0.1 ) . therefore , additional analyses after exclusion of these three variables , or exclusion of the hypertension and bmi variables after stratification of marital status ( since marital status ( p=0.013 ) , was associated with suicide death in univariate cox analysis , while bmi ( p=0.51 ) and hypertension ( p=0.36 ) were not ) were performed . subgroup analysis , analysis with different alcohol intake variables , and additional analysis with different covariables served as a sensitivity analysis . all statistical analyses were performed using sas v.9.4 ( sas inc , cary , north carolina , usa ) . the p value was calculated with two - sided tests , and a statistical significance level of 0.05 was applied . the kangwha cohort study was established in march 1985.14 15 among 9378 residents of kangwha county in korea who were 55 years or older as of february 1985 , 6372 persons ( 67.9% ) participated in the survey . participants who were lost to follow - up after the initial survey ( n=39 ) were excluded , as were those with missing information about drinking status ( n=2 ) , their body mass index ( bmi ; n=143 ) , or other covariates ( n=25 ) . in the end , a total of 6163 elderly ( 2635 men ; 3528 women ) were included as a study population . the institutional review board of yonsei university ( approval no . 4 - 2007 - 0182 ) do you drink alcohol? the drinking frequency was reported as daily , almost daily , 23 times a week , 14 times a month or 412 times a year . the question concerning the type of alcoholic beverage and the amount of alcohol consumption was , how much ( in doe ( 1800 ml ) , hop ( 180 ml ) , bottles or glasses ) do you drink of a type of alcoholic beverage? alcohol consumption ( alcohol in grams ) per week was estimated by multiplying the amount of alcohol consumption per drinking day ( ml ) by the alcohol content in the particular type of alcoholic beverage , the drinking days per week , and the specific gravity of alcohol ( 0.8 ) . a different score for drinking days per week was assigned to each drinking frequency ( daily : 7.0 ; almost daily : 5.5 ; 23 times a week : 2.5 ; 14 times a month : 0.625 ; 412 times a year : 0.163 ; non - drinking : 0 ) . participants were asked to fill in up to two types of alcoholic beverage they usually consume on one drinking day . the amount of alcohol consumption was calculated for each type and summed . among drinkers , the alcohol content and the bottle volume of each type of alcoholic beverage were determined on the basis of data from the year 1985 . soju is a distilled alcoholic beverage , a type of liquor , and makkoli is an unfiltered alcoholic beverage made from rice , both native to korea . soju had 25% alcohol and makkoli 6% alcohol at the time of the survey for the kangwha cohort study in 1985 . the amount of ethanol was 10 g in a glass of soju and 9.6 g in a glass of makkoli . the alcohol frequency was merged into four groups in further analysis : daily , 26 days a week ( almost daily and 23 times a week combined ) , 4 or fewer days a month ( 14 times a month and 412 times a year combined ) , and non - drinkers . the alcohol intake amount ( grams of alcohol ) per week was classified into four groups ( grams of alcohol per week ( drinks per week , with 1 drink as 14 g of alcohol16 ; non - drinkers , < 70 ( < 5 drinks ) ; 70209 ( 514 ) , 210419 ( 1529 ) , and 420 ( 30 ) ) . the amount consumed ( grams of alcohol ) per drinking day was classified into four groups ( grams of alcohol per drinking day ( drinks per occasion ) ; non - drinkers , < 70 ( < 5 drinks ) ; 70139 ( 59 ) , and 140 ( 10 ) ) . one standard drink was considered to be around 14 g.16 the alcohol intake frequency ( number of days ) per week , alcohol intake volume per week , and alcohol intake amount per drinking day were also analysed as continuous variables . trained investigators interviewed each participant using a structured questionnaire for sociodemographic factors and health - related factors including alcohol intake - related variables , smoking status and marital status . bmi was calculated as the weight in kilograms divided by the square of the height in metres ( kg / m ) . data on deaths from suicide from 1 january 1992 to 31 december 2008 were confirmed by national death records from the national statistical office of korea . data on those who died from march 1985 to 31 december 1991 were collected either through calls or visits of trained surveyors twice a year , or from records of burial and death certificates at local administrative branch offices in each study region . follow - up was performed through record linkage at the national level and was complete , except for the case of emigrants . the test and analysis of variance were performed to compare individual characteristics by drinking status . cox proportional hazards models were used to calculate hrs associated with alcohol intake - related variables . the covariables included in the cox model were age at enrolment ( continuous variable ) , hypertension ( hypertension , no hypertension ; an indicator for cardiovascular health6 ) , smoking ( never smoker , past smoker , or current smoker ; participants were asked to answer i quit smoking to the question , do you smoke cigarettes? ) , bmi ( kg / m ; < 18.5 , 18.524.9 ( reference ) , or 25.0 ) , self - rated health ( good or fair , or poor ) , marital status ( living with a spouse , not living with a spouse ) , education ( ever , never ) and occupation ( non - agriculture , agriculture ) . since the number of suicide deaths is small , there is a possibility of overfitting when all those potential confounders are included in the cox model ; thus , additional analysis was performed , including only those variables with the model p value < 0.2 for the wald test in the univariate cox analysis : sex , smoking status , self - rated health compared to the same age group , marital status and occupation . additionally , analysis according to the follow - up period ( 19851996 vs 19972008 ) was performed to examine whether alcohol - related suicide death may differ by period after alcohol intake assessment . hypertension , marital status and a dummy variable for bmi ( 25 kg / m ) showed some evidence of a violation of the assumption ( p<0.1 ) . therefore , additional analyses after exclusion of these three variables , or exclusion of the hypertension and bmi variables after stratification of marital status ( since marital status ( p=0.013 ) , was associated with suicide death in univariate cox analysis , while bmi ( p=0.51 ) and hypertension ( p=0.36 ) were not ) were performed . subgroup analysis , analysis with different alcohol intake variables , and additional analysis with different covariables served as a sensitivity analysis . all statistical analyses were performed using sas v.9.4 ( sas inc , cary , north carolina , usa ) . the p value was calculated with two - sided tests , and a statistical significance level of 0.05 was applied . during 88 376 person - years of follow - up ( mean : 14.3 person - years ) , 37 men and 24 women died by suicide . the average ( sd ) age at baseline of the korean elderly was 66.6 ( 7.9 ) years . current drinkers were slightly younger than non - drinkers , and they tended to be men , current smokers , healthy , living with a spouse and have more formal education , compared with non - drinkers ( table 1 ) . baseline characteristics of the korean elderly according to alcohol intake status one - way analysis of variance . measured blood pressure 140/90 mm hg or on regular medication . no formal education , not even elementary school . 1 standard drink considered to be approximately 14 g according to the national institute on alcohol abuse and alcoholism of the usa . , current drinkers had a higher hr of suicide than non - drinkers and higher frequency and amount of alcohol intake were associated with a higher hr of suicide ( table 2 ) . two or more days of alcohol intake per week , 70 g ( 5 drinks ) or more of alcohol consumption per drinking day , and 210 g ( 15 drinks ) or more alcohol consumption per week were associated with higher suicide mortality , compared with non - drinkers , in the unadjusted analysis . after adjustment for potential confounders , adjusted hrs of one drinking day increase per week , each 70 g ( 5 drinks ) higher alcohol intake per drinking day , and each 140 g ( 10 drinks ) higher alcohol intake per week , were 1.17 , 1.38 and 1.12 , respectively ( table 2 ) . current smokers and people with poor self - rated health had higher suicide mortality , after adjustment for other risk factors including alcohol intake status ( see online supplementary table s1 ) . crude death rates and hrs of suicide death by alcohol consumption - related variables in the korean elderly * death rate per 100 000 person - years . adjusted for age at enrolment ( continuous variable ) , sex ( men , women ) , smoking status ( current smoker , past smoker , never smoker ) , body mass index ( kg / m ; < 18.5 , 18.524.9 , 25 ) , hypertension ( measured blood pressure 140/90 mm hg or on regular medication ; yes , no ) , self - rated health compared to the same age group ( good or fair , poor ) , marital status ( living with a spouse , not living with a spouse ) , education ( ever , never ) and occupation ( non - agriculture , agriculture ) . 1 standard drink as around 14 g according to the national institute on alcohol abuse and alcoholism of the usa . in a stratified analysis by sex , given the same alcohol intake , women seemed to have a higher relative risk of suicide associated with alcohol consumption , compared with men ( figure 1 ) . however , p values of the interaction of sex and alcohol intake - related variables with suicide were above 0.05 , due to the small number of suicide deaths . hrs associated with alcohol consumption did not differ between the first half ( 19851996 ) and the latter half ( 19972008 ) of the follow - up ( see online supplementary figure s1 ) . the hrs of alcohol consumption variables from the sensitivity analysis with reduced covariables to deal with potential overfitting , or violation of the proportional hazards assumption , were generally the same as in the main analysis ( see online supplementary table s2 ) . covariables included in the cox model were age at enrolment ( continuous variable ) , smoking status ( current smoker , past smoker , never smoker ) , body mass index ( kg / m ; < 18.5 , 18.524.9 , 25 ) , hypertension ( measured blood pressure 140/90 mm hg or on regular medication ; yes , no ) , self - rated health compared to the same age group ( good or fair , poor ) , marital status ( living with a spouse , not living with a spouse ) , education ( ever , never ) and occupation ( non - agriculture , agriculture ) . in our community - based cohort study among the korean rural elderly , more frequent drinking , a larger amount of alcohol consumption per drinking day and a larger amount of average alcohol consumption were associated with a higher risk of death by suicide . one drinking day increase per week , a 70 g ( 5-drink ) increase in the average alcohol intake per drinking day , and a 140 g ( 10-drink ) increase in the average alcohol intake per week were associated with a 17% , 38% and 12% higher risk of death by suicide , respectively , after adjustment for age , sex , socioeconomic status , behavioural and health - related potential confounders in the korean elderly . few prospective cohort studies have ever examined the impact of the quantity of usual alcohol consumption on suicide mortality after adjustment for potential confounders.57 1113 17 among community - based cohort studies,1113 in 43 383 japanese men from a public health centre - based cohort , occasional drinkers had the lowest risk of death by suicide , while non - drinkers and heavy drinkers ( 414 g alcohol per week ) had a higher risk;11 in another public health centre - based cohort study in 22 804 japanese men ( mean age : 60 years ) , alcohol consumption was linearly associated with suicide death;13 in 128 934 participants of the northern california medical care programme in the usa , greater alcohol consumption ( especially 6 drinks per day ) was associated with higher suicide death.12 among occupation - based cohort studies,57 17 in around 49 000 norwegian conscripts , high alcohol consumption ( > 250 g / week ) was associated with a non - significantly increased risk of suicide ( hr=1.7 , 95% ci 1.0 to 2.8 ) compared to moderate consumption ( 1100 g / week);17 among 89 299 physicians and 47 654 health professionals in the usa , alcohol consumption was not associated with suicide death;5 7 among 1.3 million korean adults , mostly government and school employees , moderate alcohol intake ( 124 g / day ) , but not high alcohol intake ( 25 g / day ) , was associated with higher suicide mortality and alcohol intake was not linearly associated with suicide death.6 additionally , among nested case control studies based on prospective cohorts in the usa , the usual amount of alcohol consumption was not associated with suicide death in the community elderly aged 65 years and older , elderly in a retirement community and former college students.810 among prospective studies based on a community cohort , three studies examined the associations in the mainly elderly population with a mean age of 60 years or above.9 10 13 as noted in the previous paragraph , prospective studies have produced inconsistent results on the association between usual alcohol consumption and suicide death . however , results from community - based cohort studies , including the current study , indicated that usual alcohol consumption may be linearly associated with suicide death in the community - dwelling population , at least among current drinkers.1113 additionally , our study does not support a protective effect of moderate alcohol consumption against suicide mortality in accordance with other,12 13 but not all,11 community - based cohort studies . as for occupation - based cohort studies , usual alcohol consumption did not generally show a linear dose , participants were mostly healthier , and had a higher socioeconomic status and potentially easier access to healthcare and other resources than the general population.57 they also had a generally lower alcohol consumption . these factors combined might affect the association between usual alcohol intake and suicide mortality in previous occupation - based cohort studies . prospective cohort studies have seldom evaluated the association between usual alcohol consumption and suicide mortality among men and women separately . in this study , estimates of relative risks associated with alcohol consumption variables a one - drinking - day increase per week , a 70 g ( 5-drink ) increase of average alcohol intake per drinking day , and a 140 g ( 10-drink ) increase of average alcohol intake per week were all higher in women , compared with men . similar results have been observed in a previous study in korean adults , showing that women had a higher relative risk associated with moderate alcohol consumption ( 124 g / day ) , compared with men ( hr=1.83 , 95% ci 1.09 to 3.07 in women ; hr=1.20 , 95% ci 0.93 to 1.56 in men).6 p values of the interaction of sex were above 0.05 , partly due to the small number of suicide deaths . however , given the lower body weight as well as the lower threshold of alcohol use for better cardiovascular health in women than in men , our findings may support a lower threshold in women than in men for interventions to prevent suicide death . in this study , relative risks associated with alcohol consumption - related continuous variables , unlike depressive symptoms,18 did not decrease during the latter half of follow - up ( 19972008 ) , compared with the first half ( 19851996 ) . this finding was concordant with the previous findings in japanese men that relative risk of death by suicide associated with alcohol consumption did not decrease after exclusion of early follow - up.13 this finding suggests that alcohol consumption - related variables may be used as a risk stratification tool for a long - term suicide prevention programme . a prospective design and nearly complete long - term follow - up on suicide using national mortality data are one of the strengths of this study . a homogeneous study population with the same ethnicity and culture , who live in the same regional community , is another of its strengths . yet another strength of our study is that it includes detailed information on alcohol consumption . to the best of our knowledge , only one prospective study examining the association with suicide death collected detailed information on alcohol consumption including drinking frequency , overall average consumption and quantity of alcohol consumed on drinking days.7 this study has limitations . first , we observed only 61 suicide deaths , so statistical power may be lacking in some analyses , including the assessment of interaction by sex . however , the number of suicides in persons with high alcohol consumption such as 30 g / day was comparable to studies with twice the number of all suicide deaths.5 7 12 second , our assessment of alcohol consumption was made using a self - reported questionnaire , and it was not validated separately . however , self - report measures of alcohol consumption are known to have reasonable reliability and validity,19 and they were assessed prospectively before suicide death . therefore , potential measurement errors are most likely non - differential with respect to suicide death , and are unlikely to overestimate the risks . third , depressive symptoms and other psychiatric disorders , strong predictors of suicide death , were not adjusted for.18 however , alcohol use status has been suggested to increase the risk of death by suicide , independently of other psychiatric disorders including depression.20 fourth , other potential confounders such as suicidal ideation , detailed information on health burden , social support and socioeconomic status were not included in the analysis . fifth , our study could not distinguish between never drinkers and ex - drinkers among non - drinkers . since ex - drinkers tended to have higher suicide mortality in some,11 18 but not all,13 previous studies , the risk of suicide death among current drinkers , compared with never drinkers , may be underestimated in this study . sixth , suicide death was defined by death certificates . however , any misclassification of suicide , as mentioned earlier , is likely to have been mostly non - differential with regard to alcohol consumption ; thus , potential misclassifications generally would have been unlikely to overestimate the relative risk . seventh , homogeneity of the participants could also be a limitation of our findings generalisability . the magnitude of association of risk factors with suicide death may differ by ethnicity , culture , age and level of urbanicity.4 18 2124 therefore , some of our findings regarding the korean rural elderly individuals may have limited generalisability to other ethnic , cultural or age groups , or to urban populations . few prospective cohort studies have ever examined the impact of the quantity of usual alcohol consumption on suicide mortality after adjustment for potential confounders.57 1113 17 among community - based cohort studies,1113 in 43 383 japanese men from a public health centre - based cohort , occasional drinkers had the lowest risk of death by suicide , while non - drinkers and heavy drinkers ( 414 g alcohol per week ) had a higher risk;11 in another public health centre - based cohort study in 22 804 japanese men ( mean age : 60 years ) , alcohol consumption was linearly associated with suicide death;13 in 128 934 participants of the northern california medical care programme in the usa , greater alcohol consumption ( especially 6 drinks per day ) was associated with higher suicide death.12 among occupation - based cohort studies,57 17 in around 49 000 norwegian conscripts , high alcohol consumption ( > 250 g / week ) was associated with a non - significantly increased risk of suicide ( hr=1.7 , 95% ci 1.0 to 2.8 ) compared to moderate consumption ( 1100 g / week);17 among 89 299 physicians and 47 654 health professionals in the usa , alcohol consumption was not associated with suicide death;5 7 among 1.3 million korean adults , mostly government and school employees , moderate alcohol intake ( 124 g / day ) , but not high alcohol intake ( 25 g / day ) , was associated with higher suicide mortality and alcohol intake was not linearly associated with suicide death.6 additionally , among nested case control studies based on prospective cohorts in the usa , the usual amount of alcohol consumption was not associated with suicide death in the community elderly aged 65 years and older , elderly in a retirement community and former college students.810 among prospective studies based on a community cohort , three studies examined the associations in the mainly elderly population with a mean age of 60 years or above.9 10 13 as noted in the previous paragraph , prospective studies have produced inconsistent results on the association between usual alcohol consumption and suicide death . however , results from community - based cohort studies , including the current study , indicated that usual alcohol consumption may be linearly associated with suicide death in the community - dwelling population , at least among current drinkers.1113 additionally , our study does not support a protective effect of moderate alcohol consumption against suicide mortality in accordance with other,12 13 but not all,11 community - based cohort studies . as for occupation - based cohort studies , participants were mostly healthier , and had a higher socioeconomic status and potentially easier access to healthcare and other resources than the general population.57 they also had a generally lower alcohol consumption . these factors combined might affect the association between usual alcohol intake and suicide mortality in previous occupation - based cohort studies . prospective cohort studies have seldom evaluated the association between usual alcohol consumption and suicide mortality among men and women separately . in this study , estimates of relative risks associated with alcohol consumption variables a one - drinking - day increase per week , a 70 g ( 5-drink ) increase of average alcohol intake per drinking day , and a 140 g ( 10-drink ) increase of average alcohol intake per week were all higher in women , compared with men . similar results have been observed in a previous study in korean adults , showing that women had a higher relative risk associated with moderate alcohol consumption ( 124 g / day ) , compared with men ( hr=1.83 , 95% ci 1.09 to 3.07 in women ; hr=1.20 , 95% ci 0.93 to 1.56 in men).6 p values of the interaction of sex were above 0.05 , partly due to the small number of suicide deaths . however , given the lower body weight as well as the lower threshold of alcohol use for better cardiovascular health in women than in men , our findings may support a lower threshold in women than in men for interventions to prevent suicide death . in this study , relative risks associated with alcohol consumption - related continuous variables , unlike depressive symptoms,18 did not decrease during the latter half of follow - up ( 19972008 ) , compared with the first half ( 19851996 ) . this finding was concordant with the previous findings in japanese men that relative risk of death by suicide associated with alcohol consumption did not decrease after exclusion of early follow - up.13 this finding suggests that alcohol consumption - related variables may be used as a risk stratification tool for a long - term suicide prevention programme . a prospective design and nearly complete long - term follow - up on suicide using national mortality data are one of the strengths of this study . a homogeneous study population with the same ethnicity and culture , who live in the same regional community , is another of its strengths . yet another strength of our study is that it includes detailed information on alcohol consumption . to the best of our knowledge , only one prospective study examining the association with suicide death collected detailed information on alcohol consumption including drinking frequency , overall average consumption and quantity of alcohol consumed on drinking days.7 this study has limitations . first , we observed only 61 suicide deaths , so statistical power may be lacking in some analyses , including the assessment of interaction by sex . however , the number of suicides in persons with high alcohol consumption such as 30 g / day was comparable to studies with twice the number of all suicide deaths.5 7 12 second , our assessment of alcohol consumption was made using a self - reported questionnaire , and it was not validated separately . however , self - report measures of alcohol consumption are known to have reasonable reliability and validity,19 and they were assessed prospectively before suicide death . therefore , potential measurement errors are most likely non - differential with respect to suicide death , and are unlikely to overestimate the risks . third , depressive symptoms and other psychiatric disorders , strong predictors of suicide death , were not adjusted for.18 however , alcohol use status has been suggested to increase the risk of death by suicide , independently of other psychiatric disorders including depression.20 fourth , other potential confounders such as suicidal ideation , detailed information on health burden , social support and socioeconomic status were not included in the analysis . fifth , our study could not distinguish between never drinkers and ex - drinkers among non - drinkers . since ex - drinkers tended to have higher suicide mortality in some,11 18 but not all,13 previous studies , the risk of suicide death among current drinkers , compared with never drinkers , may be underestimated in this study . sixth , suicide death was defined by death certificates . however , any misclassification of suicide , as mentioned earlier , is likely to have been mostly non - differential with regard to alcohol consumption ; thus , potential misclassifications generally would have been unlikely to overestimate the relative risk . seventh , homogeneity of the participants could also be a limitation of our findings generalisability . the magnitude of association of risk factors with suicide death may differ by ethnicity , culture , age and level of urbanicity.4 18 2124 therefore , some of our findings regarding the korean rural elderly individuals may have limited generalisability to other ethnic , cultural or age groups , or to urban populations . among the korean rural elderly , the frequency and amount of usual alcohol consumption , per drinking day and per week , had a linear dose response relationship with suicide death . when consuming alcohol with the same frequency and amount , compared with men , women might have a higher relative risk of suicide . our findings suggest that there is no protective effect of moderate alcohol consumption on suicide death . our results also support the lower the better policy of alcohol intake , and a sex - specific alcohol threshold for any suicide prevention strategy implemented . what is already known on this subjectin a recent systematic review of the prospective studies , the magnitude of the association between alcohol use disorder and suicide death was moderate ( pooled risk ratio=1.74 , 95% ci 1.26 to 2.21).evidence that greater usual alcohol consumption is associated with higher suicide death in the prospective studies among the general population is inconclusive . what this study addsthe greater frequency and amount of usual alcohol consumption was linearly associated with higher suicide death , and there was no protective effect of moderate alcohol consumption against suicide death in the elderly persons in a rural community.when consuming alcohol with the same frequency and amount as men , women might have a higher relative risk of suicide . in a recent systematic review of the prospective studies , the magnitude of the association between alcohol use disorder and suicide death was moderate ( pooled risk ratio=1.74 , 95% ci 1.26 to 2.21 ) . evidence that greater usual alcohol consumption is associated with higher suicide death in the prospective studies among the general population is inconclusive . the greater frequency and amount of usual alcohol consumption was linearly associated with higher suicide death , and there was no protective effect of moderate alcohol consumption against suicide death in the elderly persons in a rural community . when consuming alcohol with the same frequency and amount as men , women might have a higher relative risk of suicide .

backgroundthe evidence from prospective studies on whether greater usual alcohol consumption is associated with a higher risk of death by suicide in the general population is inconclusive.methods6163 participants ( 2635 men ; 3528 women ) in a 1985 survey among rural residents in korea aged 55 years and above were followed until 2008 . a cox model was used to calculate hrs of suicide death after adjustment for demographic , socioeconomic and health - related confounders.results37 men and 24 women died by suicide . elderly persons who consumed alcohol daily , 70 g alcohol ( 5 drinks ) or more per drinking day , or 210 g alcohol ( 15 drinks ) or more per week had higher suicide mortality ( p<0.05 ) , compared with non - drinkers . an increase of one drinking day per week ( hr=1.17 , 95% ci 1.05 to 1.31 ) , 70 g ( 5 drinks ) additional alcohol intake per drinking day ( hr=1.38 , 95% ci 1.13 to 1.70 ) , and 140 g ( 10 drinks ) additional alcohol intake per week was associated with a 17% , 38% and 12% higher risk of suicide death , respectively . women had a higher relative risk of suicide death associated with alcohol consumption , compared with men.conclusionsa greater frequency and amount of usual alcohol consumption was linearly associated with higher suicide death . given the same amount of alcohol consumption , women might have a higher relative risk of suicide than men . our findings support the lower the better for alcohol intake , no protective effect of moderate alcohol consumption , and a sex - specific guideline ( lower alcohol threshold for women ) as actions to prevent suicide death .

Introduction Methods Study population Estimation of usual alcohol consumption Baseline data collection Follow-up and outcome assessment Statistical analysis Results Discussion Relationship to previous research Association in women Association according to follow-up time Strengths and limitations of the study Conclusion Supplementary Material

death by suicide is a leading cause of death,1 and initiatives to advance suicide prevention programmes are a high priority in many countries.2 harmful alcohol use has been recognised as a strong risk factor for suicide death.2 in psychological autopsy studies , alcohol use disorder was strongly associated with suicide death ( or=5.2 , 95% ci 3.3 to 8.3),3 and heavy alcohol use was also strongly linked with suicide death ( or=6.2 , 95% ci 5.6 to 6.9 in men).4 in a recent systematic review of the prospective studies , however , the magnitude of the association between alcohol use disorder and suicide death was reported to be moderate ( pooled risk ratio=1.74 , 95% ci 1.26 to 2.21 ) . furthermore , when usual alcohol consumption rather than alcohol use disorder is considered in the general population , evidence of the impact of usual alcohol consumption on suicide death is less than convincing . control studies based on a prospective cohort , have indicated that alcohol consumption was not associated with a higher risk of suicide death , compared to no alcohol consumption , even in the group with the highest consumption in each study,511 with a few exceptions.12 13 overall , the association between usual alcohol consumption and suicide death is unclear . since the majority of adults drink alcohol at some point in their life , a better understanding of usual alcohol consumption and suicide death should inform decision - making for better suicide prevention strategies in the general population . the purpose of this study was to prospectively examine the association of usual alcohol intake - related variables including alcohol intake frequency , average alcohol consumption and quantity of alcohol consumed on a drinking day with suicide death in a community dwelling elderly population in korea . we tried to elucidate questions such as whether greater usual alcohol consumption is associated with a greater risk of suicide death ( dose response relationship ) , and whether moderate alcohol consumption ( below 5 drinks / week , or 4 or fewer drinking days a month ) has a protective effect against suicide death compared to abstaining completely . in the end , a total of 6163 elderly ( 2635 men ; 3528 women ) were included as a study population . alcohol consumption ( alcohol in grams ) per week was estimated by multiplying the amount of alcohol consumption per drinking day ( ml ) by the alcohol content in the particular type of alcoholic beverage , the drinking days per week , and the specific gravity of alcohol ( 0.8 ) . the alcohol frequency was merged into four groups in further analysis : daily , 26 days a week ( almost daily and 23 times a week combined ) , 4 or fewer days a month ( 14 times a month and 412 times a year combined ) , and non - drinkers . the alcohol intake amount ( grams of alcohol ) per week was classified into four groups ( grams of alcohol per week ( drinks per week , with 1 drink as 14 g of alcohol16 ; non - drinkers , < 70 ( < 5 drinks ) ; 70209 ( 514 ) , 210419 ( 1529 ) , and 420 ( 30 ) ) . the amount consumed ( grams of alcohol ) per drinking day was classified into four groups ( grams of alcohol per drinking day ( drinks per occasion ) ; non - drinkers , < 70 ( < 5 drinks ) ; 70139 ( 59 ) , and 140 ( 10 ) ) . cox proportional hazards models were used to calculate hrs associated with alcohol intake - related variables . in the end , a total of 6163 elderly ( 2635 men ; 3528 women ) were included as a study population . alcohol consumption ( alcohol in grams ) per week was estimated by multiplying the amount of alcohol consumption per drinking day ( ml ) by the alcohol content in the particular type of alcoholic beverage , the drinking days per week , and the specific gravity of alcohol ( 0.8 ) . the alcohol intake amount ( grams of alcohol ) per week was classified into four groups ( grams of alcohol per week ( drinks per week , with 1 drink as 14 g of alcohol16 ; non - drinkers , < 70 ( < 5 drinks ) ; 70209 ( 514 ) , 210419 ( 1529 ) , and 420 ( 30 ) ) . the amount consumed ( grams of alcohol ) per drinking day was classified into four groups ( grams of alcohol per drinking day ( drinks per occasion ) ; non - drinkers , < 70 ( < 5 drinks ) ; 70139 ( 59 ) , and 140 ( 10 ) ) . cox proportional hazards models were used to calculate hrs associated with alcohol intake - related variables . during 88 376 person - years of follow - up ( mean : 14.3 person - years ) , 37 men and 24 women died by suicide . current drinkers were slightly younger than non - drinkers , and they tended to be men , current smokers , healthy , living with a spouse and have more formal education , compared with non - drinkers ( table 1 ) . , current drinkers had a higher hr of suicide than non - drinkers and higher frequency and amount of alcohol intake were associated with a higher hr of suicide ( table 2 ) . two or more days of alcohol intake per week , 70 g ( 5 drinks ) or more of alcohol consumption per drinking day , and 210 g ( 15 drinks ) or more alcohol consumption per week were associated with higher suicide mortality , compared with non - drinkers , in the unadjusted analysis . after adjustment for potential confounders , adjusted hrs of one drinking day increase per week , each 70 g ( 5 drinks ) higher alcohol intake per drinking day , and each 140 g ( 10 drinks ) higher alcohol intake per week , were 1.17 , 1.38 and 1.12 , respectively ( table 2 ) . crude death rates and hrs of suicide death by alcohol consumption - related variables in the korean elderly * death rate per 100 000 person - years . adjusted for age at enrolment ( continuous variable ) , sex ( men , women ) , smoking status ( current smoker , past smoker , never smoker ) , body mass index ( kg / m ; < 18.5 , 18.524.9 , 25 ) , hypertension ( measured blood pressure 140/90 mm hg or on regular medication ; yes , no ) , self - rated health compared to the same age group ( good or fair , poor ) , marital status ( living with a spouse , not living with a spouse ) , education ( ever , never ) and occupation ( non - agriculture , agriculture ) . in a stratified analysis by sex , given the same alcohol intake , women seemed to have a higher relative risk of suicide associated with alcohol consumption , compared with men ( figure 1 ) . the hrs of alcohol consumption variables from the sensitivity analysis with reduced covariables to deal with potential overfitting , or violation of the proportional hazards assumption , were generally the same as in the main analysis ( see online supplementary table s2 ) . covariables included in the cox model were age at enrolment ( continuous variable ) , smoking status ( current smoker , past smoker , never smoker ) , body mass index ( kg / m ; < 18.5 , 18.524.9 , 25 ) , hypertension ( measured blood pressure 140/90 mm hg or on regular medication ; yes , no ) , self - rated health compared to the same age group ( good or fair , poor ) , marital status ( living with a spouse , not living with a spouse ) , education ( ever , never ) and occupation ( non - agriculture , agriculture ) . in our community - based cohort study among the korean rural elderly , more frequent drinking , a larger amount of alcohol consumption per drinking day and a larger amount of average alcohol consumption were associated with a higher risk of death by suicide . one drinking day increase per week , a 70 g ( 5-drink ) increase in the average alcohol intake per drinking day , and a 140 g ( 10-drink ) increase in the average alcohol intake per week were associated with a 17% , 38% and 12% higher risk of death by suicide , respectively , after adjustment for age , sex , socioeconomic status , behavioural and health - related potential confounders in the korean elderly . few prospective cohort studies have ever examined the impact of the quantity of usual alcohol consumption on suicide mortality after adjustment for potential confounders.57 1113 17 among community - based cohort studies,1113 in 43 383 japanese men from a public health centre - based cohort , occasional drinkers had the lowest risk of death by suicide , while non - drinkers and heavy drinkers ( 414 g alcohol per week ) had a higher risk;11 in another public health centre - based cohort study in 22 804 japanese men ( mean age : 60 years ) , alcohol consumption was linearly associated with suicide death;13 in 128 934 participants of the northern california medical care programme in the usa , greater alcohol consumption ( especially 6 drinks per day ) was associated with higher suicide death.12 among occupation - based cohort studies,57 17 in around 49 000 norwegian conscripts , high alcohol consumption ( > 250 g / week ) was associated with a non - significantly increased risk of suicide ( hr=1.7 , 95% ci 1.0 to 2.8 ) compared to moderate consumption ( 1100 g / week);17 among 89 299 physicians and 47 654 health professionals in the usa , alcohol consumption was not associated with suicide death;5 7 among 1.3 million korean adults , mostly government and school employees , moderate alcohol intake ( 124 g / day ) , but not high alcohol intake ( 25 g / day ) , was associated with higher suicide mortality and alcohol intake was not linearly associated with suicide death.6 additionally , among nested case control studies based on prospective cohorts in the usa , the usual amount of alcohol consumption was not associated with suicide death in the community elderly aged 65 years and older , elderly in a retirement community and former college students.810 among prospective studies based on a community cohort , three studies examined the associations in the mainly elderly population with a mean age of 60 years or above.9 10 13 as noted in the previous paragraph , prospective studies have produced inconsistent results on the association between usual alcohol consumption and suicide death . however , results from community - based cohort studies , including the current study , indicated that usual alcohol consumption may be linearly associated with suicide death in the community - dwelling population , at least among current drinkers.1113 additionally , our study does not support a protective effect of moderate alcohol consumption against suicide mortality in accordance with other,12 13 but not all,11 community - based cohort studies . as for occupation - based cohort studies , usual alcohol consumption did not generally show a linear dose , participants were mostly healthier , and had a higher socioeconomic status and potentially easier access to healthcare and other resources than the general population.57 they also had a generally lower alcohol consumption . in this study , estimates of relative risks associated with alcohol consumption variables a one - drinking - day increase per week , a 70 g ( 5-drink ) increase of average alcohol intake per drinking day , and a 140 g ( 10-drink ) increase of average alcohol intake per week were all higher in women , compared with men . similar results have been observed in a previous study in korean adults , showing that women had a higher relative risk associated with moderate alcohol consumption ( 124 g / day ) , compared with men ( hr=1.83 , 95% ci 1.09 to 3.07 in women ; hr=1.20 , 95% ci 0.93 to 1.56 in men).6 p values of the interaction of sex were above 0.05 , partly due to the small number of suicide deaths . however , given the lower body weight as well as the lower threshold of alcohol use for better cardiovascular health in women than in men , our findings may support a lower threshold in women than in men for interventions to prevent suicide death . in this study , relative risks associated with alcohol consumption - related continuous variables , unlike depressive symptoms,18 did not decrease during the latter half of follow - up ( 19972008 ) , compared with the first half ( 19851996 ) . this finding was concordant with the previous findings in japanese men that relative risk of death by suicide associated with alcohol consumption did not decrease after exclusion of early follow - up.13 this finding suggests that alcohol consumption - related variables may be used as a risk stratification tool for a long - term suicide prevention programme . third , depressive symptoms and other psychiatric disorders , strong predictors of suicide death , were not adjusted for.18 however , alcohol use status has been suggested to increase the risk of death by suicide , independently of other psychiatric disorders including depression.20 fourth , other potential confounders such as suicidal ideation , detailed information on health burden , social support and socioeconomic status were not included in the analysis . since ex - drinkers tended to have higher suicide mortality in some,11 18 but not all,13 previous studies , the risk of suicide death among current drinkers , compared with never drinkers , may be underestimated in this study . few prospective cohort studies have ever examined the impact of the quantity of usual alcohol consumption on suicide mortality after adjustment for potential confounders.57 1113 17 among community - based cohort studies,1113 in 43 383 japanese men from a public health centre - based cohort , occasional drinkers had the lowest risk of death by suicide , while non - drinkers and heavy drinkers ( 414 g alcohol per week ) had a higher risk;11 in another public health centre - based cohort study in 22 804 japanese men ( mean age : 60 years ) , alcohol consumption was linearly associated with suicide death;13 in 128 934 participants of the northern california medical care programme in the usa , greater alcohol consumption ( especially 6 drinks per day ) was associated with higher suicide death.12 among occupation - based cohort studies,57 17 in around 49 000 norwegian conscripts , high alcohol consumption ( > 250 g / week ) was associated with a non - significantly increased risk of suicide ( hr=1.7 , 95% ci 1.0 to 2.8 ) compared to moderate consumption ( 1100 g / week);17 among 89 299 physicians and 47 654 health professionals in the usa , alcohol consumption was not associated with suicide death;5 7 among 1.3 million korean adults , mostly government and school employees , moderate alcohol intake ( 124 g / day ) , but not high alcohol intake ( 25 g / day ) , was associated with higher suicide mortality and alcohol intake was not linearly associated with suicide death.6 additionally , among nested case control studies based on prospective cohorts in the usa , the usual amount of alcohol consumption was not associated with suicide death in the community elderly aged 65 years and older , elderly in a retirement community and former college students.810 among prospective studies based on a community cohort , three studies examined the associations in the mainly elderly population with a mean age of 60 years or above.9 10 13 as noted in the previous paragraph , prospective studies have produced inconsistent results on the association between usual alcohol consumption and suicide death . however , results from community - based cohort studies , including the current study , indicated that usual alcohol consumption may be linearly associated with suicide death in the community - dwelling population , at least among current drinkers.1113 additionally , our study does not support a protective effect of moderate alcohol consumption against suicide mortality in accordance with other,12 13 but not all,11 community - based cohort studies . as for occupation - based cohort studies , participants were mostly healthier , and had a higher socioeconomic status and potentially easier access to healthcare and other resources than the general population.57 they also had a generally lower alcohol consumption . in this study , estimates of relative risks associated with alcohol consumption variables a one - drinking - day increase per week , a 70 g ( 5-drink ) increase of average alcohol intake per drinking day , and a 140 g ( 10-drink ) increase of average alcohol intake per week were all higher in women , compared with men . similar results have been observed in a previous study in korean adults , showing that women had a higher relative risk associated with moderate alcohol consumption ( 124 g / day ) , compared with men ( hr=1.83 , 95% ci 1.09 to 3.07 in women ; hr=1.20 , 95% ci 0.93 to 1.56 in men).6 p values of the interaction of sex were above 0.05 , partly due to the small number of suicide deaths . however , given the lower body weight as well as the lower threshold of alcohol use for better cardiovascular health in women than in men , our findings may support a lower threshold in women than in men for interventions to prevent suicide death . in this study , relative risks associated with alcohol consumption - related continuous variables , unlike depressive symptoms,18 did not decrease during the latter half of follow - up ( 19972008 ) , compared with the first half ( 19851996 ) . this finding was concordant with the previous findings in japanese men that relative risk of death by suicide associated with alcohol consumption did not decrease after exclusion of early follow - up.13 this finding suggests that alcohol consumption - related variables may be used as a risk stratification tool for a long - term suicide prevention programme . third , depressive symptoms and other psychiatric disorders , strong predictors of suicide death , were not adjusted for.18 however , alcohol use status has been suggested to increase the risk of death by suicide , independently of other psychiatric disorders including depression.20 fourth , other potential confounders such as suicidal ideation , detailed information on health burden , social support and socioeconomic status were not included in the analysis . since ex - drinkers tended to have higher suicide mortality in some,11 18 but not all,13 previous studies , the risk of suicide death among current drinkers , compared with never drinkers , may be underestimated in this study . among the korean rural elderly , the frequency and amount of usual alcohol consumption , per drinking day and per week , had a linear dose response relationship with suicide death . when consuming alcohol with the same frequency and amount , compared with men , women might have a higher relative risk of suicide . our findings suggest that there is no protective effect of moderate alcohol consumption on suicide death . our results also support the lower the better policy of alcohol intake , and a sex - specific alcohol threshold for any suicide prevention strategy implemented . what is already known on this subjectin a recent systematic review of the prospective studies , the magnitude of the association between alcohol use disorder and suicide death was moderate ( pooled risk ratio=1.74 , 95% ci 1.26 to 2.21).evidence that greater usual alcohol consumption is associated with higher suicide death in the prospective studies among the general population is inconclusive . what this study addsthe greater frequency and amount of usual alcohol consumption was linearly associated with higher suicide death , and there was no protective effect of moderate alcohol consumption against suicide death in the elderly persons in a rural community.when consuming alcohol with the same frequency and amount as men , women might have a higher relative risk of suicide . in a recent systematic review of the prospective studies , the magnitude of the association between alcohol use disorder and suicide death was moderate ( pooled risk ratio=1.74 , 95% ci 1.26 to 2.21 ) . evidence that greater usual alcohol consumption is associated with higher suicide death in the prospective studies among the general population is inconclusive . the greater frequency and amount of usual alcohol consumption was linearly associated with higher suicide death , and there was no protective effect of moderate alcohol consumption against suicide death in the elderly persons in a rural community . when consuming alcohol with the same frequency and amount as men , women might have a higher relative risk of suicide .

protein glycosylation is one of the most frequent and relevant post - translational modifications . it is present in more than half of all proteins in nature and plays a key role in the modulation of protein properties and function . one of the fastest growing areas in the pharmaceutical industry is the field of therapeutic glycoproteins . they are currently being marketed as natural mixtures of glycoforms ( i.e. , conserved peptide backbone but highly variable as to the site and pattern of glycosylation ) . our laboratory has a long - standing interest in the synthesis of homogeneous glycoproteins , as exhibited in our recently reported chemical synthesis of an erythropoietin glycoform with the wild - type polypeptide backbone and well - defined n- and o - glycosides . we have also been interested in the human glycoprotein hormones ( hgph ) , which are composed of two noncovalently associated subunits , a common -subunit ( -hgph ) and a hormone - specific -subunit , each containing diverse oligosaccharides at defined glycosylation sites . recently , we described the synthesis of the - and -subunits of human follicle - stimulating hormone ( hfsh or follitropin ) . herein , we have focused our synthetic efforts on the gonadotropins , human luteinizing hormone ( hlh or lutropin ) and human chorionic gonadotropin ( hcg ) . the gonadotropic hormones hlh and hcg are required for normal development and secretory activity of the gonads and stimulate the endocrine and gametogenic functions . hlh is produced by gonadotroph cells in the pituitary gland and plays a key role in the reproductive process , including regulating the menstrual cycle , triggering ovulation and development of the corpus luteum , and stimulating the production of testosterone . hcg is produced primarily in the human placenta and has similar physiological functions to hlh ( i.e. , upregulating progesterone and testosterone production and inducing ovulation and spermatogenesis ) . it also plays a role in the protection of the fertilized embryo and development of the fetus during pregnancy . in clinical settings , marketed forms of these hormones ( often co - administered ) are used in human reproductive medicine for the treatment of infertility and in in vitro fertilization . interestingly , the -subunit of hcg ( -hcg ) is also overexpressed as particular glycoforms in certain types of tumors ( epithelial , pancreatic , colorectal ) and has been found to inhibit apoptosis in cancer tissues and promote metastasis . clinical trials using anti--hcg cancer vaccines have been carried out in patients with epithelial , pancreatic , and colorectal cancer . thus , -hcg constitutes a promising target for cancer immunotherapy and antitumor vaccine development . furthermore , antibodies against -hcg and one type of hcg glycoform ( hyperglycosylated hcg ) have been shown to inhibit tumor cell growth and metastasis . there have also been studies , though conflicting , on the antiproliferative properties of hcg against the kaposi s sarcoma ( ks ) in hiv patients as well as its biological activity against hiv-1 . these conflicting studies may be due to the heterogeneity of different clinical - grade preparations of hcg , which contain mixtures of complex glycoforms . since certain glycoforms are more active than others , access to specific , discrete glycoforms by chemical synthesis can confer significant therapeutic advantages . moreover , the synthesis of chemically pure glycoproteins bearing defined glycans can help determine the specific roles of glycosylation in biosynthesis and function . structurally , the -hlh subunit consists of 121 residues with one n - linked glycan at asn30 ( figure 1a ) . the more complex -subunit of hcg shares 85% sequence homology with -hlh . however , it has a unique serine - rich , 30 amino acid carboxy terminal extension with 4 additional sites of o - glycosylation at these residues . thus , -hcg is made up of a 145-amino acid protein backbone bearing two n - linked glycans at asn13 and asn30 and four o - linked glycans at ser121 , ser127 , ser132 , and ser138 ( figure 1b ) . the carbohydrate content represents about one - third of the molecular weight of hcg , and glycosylation is of structural and functional importance , affecting both its half - life in circulation and signal transduction induced by this hormone . numerous investigations , mostly on hcg , suggest that the sugar chains are not required for hormone - receptor binding but play a critical role in receptor activation . the major structural differences in the asn - linked carbohydrates of hlh and hcg are in the terminal sugar moieties of the sialylated , biantennary , complex - type n - glycans ( figure 1c ) . the observed diversity of terminal glycosylation sequences suggests that the peripheral sugar residues are not essential for the biological action of these hormones . this observation , together with the fact that structures lacking n - glycosylation exhibit almost complete loss of hormone function , indicates that the underlying core structures may play a more relevant role in the biological activity of both hormones . however , the entire -hcg carboxy - terminus , which contains four o - glycosylation sites with mucin - type o - glycans ( figure 1d ) , has been found to be a common epitope for hcg - based monoclonal antibodies , which can inhibit tumor cell growth and metastasis . detection of hcg isoforms by these antibodies has been used clinically to screen for down syndrome , to identify pregnancy disorders , and to monitor trophoblastic disease . in this work , we report the first total synthesis of the two longest -subunits of the human glycoprotein hormones ( -hlh and -hcg ) , which were accessed as pure glycoproteins bearing defined carbohydrate structures . the successful approach is exemplified herein with the use of the disaccharide chitobiose at the two n - glycosylation sites and the monosaccharide n - acetyl - galactosamine ( galnac ) at the four o - glycosylation sites as model glycans for our glycoprotein assembly . the system has been synthesized with protected cysteines anticipating folding and its association with the previously synthesized , common -subunit of the human glycoprotein hormones . based on our extensive experience with similar glycoprotein targets , final cysteine deprotection and subsequent folding of the corresponding -subunits are expected to be successfully accomplished in forthcoming studies to provide fully synthetic materials for future biological evaluation . the complexity of the hcg -subunit stems not only from its size ( the longest of all human glycoprotein hormones ) and the presence of the two n - glycosylation sites but also from the position of its unique , closely spaced four o - glycosylation sites at the carboxy - terminus of the molecule . fortunately , the relatively high content of cysteine residues in the peptide backbone at fairly regular intervals enables the assembly of both full - length glycoproteins from appropriately selected peptide fragments using the native chemical ligation ( ncl ) reaction . our plan for the construction of -hlh and -hcg was based on maximum convergence , and thus , we identified the key ligation sites depicted in figure 1 . -hlh was envisioned to arise from three fragments of between 30 and 50 amino acids long , whereas our synthetic strategy for hcg relied on the assembly of five peptide segments , each bearing the corresponding n-/o - linked glycans in a highly modular fashion . ( a ) fully elaborated -subunit of hlh with chitobiose at the n - glycosylation site ( asn30 ) and proposed disconnection positions . ( b ) homogeneous full - length sequence of -hcg bearing two chitobiose ( asn13 and asn30 ) and four galnac moieties ( ser121 , ser127 , ser132 , and ser138 ) at the n- and o - glycosylation sites , respectively . key ligation sites and envisioned peptide fragments for final assembly are also indicated . ( c ) structure of n - linked carbohydrates at defined glycosylation sites on hlh and hcg polypeptide backbone ; elaborated dodecasaccharide ( left ) and chitobiose as model glycan ( right ) . ( d ) structure of representative o - linked glycans present in hcg peptide sequence : glycophorin tetrasaccharide ( left ) and simpler galnac . given the high sequence homology between both targets and the relatively simpler structure of -hlh , in comparison with -hcg , we chose to start with the synthesis of the former , en route to -hcg . in addition to providing access to hlh as one of the important glycoproteins within the family , this synthetic sequence could also serve as a proof of concept for the feasibility of the approach in terms of the following strategic steps : glycosylation via lansbury aspartylation , global deprotection of the glycopeptide segments , and the final union of these fragments by ncl . for the preparation of -hlh , we initially envisioned a retrosynthetic disconnection of the molecule in three fragments : -hlh(chitobiose)[s1y37 ] , -hlh[c38g71 ] , and -hlh[c72l121 ] ( figure 1a ) . the corresponding peptide fragments were obtained by fmoc - based solid - phase peptide synthesis ( spps ) with acid - labile protecting groups on the amino acid side chains , with the exception of the non - ncl - participating cysteine residues , in which case the acetamidomethyl ( acm ) group was chosen . for the glycopeptide segment ( -hlh(chitobiose)[s1y37 ] ) , a pseudoproline dipeptide ( ile27-thr28 ) was introduced in the sequence to improve the synthetic efficiency of the subsequent glycosylation at asn30 under lansbury conditions . in addition , the aspartic acid that will bear the chitobiose moiety was protected with a quasi - orthogonal o-2-phenylisopropyl ester ( o-2-phpr or opp ) group that can be removed selectively in the presence of tbu - based protecting groups by treatment with 12% trifluoroacetic acid ( tfa ) in dichloromethane . thus , after cleavage from the resin , the c - terminal carboxylic acid of the protected peptide was first coupled to tyrosine phenylthioester ( hclh - tyr - sph ) under sakakibara conditions , which are known to be epimerization free , and subsequent selective deprotection of asp30 provided the free carboxylic acid side chain ( pf1:-hlh[s1y37 ] ) . next , hatu - mediated lansbury aspartylation with chitobiose glycosyl amine followed by treatment with cocktail b ( 88% tfa , 5% h2o , 5% phenol , 2% tipsh ) for removal of all acid - labile protecting groups on the amino acid side chains afforded the corresponding glycopeptide fragment -hlh(chitobiose)[s1y37 ] in 22% yield ( over four steps ) after only one hplc purification ( scheme 1a ) . for the synthesis of the second fragment -hlh[z38g71 ] , the n - terminal cysteine was protected as a thiazolidine ( thz , z ) , and the c - terminal glycine residue was converted to glycine phenylthioester using pybop as a coupling agent . finally , global deprotection under acidic conditions with cocktail b provided -hlh[z38g71 ] in 72% yield ( over two steps ) ( scheme 1b ) . amino acid residues bearing acid - labile protecting groups shown in deep red , pseudoproline dipeptides shown in orange ( underlined ) , and acm - protected cys residues shown in purple . cocktail b : 88% tfa , 5% h2o , 5% phenol ( phoh ) , 2% triisopropylsilane ( tipsh ) . the last segment required for the assembly was -hlh[c72l121 ] ( scheme 2 ) . this fragment is devoid of glycosylation sites , and our initial approach was to synthesize it entirely by spps . however , despite the incorporation of three pseudoproline dipeptides and the use of the hmb ( 2-hydroxy-4-methoxybenzyl ) protecting group , aspartimide formation was predominant , and we were unable to obtain useful yields of this peptide on solid support . we therefore approached this synthesis through ncl of two smaller peptide fragments of roughly similar size . thus , protected peptides -hlh[z72s98 ] and -hlh[z100l121 ] were accessed using spps following cleavage from the resin . in the first case , the terminal serine residue ( ser98 ) was coupled to allyl - protected aspartic acid ethylthioester [ hclh - asp(oallyl)-set ] under sakakibara conditions , and subsequent treatment with cocktail b gave -hlh[z72d99 ] in 58% yield ( over two steps ) . the fully deprotected peptide -hlh[c100l121 ] was obtained in two steps ( 64% ) , involving global acid deprotection ( cocktail b ) and conversion of the n - terminal thiazolidine ( thz100 ) to cysteine using methoxyamine hydrochloride ( meonh2hcl ) at ph 4.04.5 . the two small fragments were then coupled under ncl conditions ( phosphate buffer solution containing guanidinehcl and tcephcl at ph 7.27.4 ) with 4-mercaptophenylacetic acid ( mpaa ) as an additive , and the allyl protecting group on the asp99 side chain was subsequently removed [ pdcl2(dppf ) , phenylsilane , dmso ] . finally , the n - terminal thz group was cleaved using a buffer solution of meonh2hcl and guanidinehcl in water at ph 4.04.5 to provide -hlh[c72l121 ] in 55% yield ( over three steps ) ( scheme 2 ) . amino acid residues bearing acid - labile protecting groups shown in deep red , pseudoproline dipeptides shown in orange ( underlined ) , acm - protected cys residues shown in purple , and thioester functionality shown in green . ncl buffer : guanidinehcl ( 6 m ) , na2hpo4 ( 0.2 m ) , tris(2-carboxyethyl)phosphine hydrochloride ( tcephcl ) ( 0.02 m ) , 4-mercaptophenylacetic acid ( mpaa ) ( 0.2 m ) , aqueous tcep solution ( 0.5 m ) , ph 7.27.4 . thz - deprotection buffer : meonh2hcl ( 0.3 m ) , guanidinehcl ( 6 m ) , ph 4.04.5 . cocktail b : 88% tfa , 5% h2o , 5% phenol ( phoh ) , 2% triisopropylsilane ( tipsh ) . the final assembly of the individual peptide fragments commenced with the coupling of segment -hlh[c72l121 ] with -hlh[z38g71 ] through ncl , followed by removal of the thz group to free the n - terminal cysteine required for the final ligation , in a one - flask procedure . purification by hplc provided the desired peptide -hlh[c38l121 ] in 43% yield ( two steps ) . finally , -hlh[c38l121 ] was coupled to the chitobiose - containing glycopeptide -hlh(chitobiose)[s1y37 ] under ncl conditions to afford the full - length -subunit of hlh -hlh(chitobiose)[s1l121 ] in 35% yield following hplc purification ( scheme 3 ) . ncl buffer : guanidinehcl ( 6 m ) , na2hpo4 ( 0.2 m ) , tris(2-carboxyethyl)phosphine hydrochloride ( tcephcl ) ( 0.02 m ) , 4-mercaptophenylacetic acid ( mpaa ) ( 0.2 m ) , aqueous tcep solution ( 0.5 m ) ph 7.27.4 . having identified a successful route that featured ligation steps proceeding with complete conversion in reasonable reaction time frames , we set out to synthesize the larger and more complex -subunit of hcg incorporating two n - linked glycans and four closely spaced o - glycosylation sites . our initial approach to -hcg relied on the key disconnections shown in figure 1b , whereby five simpler fragments , each bearing the corresponding n-/o - glycans could be merged together via ncl . this highly modular strategy would allow us to investigate the effect of introducing various defined carbohydrates at different sites on the protein scaffold . as for the glycosylation points , each of the n - linked glycans was coupled to the complementary peptide fragment via lansbury aspartylation , as shown earlier for -hlh . interestingly , a more challenging approach was also considered , consisting of the double incorporation , simultaneously , of the two n - linked carbohydrates on a longer peptide backbone comprising both n - glycosylation sites . with respect to the more complicated o - glycopeptide fragment , we envisioned the installation of all four o - linked glycans on solid support as glycosyl - serine cassettes during the synthesis of the carboxy - terminal segment . our initial target glycoform presents the readily available disaccharide chitobiose as a model n - linked glycan for the native complex bianntenary dodecasaccharide ( figure 1b and c ) , similar to that previously described for hlh . we began our approach with the synthesis of protected pf1:-hcg[s1g22 ] and pf2:-hcg[z23y37 ] using standard fmoc - based spps followed by cleavage from resin , c - terminus derivatization , and selective aspartic acid deprotection ( scheme 4a and b ) . in the event , the glycine residue ( gly22 ) in fragment 1 was reacted with phsh under pybop - mediated coupling conditions to give the corresponding c - terminal thioester , whereas fragment 2 was subjected to single amino acid attachment with hclh - tyr - sph under epimerization - free sakakibara conditions ( edc , hoobt ) . in both cases , selective removal of the opp protecting group ( at asp 13 and asp30 , respectively ) revealed the free aspartic acid to be coupled with chitobiose amine . thus , pf1:-hcg[s1g22 ] and pf2:-hcg[z23y37 ] were then subjected to the two - step lansbury aspartylation / cocktail b deprotection protocol described earlier to afford glycopeptide fragments -hcg(chitobiose)[s1g22 ] and -hcg[z23y37 ] in 28% and 25% yield , respectively , ( over four steps ) after hplc purification . since the same n - glycan ( chitobiose ) was to be installed at both fragments , a double lansbury aspartylation strategy was also explored for the synthesis of the entire bis - glycosylated segment -hcg(chitobiose)2[s1y37 ] ( scheme 4c ) . first , spps of the protected fragment , followed by cleavage from the resin , c - terminal thioesterification ( phsh , pybop ) , and subsequent selective asp(opp ) deprotection ( 2% tfa / dcm ) provided peptide segment pf1.2:-hcg[s1y37 ] having the two aspartic acid ( asp13 and asp30 ) side chains free for further coupling with two chitobiose units . in the event , 6 equiv of chitobiose amine , with respect to the peptide , and 6 equiv of hatu as the coupling agent were employed to give an encouraging 18% yield ( over four steps ) after global deprotection with cocktail b. a 9% yield of monoglycosylated product was also isolated under these reaction conditions . importantly , in the case of incorporating the same glycan unit , this approach represents a significant improvement over the synthesis of the two smaller individual fragments in terms of convergency and overall synthetic efficiency . thus , this strategy was preferentially applied to access the corresponding -hcg(chitobiose)2[s1y37 ] fragment ( scheme 4c ) . amino acid residues bearing acid - labile protecting groups shown in deep red , pseudoproline dipeptides shown in orange ( underlined ) , and acm - protected cys residues shown in purple . cocktail b : 88% tfa , 5% h2o , 5% phenol ( phoh ) , 2% triisopropylsilane ( tipsh ) . fragment 3 was prepared in a very straightforward manner , beginning with spps , followed by cleavage from the resin and thioesterification of the c - terminus with phsh . after deprotection with cocktail b , -hcg[z38g71 ] was isolated in 75% yield over two steps ( 52% overall from resin ) ( scheme 5a ) . the synthesis of fragment 4 , -hcg[z72t109 ] , was initially hampered by significant aspartimide formation . fortunately , the use of a piperidine / oxyma pure cocktail for the fmoc - deprotection during the spps entirely suppressed this problem , and after single amino acid attachment ( hclh - thr - sph ) at the c - terminus , followed by subsequent treatment with cocktail b , the desired peptide fragment -hcg[z72t109 ] was obtained in 45% yield ( over two steps ) with no aspartimide formation ( scheme 5b ) . amino acid residues bearing acid - labile protecting groups shown in deep red , pseudoproline dipeptides shown in orange ( underlined ) , acm - protected cys residues shown in purple , and thioester functionalities shown in green . cocktail b : 88% tfa , 5% h2o , 5% phenol ( phoh ) , 2% triisopropylsilane ( tipsh ) . finally , access to the last segment -hcg(ac3-galnac)4[c110q145 ] required a dramatically different approach than that described above . the presence of four o - linked glycans prohibits the late - stage attachment of these carbohydrates to the fully elaborated peptide . approach was followed , wherein the o - glycan , linked to a properly protected serine residue , is directly introduced in the peptide sequence as a conveniently protected glycosyl amino acid building block ( cassette ) , during solid - supported synthesis ( scheme 6a ) . this process not only allowed for assembly of this complex glycopeptide fragment but also , in principle , enables the installation of different o - linked glycans by simply applying the desired cassette during peptide backbone elongation on spps . we then targeted the synthesis of this fragment bearing the -n - acetylgalactosamine moiety ( galnac ) on all four serine residues , which corresponds to the tn antigen structure ( scheme 6b ) . this system is the minimal common constituent of larger o - linked glycans and has been shown to be relatively abundant at ser121 , 127 , 132 , and 138 in wild - type -hcg . following known procedures , we synthesized the conveniently protected , glycosylated fmoc - ser building block bearing o - acetyl groups in the galnac residue and having a free carboxylic acid on the serine . with this cassette in hand , we performed the synthesis of the required fragment via automated spps , with manual coupling of the corresponding glycosyl - serine residue ( 1.5 equiv ) under pyaop / hoat coupling conditions . gratifyingly , after treatment with cocktail b , we were able to obtain the entire fragment -hcg(ac3-galnac)4[c110q145 ] with o - acetyl protected sugars in a single spps with great purity in 22% overall yield after hplc purification ( scheme 6b ) . importantly , this successful synthesis was amenable to scale - up , which afforded up to more than 50 mg of the most complex fragment of this synthetic version of -hcg bearing four closely spaced tn antigen structures . this strategy is likely to be applicable to the installation of alternative o - linked glycan cassettes at the four o - glycosylation sites for the assembly of different glycoforms . in this regard , a number of glycosyl amino acids incorporating more elaborated glycans , such as tumor - associated carbohydrate antigens ( tf , stn , stf ) or mucin - related core structures 14 , have been synthesized through the cassette approach . importantly , using these o - linked glycosylated building blocks , the cassette methodology has been successfully applied for the solution- and solid - phase synthesis of a series of mucin - derived o - glycopeptides bearing larger oligosaccharides . these examples constitute an important precedent for the applicability of this strategy toward more complex glycosylated versions of -hcg[c110-q145 ] . practical synthetic access to the carboxy - terminus fragment of -hcg is an important step in itself as it has been found to be a common epitope for hcg - based monoclonal antibodies . screening of the specificity of different glycoforms and their role in binding to these antibodies or the development of new glycoforms could be used as a powerful tool in important clinical applications . with all the prerequisite fragments in hand , assembly of the -subunit of hcg relied on the coupling of the individual peptide segments by ncl , starting with fragments -hcg[z72t109 ] and -hcg(ac3-galnac)4[c110q145 ] . thus , these two fragments were joined together under standard ncl conditions , using mpaa as an additive . upon completion of the reaction as monitored by ultraperformance liquid chromatography ( uplc ) , the terminal thz ( z ) protecting group was removed using meonh2hcl in a one - flask procedure . subsequently , size - exclusion centrifugal filtration of the previous crude mixture followed by treatment with 5% aqueous hydrazine led to clean and complete removal of the acetate groups to provide -hcg(galnac)4[c72q145 ] in 50% yield over three steps ( scheme 7 ) . with the in situ ligation and thz opening followed by acetate deprotection successfully performed in the synthesis of half of the full - length glycoprotein , we next used another ncl to bring -hcg(galnac)4[c72q145 ] and -hcg[z38g71 ] together . the coupling reaction was completed within 3 h with no starting material remaining , as assessed by lc - ms . then , meonh2hcl was added , and the ph was adjusted to 4.04.5 to induce conversion of the n - terminal thz to cysteine . happily , after size - exclusion centrifugal filtration , this procedure enabled good recovery of -hcg(galnac)4[c38q145 ] for the final ligation reaction . in the critical ligation event , o - glycosylated fragment -hcg(galnac)4[c38q145 ] was combined with n - glycopeptide -hcg(chitobiose)2[s1y37 ] under standard ncl conditions to give the target glycoprotein -hcg(chitobiose)2(galnac)4[s1q145 ] after 4 h. upon hplc purification , the primary sequence of the 145-residue -subunit of hcg containing homogeneous n- and o - linked glycans was obtained in a gratifying 33% yield ( scheme 8) [ see figure 2 for mass spectrum and lc trace ( uv ) of -hcg containing chitobiose at asn13 and asn30 , and n - acetylgalactosamine at ser121 , ser127 , ser132 , and ser138 ] . mass spectrum and uv trace of -hcg(chitobiose)2(galnac)4[s1q145 ] glycoprotein . calcd for c759h1239n213o252s13 , 17797.08 da ( average isotopes ) [ m + 9h]m / z 1978.45 , found 1979.24 ; [ m + 10h]m / z 1780.71 , found 1781.27 ; [ m + 11h]m / z 1618.92 , found 1619.29 ; [ m + 12h]m / z 1484.09 , found 1484.67 ; [ m + 13h]m / z 1370.01 , found 1370.42 ; [ m + 14h]m / z 1272.22 , found 1272.55 ; [ m + 15h]m / z 1187.47 , found 1187.79 ; [ m + 16h]m / z 1113.32 , found 1113.54 ; [ m + 17h]m / z 1047.89 , found 1048.16 ; [ m + 18h]m / z 989.73 , found 990.02 ; [ m + 19h]m / z 937.69 , found 938.02 ; [ m + 20h]m / z 890.85 , found 891.14 . thus , compound -hcg(chitobiose)2(galnac)4[s1q145 ] , bearing not only two n - linked carbohydrates but also , and even more challenging , four o - linked sugars , represents the largest human glycoprotein hormone to have been synthesized in homogeneous form using strictly chemical means . while the current work has involved chitobiose and galnac as representative n-/o - linked model glycans , this demonstration of feasibility through the modular glycoform assembly presented herein opens the door to the application of this technology to the synthesis of a library of homogeneous glycoproteins via chemical synthesis by installing alternative glycans on the corresponding peptide fragments in summary , we have synthesized the glycosylated primary sequence of two complex glycoprotein hormones ( -hlh and -hcg ) in homogeneous form using the current innovations of peptide and glycopeptide chemistry . the first approach described for the preparation of -hlh served to validate the synthetic strategy en route to the more complex hcg -subunit . first , a double lansbury glycosylation was accomplished to provide the n - terminal fragment of the molecule in a highly modular fashion . second , practical access to the carboxy - terminus was gained by sequential installation of four o - linked glycosyl - amino acid cassettes into closely spaced o - glycosylation sites in a single and high - yielding solid - supported synthesis . this o - linked glycopeptide fragment was then successfully advanced by further coupling , deprotection , and subsequent ligations with the remaining peptide segments to provide the full - length -subunit of hcg . interestingly , the highly modular assembly exemplified herein sets the stage for accessing more complex , chemically pure -hcg glycoforms by synthesizing collections of each individual fragment containing a number of different , elaborated glycans and bringing them together following the successful ligation strategy outlined in this work . while the late - stage processes involving final acm removal and oxidation / folding remain to be validated for -hlh and -hcg , building on our previous successes with erythropoietin and other glycoprotein targets , we have confidence in the ability to generate correctly folded -subunits of these glycoprotein hormones . this important prospect should allow future biological studies of these well - defined glycoforms to better understand the specific roles of certain glycans in the function and bioactivity of hcg in clinical settings . this knowledge may , in turn , lead to the development of more efficacious and improved therapeutics .

human luteinizing hormone ( hlh ) and human chorionic gonadotropin ( hcg ) are human glycoprotein hormones each consisting of two subunits , an identical -subunit and a unique -subunit , that form noncovalent heterodimers . structurally , -hcg shares a high degree of sequence similarity with -hlh , including a common n - glycosylation site at the n - terminus but differs mainly in the presence of an extended c - terminal portion incorporating four closely spaced o - linked glycans . these glycoproteins play important roles in reproduction and are used clinically in the treatment of infertility . in addition , the role of hcg as a tumor marker in a variety of cancers has also attracted significant interest for the development of cancer vaccines . in clinical applications , these hormones are administered as mixtures of glycoforms due to limitations of biological methods in producing homogeneous samples of these glycoproteins . using the powerful tools of chemical synthesis , the work presented herein focuses on the highly convergent syntheses of homogeneous -hlh and -hcg bearing model glycans at all native glycosylation sites . key steps in these syntheses include a successful double lansbury glycosylation en route to the n - terminal fragment of -hcg and the sequential installation of four o - linked glycosyl - amino acid cassettes into closely spaced o - glycosylation sites in a single , high - yielding solid - supported synthesis to access the c - terminal portion of the molecule . the final assembly of the individual glycopeptide fragments involved a stepwise native chemical ligation strategy to provide the longest and most complex human glycoprotein hormone ( -hcg ) as well as its closely related homologue ( -hlh ) as discrete glycoforms .

Introduction Results and Discussion Conclusion

our laboratory has a long - standing interest in the synthesis of homogeneous glycoproteins , as exhibited in our recently reported chemical synthesis of an erythropoietin glycoform with the wild - type polypeptide backbone and well - defined n- and o - glycosides . we have also been interested in the human glycoprotein hormones ( hgph ) , which are composed of two noncovalently associated subunits , a common -subunit ( -hgph ) and a hormone - specific -subunit , each containing diverse oligosaccharides at defined glycosylation sites . herein , we have focused our synthetic efforts on the gonadotropins , human luteinizing hormone ( hlh or lutropin ) and human chorionic gonadotropin ( hcg ) . hlh is produced by gonadotroph cells in the pituitary gland and plays a key role in the reproductive process , including regulating the menstrual cycle , triggering ovulation and development of the corpus luteum , and stimulating the production of testosterone . in clinical settings , marketed forms of these hormones ( often co - administered ) are used in human reproductive medicine for the treatment of infertility and in in vitro fertilization . interestingly , the -subunit of hcg ( -hcg ) is also overexpressed as particular glycoforms in certain types of tumors ( epithelial , pancreatic , colorectal ) and has been found to inhibit apoptosis in cancer tissues and promote metastasis . there have also been studies , though conflicting , on the antiproliferative properties of hcg against the kaposi s sarcoma ( ks ) in hiv patients as well as its biological activity against hiv-1 . these conflicting studies may be due to the heterogeneity of different clinical - grade preparations of hcg , which contain mixtures of complex glycoforms . structurally , the -hlh subunit consists of 121 residues with one n - linked glycan at asn30 ( figure 1a ) . however , it has a unique serine - rich , 30 amino acid carboxy terminal extension with 4 additional sites of o - glycosylation at these residues . thus , -hcg is made up of a 145-amino acid protein backbone bearing two n - linked glycans at asn13 and asn30 and four o - linked glycans at ser121 , ser127 , ser132 , and ser138 ( figure 1b ) . the major structural differences in the asn - linked carbohydrates of hlh and hcg are in the terminal sugar moieties of the sialylated , biantennary , complex - type n - glycans ( figure 1c ) . however , the entire -hcg carboxy - terminus , which contains four o - glycosylation sites with mucin - type o - glycans ( figure 1d ) , has been found to be a common epitope for hcg - based monoclonal antibodies , which can inhibit tumor cell growth and metastasis . in this work , we report the first total synthesis of the two longest -subunits of the human glycoprotein hormones ( -hlh and -hcg ) , which were accessed as pure glycoproteins bearing defined carbohydrate structures . the successful approach is exemplified herein with the use of the disaccharide chitobiose at the two n - glycosylation sites and the monosaccharide n - acetyl - galactosamine ( galnac ) at the four o - glycosylation sites as model glycans for our glycoprotein assembly . the complexity of the hcg -subunit stems not only from its size ( the longest of all human glycoprotein hormones ) and the presence of the two n - glycosylation sites but also from the position of its unique , closely spaced four o - glycosylation sites at the carboxy - terminus of the molecule . fortunately , the relatively high content of cysteine residues in the peptide backbone at fairly regular intervals enables the assembly of both full - length glycoproteins from appropriately selected peptide fragments using the native chemical ligation ( ncl ) reaction . -hlh was envisioned to arise from three fragments of between 30 and 50 amino acids long , whereas our synthetic strategy for hcg relied on the assembly of five peptide segments , each bearing the corresponding n-/o - linked glycans in a highly modular fashion . ( a ) fully elaborated -subunit of hlh with chitobiose at the n - glycosylation site ( asn30 ) and proposed disconnection positions . ( b ) homogeneous full - length sequence of -hcg bearing two chitobiose ( asn13 and asn30 ) and four galnac moieties ( ser121 , ser127 , ser132 , and ser138 ) at the n- and o - glycosylation sites , respectively . ( c ) structure of n - linked carbohydrates at defined glycosylation sites on hlh and hcg polypeptide backbone ; elaborated dodecasaccharide ( left ) and chitobiose as model glycan ( right ) . ( d ) structure of representative o - linked glycans present in hcg peptide sequence : glycophorin tetrasaccharide ( left ) and simpler galnac . given the high sequence homology between both targets and the relatively simpler structure of -hlh , in comparison with -hcg , we chose to start with the synthesis of the former , en route to -hcg . in addition to providing access to hlh as one of the important glycoproteins within the family , this synthetic sequence could also serve as a proof of concept for the feasibility of the approach in terms of the following strategic steps : glycosylation via lansbury aspartylation , global deprotection of the glycopeptide segments , and the final union of these fragments by ncl . for the preparation of -hlh , we initially envisioned a retrosynthetic disconnection of the molecule in three fragments : -hlh(chitobiose)[s1y37 ] , -hlh[c38g71 ] , and -hlh[c72l121 ] ( figure 1a ) . the corresponding peptide fragments were obtained by fmoc - based solid - phase peptide synthesis ( spps ) with acid - labile protecting groups on the amino acid side chains , with the exception of the non - ncl - participating cysteine residues , in which case the acetamidomethyl ( acm ) group was chosen . in addition , the aspartic acid that will bear the chitobiose moiety was protected with a quasi - orthogonal o-2-phenylisopropyl ester ( o-2-phpr or opp ) group that can be removed selectively in the presence of tbu - based protecting groups by treatment with 12% trifluoroacetic acid ( tfa ) in dichloromethane . thus , after cleavage from the resin , the c - terminal carboxylic acid of the protected peptide was first coupled to tyrosine phenylthioester ( hclh - tyr - sph ) under sakakibara conditions , which are known to be epimerization free , and subsequent selective deprotection of asp30 provided the free carboxylic acid side chain ( pf1:-hlh[s1y37 ] ) . for the synthesis of the second fragment -hlh[z38g71 ] , the n - terminal cysteine was protected as a thiazolidine ( thz , z ) , and the c - terminal glycine residue was converted to glycine phenylthioester using pybop as a coupling agent . the fully deprotected peptide -hlh[c100l121 ] was obtained in two steps ( 64% ) , involving global acid deprotection ( cocktail b ) and conversion of the n - terminal thiazolidine ( thz100 ) to cysteine using methoxyamine hydrochloride ( meonh2hcl ) at ph 4.04.5 . finally , the n - terminal thz group was cleaved using a buffer solution of meonh2hcl and guanidinehcl in water at ph 4.04.5 to provide -hlh[c72l121 ] in 55% yield ( over three steps ) ( scheme 2 ) . the final assembly of the individual peptide fragments commenced with the coupling of segment -hlh[c72l121 ] with -hlh[z38g71 ] through ncl , followed by removal of the thz group to free the n - terminal cysteine required for the final ligation , in a one - flask procedure . having identified a successful route that featured ligation steps proceeding with complete conversion in reasonable reaction time frames , we set out to synthesize the larger and more complex -subunit of hcg incorporating two n - linked glycans and four closely spaced o - glycosylation sites . as for the glycosylation points , each of the n - linked glycans was coupled to the complementary peptide fragment via lansbury aspartylation , as shown earlier for -hlh . interestingly , a more challenging approach was also considered , consisting of the double incorporation , simultaneously , of the two n - linked carbohydrates on a longer peptide backbone comprising both n - glycosylation sites . with respect to the more complicated o - glycopeptide fragment , we envisioned the installation of all four o - linked glycans on solid support as glycosyl - serine cassettes during the synthesis of the carboxy - terminal segment . our initial target glycoform presents the readily available disaccharide chitobiose as a model n - linked glycan for the native complex bianntenary dodecasaccharide ( figure 1b and c ) , similar to that previously described for hlh . in the event , the glycine residue ( gly22 ) in fragment 1 was reacted with phsh under pybop - mediated coupling conditions to give the corresponding c - terminal thioester , whereas fragment 2 was subjected to single amino acid attachment with hclh - tyr - sph under epimerization - free sakakibara conditions ( edc , hoobt ) . since the same n - glycan ( chitobiose ) was to be installed at both fragments , a double lansbury aspartylation strategy was also explored for the synthesis of the entire bis - glycosylated segment -hcg(chitobiose)2[s1y37 ] ( scheme 4c ) . first , spps of the protected fragment , followed by cleavage from the resin , c - terminal thioesterification ( phsh , pybop ) , and subsequent selective asp(opp ) deprotection ( 2% tfa / dcm ) provided peptide segment pf1.2:-hcg[s1y37 ] having the two aspartic acid ( asp13 and asp30 ) side chains free for further coupling with two chitobiose units . fragment 3 was prepared in a very straightforward manner , beginning with spps , followed by cleavage from the resin and thioesterification of the c - terminus with phsh . fortunately , the use of a piperidine / oxyma pure cocktail for the fmoc - deprotection during the spps entirely suppressed this problem , and after single amino acid attachment ( hclh - thr - sph ) at the c - terminus , followed by subsequent treatment with cocktail b , the desired peptide fragment -hcg[z72t109 ] was obtained in 45% yield ( over two steps ) with no aspartimide formation ( scheme 5b ) . the presence of four o - linked glycans prohibits the late - stage attachment of these carbohydrates to the fully elaborated peptide . approach was followed , wherein the o - glycan , linked to a properly protected serine residue , is directly introduced in the peptide sequence as a conveniently protected glycosyl amino acid building block ( cassette ) , during solid - supported synthesis ( scheme 6a ) . this process not only allowed for assembly of this complex glycopeptide fragment but also , in principle , enables the installation of different o - linked glycans by simply applying the desired cassette during peptide backbone elongation on spps . importantly , this successful synthesis was amenable to scale - up , which afforded up to more than 50 mg of the most complex fragment of this synthetic version of -hcg bearing four closely spaced tn antigen structures . this strategy is likely to be applicable to the installation of alternative o - linked glycan cassettes at the four o - glycosylation sites for the assembly of different glycoforms . importantly , using these o - linked glycosylated building blocks , the cassette methodology has been successfully applied for the solution- and solid - phase synthesis of a series of mucin - derived o - glycopeptides bearing larger oligosaccharides . practical synthetic access to the carboxy - terminus fragment of -hcg is an important step in itself as it has been found to be a common epitope for hcg - based monoclonal antibodies . screening of the specificity of different glycoforms and their role in binding to these antibodies or the development of new glycoforms could be used as a powerful tool in important clinical applications . with all the prerequisite fragments in hand , assembly of the -subunit of hcg relied on the coupling of the individual peptide segments by ncl , starting with fragments -hcg[z72t109 ] and -hcg(ac3-galnac)4[c110q145 ] . upon completion of the reaction as monitored by ultraperformance liquid chromatography ( uplc ) , the terminal thz ( z ) protecting group was removed using meonh2hcl in a one - flask procedure . then , meonh2hcl was added , and the ph was adjusted to 4.04.5 to induce conversion of the n - terminal thz to cysteine . in the critical ligation event , o - glycosylated fragment -hcg(galnac)4[c38q145 ] was combined with n - glycopeptide -hcg(chitobiose)2[s1y37 ] under standard ncl conditions to give the target glycoprotein -hcg(chitobiose)2(galnac)4[s1q145 ] after 4 h. upon hplc purification , the primary sequence of the 145-residue -subunit of hcg containing homogeneous n- and o - linked glycans was obtained in a gratifying 33% yield ( scheme 8) [ see figure 2 for mass spectrum and lc trace ( uv ) of -hcg containing chitobiose at asn13 and asn30 , and n - acetylgalactosamine at ser121 , ser127 , ser132 , and ser138 ] . thus , compound -hcg(chitobiose)2(galnac)4[s1q145 ] , bearing not only two n - linked carbohydrates but also , and even more challenging , four o - linked sugars , represents the largest human glycoprotein hormone to have been synthesized in homogeneous form using strictly chemical means . while the current work has involved chitobiose and galnac as representative n-/o - linked model glycans , this demonstration of feasibility through the modular glycoform assembly presented herein opens the door to the application of this technology to the synthesis of a library of homogeneous glycoproteins via chemical synthesis by installing alternative glycans on the corresponding peptide fragments in summary , we have synthesized the glycosylated primary sequence of two complex glycoprotein hormones ( -hlh and -hcg ) in homogeneous form using the current innovations of peptide and glycopeptide chemistry . the first approach described for the preparation of -hlh served to validate the synthetic strategy en route to the more complex hcg -subunit . first , a double lansbury glycosylation was accomplished to provide the n - terminal fragment of the molecule in a highly modular fashion . second , practical access to the carboxy - terminus was gained by sequential installation of four o - linked glycosyl - amino acid cassettes into closely spaced o - glycosylation sites in a single and high - yielding solid - supported synthesis . this o - linked glycopeptide fragment was then successfully advanced by further coupling , deprotection , and subsequent ligations with the remaining peptide segments to provide the full - length -subunit of hcg . interestingly , the highly modular assembly exemplified herein sets the stage for accessing more complex , chemically pure -hcg glycoforms by synthesizing collections of each individual fragment containing a number of different , elaborated glycans and bringing them together following the successful ligation strategy outlined in this work . while the late - stage processes involving final acm removal and oxidation / folding remain to be validated for -hlh and -hcg , building on our previous successes with erythropoietin and other glycoprotein targets , we have confidence in the ability to generate correctly folded -subunits of these glycoprotein hormones . this important prospect should allow future biological studies of these well - defined glycoforms to better understand the specific roles of certain glycans in the function and bioactivity of hcg in clinical settings . this knowledge may , in turn , lead to the development of more efficacious and improved therapeutics .

as the third commonest cancer in males and females and the second commonest cause of cancer death , colorectal cancer ( crc ) is an important health problem in the world . there are estimated to be 334 000 new cases of crc in europe and 142 570 new cases in the united states , and 36% of these patients will succumb to their crc . of all patients with crc , approximately 65% develop distant metastasis , and the commonest location ( 40% ) is the liver . the proportion of patients who presented with synchronous versus metachronous colorectal liver metastases ( crlms ) in france was equal in a recent epidemiologic study : the proportion of patients with synchronous crlm and the 5-year rate of metachronous crlm were both 14% . unfortunately , only 25% of patients with crlm are amenable to curative - intent treatment [ 3 , 57 ] . since the first report of a laparoscopic liver resection ( lr ) in 1992 ( for crlm ) [ 8 , 9 ] , the field of minimally invasive liver resection ( milr ) has seen tremendous advances , paralleling those of open liver resection ( olr ) , with increasing safety and efficacy . although most early milrs were for benign disease , the first report notwithstanding , an increasing volume of nonrandomized data suggests no oncologic disadvantage to performing milr compared to olr . transabdominal us is a widely available , inexpensive , and noninvasive technique of evaluating for crlm but , compared with other modalities , has the lowest sensitivity and negative predictive value , excepting contrast - enhanced ultrasound , which by some studies is as sensitive as ct but which is not available in the united states . consequently , ct and mri are among the most commonly employed modalities used to evaluate the liver for crlm . a recent meta - analysis of diagnostic imaging of crlm , evaluating 39 articles ( 3391 patients ) , found mri to be the optimal first - line modality , with a per - patient sensitivity of 88% . while early ( 1990s ) studies showed staging laparoscopy ( sl ) to be superior to preoperative imaging in detecting unresectable or extrahepatic disease , thereby sparing as many as 34% of patients a laparotomy , when combined with intraoperative ultrasound ( ious ) , even in an era of markedly improved axial imaging ( 2000s ) , sl was able to detect unresectable disease and potentially prevent unnecessary laparotomies in 1025% of patients with primary and secondary hepatic malignancies [ 1416 ] . increasing quality in preoperative axial imaging technology , however , has challenged the use of laparoscopic ious . in a prospective study of 194 patients undergoing lr for crlm published in 2008 , tamandl et al . compared data from preoperative imaging using multidetector ct ( mdct ) and mri to intraoperative findings using ious and bimanual palpation and found that ious provided useful information regarding additional crlm in only 2.6% of patients . other groups , however , have consistently found that 10% of additional small tumors that were missed by axial imaging may be detected [ 18 , 19 ] and the preoperative treatment plan may change in nearly half of cases when a complete evaluation is performed , including exposure of the entire surface of the liver and porta hepatis , an ious scan of all 8 liver segments , porta hepatis and the paraceliac nodal bed , and a thorough evaluation of the entire peritoneal cavity to detect extrahepatic metastases . whether done laparoscopically or open , a complete us evaluation of the liver should include four steps : ( 1 ) an identification of intrahepatic vascular anatomy , ( 2 ) identification and characterization of known lesions , ( 3 ) a search for previously unrecognized lesions , and ( 4 ) the planning of a treatment strategy , which may include resection , ablation , or both . intrahepatic tumors are evaluated for size , number , location , relationship to biliary and vascular structures , and echogenicity ( most crlms are hypoechoic ( 42% ) or isoechoic ( 43% ) , while a minority ( 15% ) are hyperechoic ) . echogenicity should be noted because it has been shown to correlate with long - term survival : in a prospective evaluation of 147 patients at johns hopkins hospital , the 5-year survival for patients with hypo- , iso- , and hyperechoic crlm was 14% , 37% , and 46% , respectively . standard ablative techniques include both chemical and thermal ablation and are increasingly used , both in isolation for patients with unresectable crlm and in combination with lr . chemical ablation with ethanol or acetic acid has been performed for crlm but is less effective for crlm than for hepatocellular carcinoma [ 2123 ] . thermal ablation is therefore the preferred treatment for crlm not amenable to surgical resection . thermal ablation includes radiofrequency ablation ( rfa ) , microwave , laser , and cryoablation . in these techniques , focal heating or freezing of tumor cells causes local tumor destruction while preserving surrounding hepatic parenchyma . an emerging and still poorly studied technique is the nonthermal , nonchemical technique of electrical ablation using irreversible electroporation . although operative ablation of crlm is often done in combination with both milr and olr , percutaneous ablation of crlm is appropriate in cases in which no resection is planned and offers an attractive minimally invasive treatment option for such patients . however administered , rfa is relatively safe and less invasive than formal hepatic resection , but several notable complications may occur , including hepatic failure , hydrothorax , intraperitoneal bleeding , hepatic abscess , bile duct leaks , and tumor seeding [ 2428 ] . the reported procedure - related morbidity ranges from 2% to 12% and the mortality rate from 0% to 4.3% [ 24 , 2629 ] . many of these reports , however , include a variety of approaches , including percutaneous , laparoscopic , and open , and include more cases of hcc than crlm [ 24 , 2729 ] . given the frequent association of hcc with cirrhosis , complications such as bleeding , liver failure , and death may be more common following ablation of hcc than crlm [ 24 , 27 ] . in a recent report of 100 patients undergoing rfa for crlm , there was no procedure - related morality and the major complication rate was 8% . another study of 100 patients undergoing rfa ( 146 treatments ) for crlm revealed a major complication rate of 4.8% , including 1 death from liver failure . gillams and lees similarly found a major complication rate of 4.0% in a series of 167 patients undergoing rfa for crlm . microwave ablation ( mwa ) is an emerging technology that can also be performed open , percutaneously , or laparoscopically . like with rfa , however , no randomized trials support its use over other techniques . the primary theoretical advantages of mwa are the ability to ablate larger lesions and to do so faster . these advantages likely derive from the fact that mwa , unlike rfa , does not rely on electric current and so is not impeded by tissue desiccation and charring , both of which decrease electrical conductivity . in addition , there is no so - called heat sink effect , or heat loss from adjacent blood vessels , which in the case of rfa decreases the effectiveness and increases the time required for ablation . most reported experiences have demonstrated safe and effective mwa , with complications and local recurrence rates comparable to rfa [ 3335 ] . theoretical disadvantages include the inadvertent injury to adjacent structures and the risk of such collateral damage makes tumor location important in the decision of which modality ( e.g. , rfa versus mwa ) and route ( e.g. , open versus laparoscopic ) to choose . ablation of dome lesions or left lateral segment lesions , for instance , could expose the diaphragm and heart to thermal injury and serious morbidity , and such lesion may be better treated with an open or laparoscopic as opposed to percutaneous technique . it offers many of the same benefits of rfa and mwa , such as preservation of liver parenchyma , but at a potentially increased cost , given that the complication rates may be higher compared with rfa : morbidity 1040% and mortality 05% . complications include hepatic / iceball fracture ( 19% ) , hemorrhage ( 3.7% ) , coagulopathy ( 3.8% ) , biliary fistula ( 2.9% ) , and organ failure . furthermore , animal models have demonstrated that there is a more severe systemic response following cryotherapy than following rfa . irreversible electroporation ( ire ) is an emerging , nonthermal , nonchemical technique that uses electrical current to destroy cells . its reversible counterpart , reversible electroporation , is a common laboratory technique that has been employed for decades to transiently render cells widely permeable to allow entry of large molecules such as drugs and genes by applying an electrical field to cells that are otherwise not permeable to the molecules of interest . ire , previously viewed as an undesireable upper limit of reversible electroporation because it rendered the cells permanently permeable and therefore nonviable , is now used clinically as a form of nonthermal ablation . while supported by animal models [ 3941 ] and available commercially for use in patients , ire has not been well studied in patients . it does have the theoretical advantages , however , of being very fast ( micro- to milliseconds ) , preserving connective tissue architecture thereby allowing ablation close to vital structures in the liver hilum for example , and unlike thermal techniques it is not likely affected by blood flow . as with many new techniques , all cases of ire performed should be registered to document the role and safety of ire , as well as to identify important questions for study in clinical trials , and indeed such a registry is underway at the university of louisville . surgical resection is the standard of care for crlm , but rfa has produced comparable outcomes for limited disease , with some important caveats . although rfa has demonstrated 5-year survival rates as high as 30% in some studies [ 31 , 32 ] and numerous other studies have attempted to compare resection and ablation [ 4347 ] , recent analyses using propensity score methodology [ 48 , 49 ] have shown that comparison of survival rates following rfa and resection is not reliable , due to major differences in clinicopathologic characteristics , rfa technologies and expertise , and analysis of margin status ( namely , lack of pathologic analysis after rfa since no specimen is available ) . until randomized controlled trials comparing rfa and surgical resection are available , the question of their comparability will likely remain unanswered . in a recent systematic review performed by the american society of clinical oncology in 2009 regarding rfa for crlm , over 400 published articles were reviewed , 46 identified as having over 10 patients with adequate followup and perioperative data to analyze . there was a wide variability in the reported 5-year survival ( 1455% ) and local tumor recurrence ( 3.660% ) . while comparisons to resection may be unreliable and largely unrevealing , comparisons within rfa series for instance , it has become clear that increasing crlm size especially size > 3 cm is directly proportional to shorter survival and higher rates of recurrence [ 5254 ] . while some studies have found this to be true regardless of the approach ( open , laparoscopic , or percutaneous ) , other studies have found worse local tumor recurrence and disease - free survival in percutaneously treated patients compared with open cases . prognostic factors that contribute to overall outcome include node status of the primary crc resection , synchronous versus metachronous disease , number and size of lesions , margin status of resected hepatic lesions , cea levels , the presence of extrahepatic disease , satellite lesions and systemic treatment . rfa is an operator - dependent procedure , whether open , laparoscopic , or percutaneous , and requires careful technique and patient selection to achieve optimal outcomes . although initial laparoscopic liver surgery was typically limited to wedge resections of benign lesions that were easily accessible [ 8 , 9 , 56 ] , more advanced liver resections are now performed laparoscopically , including totally laparoscopic right , left , and central hepatectomies , extended right and left hepatectomies , and posterosuperior resections [ 5763 ] . many surgeons wishing to perform milr begin a stepwise process of beginning with easy minor resections ( figure 1 ) , later using a hand port [ 6467 ] or hybrid technique for complex resections before finally attempting totally laparoscopic major resections . however , even when the dissection and transection of the liver is totally laparoscopic in cases of extended hepatectomies , the specimen is large and requires an incision for removal [ 58 , 59 ] . this incision is often as long as that required for a hand port , which is one reason that many have advocated for maintaining and in some cases returning to a hand - assisted as opposed to a totally laparoscopic approach [ 66 , 67 ] . the main advantages of hals are tactile feedback , including ability to palpate the liver , tumor , and nodes , facile liver mobilization and retraction , quick and easy hemostasis with digital compression in cases of unexpected hemorrhage , and the multiple additional uses of the hand - port incision , including placement of additional instruments and removal of the specimen . the hybrid technique , combining the relatively basic laparoscopic skills of liver mobilization with the more advanced but open dissection of the liver hilum and transection of the liver parenchyma through a minilaparotomy , the small size of which is made possible by the laparoscopic mobilization , is another effort to shorten the learning curve ( see below ) associated with milr , thereby increasing the numbers of patients who may benefit from milr . the hybrid approach , like the hals approach , is meant to combine the advantages of laparoscopic surgery ( decreased postoperative pain and improved cosmesis , largely due to the avoidance of a subcostal incision ) with the safety , ease , and accessibility provided in open procedures . robotic surgery overcomes certain limitations encountered with the laparoscopic technique , such as 2-dimensional images and linear instruments , and offers clear advantages such as 3-dimensional imaging , tremor filtration , higher magnification , and articulating instruments with seven degrees of freedom . these advantages allow improved visualization and improved surgeon dexterity for fine movements , especially intracorporeal suturing . disadvantages inherent in the complex robotic technology include significant expense of the acquisition and maintenance of a robotic system , longer operating times , a significant learning curve to becoming proficient with the setup and utilization of the instruments , a loss of tactile feedback , and the requirement of a skilled assistant , as both the assistant and console surgeon perform integral parts of the hepatic transection [ 69 , 70 ] . the worldwide experience with robotic lr is somewhat limited with approximately 100 reported cases , but the approach appears to be safe in experienced hands [ 7174 ] . the largest series from giulianotti et al . reported 70 patients , 66 of whom underwent successful robotic resection and 16 of whom had colorectal metastases ( 4 minor resections and 12 major ) . the average surgical margin for colorectal metastases was 25 mm and the overall morbidity was 21% with a mortality of 0% and a median length of stay ( los ) of 7 days . two smaller series of robotic hepatectomies for crlm and other diagnoses berber et al . and ji et al . appear to have similar outcomes with 9 patients and 13 patients , respectively . the margins were not compromised in either group , with morbidity ranging from 7.8 to 11% , with no mortality [ 73 , 74 ] . even very complex cases , such as single - stage , combined liver and colon resection for synchronous crlm , have been performed with robotassisted laparoscopy . given that a number of studies suggest that regional ( as opposed to systemic ) chemotherapy may improve the response crlm and possibly improve survival in patients with resectable and unresectable crlm [ 7680 ] , the principle rational for hai catheter is that crlms derive 80% to 100% of their blood supply from the hepatic artery , as opposed to the portal vein . this allows for a high concentration of chemotherapeutic agents to be delivered directly into the tumors , with maximal effect on the metastatic lesion , minimal parenchymal and systemic toxicity , and minimal loss of activity due to the first - pass hepatic extraction . the laparoscopic approach provides a minimally invasive way to obtain such an access to the hepatic artery by avoiding the morbidity and mortality associated with standard laparotomy procedures . multiple studies have shown that laparoscopic hac placement is a feasible and safe procedure [ 8385 ] . hepatic artery infusion is only one of several ways to deliver therapies transarterially to the liver , thereby minimizing systemic toxicities . other transarterial therapies include transarterial embolization ( tae ) , transarterial chemoembolization ( tace ) , and intra - arterial radiotherapy ( iart ) , among others , all of which share with hai the advantages associated with delivery of therapy via the hepatic arterial system . there is not an overabundance of high - quality data supporting either tae or tace over the other , and , when compared , both have produced median survival results of 812 months [ 86 , 87 ] . nevertheless , since tace provides two therapies in one both an ischemic and a toxic insult to the crlm it is generally preferred . recently evaluated 121 patients undergoing 245 tace treatments and found a 27-month overall median survival using cisplatin , doxorubicin , mitomycin c , ethiodized oil , and polyvinyl alcohol particles . neither tace nor tae is as well studied as the treatment of crlm compared with hepatocellular carcinoma , and more data are needed to define this role . similarly , iart is the embolization of a radiation - emitter into the hepatic arteries feeding the crlm . the only fda - approved agent is yttrium-90 , a radioisotope that emits high - energy beta - particles , which was recently compared in a small phase - i study with folfox for unresectable crlm . a radiographic partial response was observed in 90% ( 18/20 ) of patients and stable disease in 10% ( 2/20 ) , with a progression - free survival of 9.3 months . as with tae and tace , a lack of high - quality data exists , as evidenced by a recent cochrane review that included only one randomized study : van hazel et al . compared iart plus systemic chemotherapy and systemic chemotherapy alone and found significantly longer progression - free survival following iart . as with tace and tae , more data are needed to improve decision - making in patients with advanced crlm . as minimally invasive techniques , surgeon familiarity with those techniques , and minimally invasive instruments particularly coagulation and stapling devices have improved , the number of patients undergoing milr has increased exponentially in the recent years ( figure 2 ) . although early series reported resections that were typically minor and performed for benign disease , increased experience has lead to more aggressive milr including malignant lesions and major hepatic resections . indeed , similar concerns were weighed against laparoscopic colon resection until randomized trials supported equivalent safety , negative - margins status , and disease - free survival [ 94 , 95 ] . a large and growing global experience ( but no randomized controlled trials ) suggests that laparoscopic resection is safe and effective for the management of liver lesions while maintaining oncologic integrity [ 64 , 65 , 92 ] . in the absence of randomized controlled trials comparing milr with olr , several observational studies have compared these modalities , including cohort studies , case - controlled studies , and intention - to - treat studies [ 96103 ] ; although they show similar rates of complications and survival , many of these contain mixed patient populations including benign and various malignant histologies . nguyen and colleagues reviewed all laparoscopic resections performed exclusively for crlm from 2000 to 2008 . they identified 109 cases , 97% of which were completed laparoscopically with a 94% negative - margin rate . forty - five percent of patients underwent a major ( 3 segments ) hepatectomy , with no mortalities and a 12% complication rate ; actuarial survival at 1 , 3 , and 5 years was 88% , 69% , and 50% , respectively , although no comparison to an open cohort was made . castaing et al . compared matched cases of laparoscopic and open hepatectomy performed for crlm and reported comparable 5-year survival rates of 64% and 56% for laparoscopic and open lr , respectively , thus supporting comparable oncologic outcomes . while the majority of the laparoscopic resections reported in the literature are minor ( <3 segments ) , dagher and colleagues studied a series of 210 major hepatectomies from 5 institutions , with 114 for malignant disease , reporting a mortality of 1% ( non - liver - related ) and an overall morbidity of 22% ( 8.1% liver - related : 6.2% bile leak , 1.4% ascites , and 0.5% hemorrhage ) . positive margins were identified in only 3 patients ( 2.6% ) , which compares favorably to the rate of margin positivity in olr for crlm which has had a wide variance from 5 to 24% [ 107109 ] . although margin status and oncologic outcomes appear favorable these results should be interpreted with caution , since selection bias is inherent in nonrandomized studies and patients deemed laparoscopic candidates may represent a different and more favorable group . in the castaing series , for instance , the operative details are variable , with vascular clamping occurring in 92% of the open versus 17% in the laparoscopic group . an interesting study performed by welsh et al . reviewed all patients who underwent olr for crlm and divided them into 2 groups , one suitable for milr and another deemed best candidates for olr . even though all resections were done open , those identified as milr candidates had a lower positive - margin rate ( 4.5% versus 15% ) and better 5-year survival ( 44% versus 37% ) . randomized data comparing milr and olr are , unfortunately , unlikely to be obtained because of difficulty in defining end points ( safety versus cost versus efficacy ) , none of which were deemed good end points for such a trial in the recent international consensus louisville statement , because of heterogeneity of the patient population and because of the length of time needed to accumulate enough patients . the louisville statement did support a prospective registry with preoperative enrollment , the creation of which would help track the dissemination of procedures aimed at improving patient safety . although initial nonrandomized data suggest that milr is likely a safe , oncologically sound means of managing crlm , ongoing critical review , multidisciplinary teams and participation in prospective data collection or trials will continue to define optimal approach to crlm . examination of cost has been reported by several groups in retrospective series . in a subgroup analysis from the university of louisville , buell et al . retrospectively reviewed and compared 29 laparoscopic and 34 open resections , finding significant actual cost savings for a laparoscopic approach to major resections ( $ 21,131 versus $ 36,821 ; p < 0.01 ) before , but not after , adjustment for changes in diagnosis - related group coding ( $ 25,457 versus $ 23,691 ; p < 0.2 ) . in a retrospective comparison of open versus laparoscopic left lateral segmentectomy , vanounou et al . at the university of pittsburgh used deviation - based cost modeling to analyze open and laparoscopic liver resections ( for both benign and malignant diseases ) and found that the overall los was 2 days shorter for the laparoscopic cases , which was associated with a per - patient cost reduction of $ 2939 compared to open cases ; when only malignant cases were considered , however , that cost reduction was attenuated at $ 1527 per patient . although both the louisville and the pittsburgh studies showed significantly shorter los for milr versus olr , other data , derived from comparing standard recovery pathway versus a fast - track recovery pathway following olr , have found a similar reduction in los ( 2 days ) following fast - track recovery . indeed , it has been argued [ 110 , 114 , 115 ] that los is determined more by extent of resection than by operative approach and may be as little as 24 days with either laparoscopic or open approach . broad surgical experience , corroborated by many studies [ 116118 ] , demonstrates the learning curve for milr . simillis et al . , for instance , showed that only in studies published after 2003 and in studies including 20 laparoscopic procedures did the operative blood loss , los , and complications decrease for laparoscopic liver surgery when compared to open lr . as the number of milr cases performed increases worldwide at a tremendous rate ( figure 2 ) and as the milr community increasingly recognizes the infeasibility of performing randomized trials of milr , there have been many calls for an international registry [ 64 , 92 ] of milr to maintain a record for monitoring outcomes of efficacy and patient safety , as has already been achieved for natural orifice transluminal endoscopic surgery , an experimental modality much more widely used for cholecystectomy ( 85% of all notes procedures ) than for milr [ 120 , 121 ] . training and credentialing of milr are currently performed at the local level and left to individual institutions . at a minimum a strong combined expertise in major laparoscopic surgery and advanced hepatobiliary techniques , including knowledge and skill in the use of intraoperative ultrasound , is required of a surgeon who wishes to begin the learning curve for milr [ 64 , 117 ] . currently , however , there are no clear , widely accepted criteria that define this required expertise . a certification process for milr has also yet to be defined and the american hepato - pancreato - biliary association does not currently require a specific number of milr cases for the training of hepatobiliary surgeons . many surgeons , however , recognize the need for a systematic progression from basic to advanced laparoscopic and hepatobiliary skills then to combined advanced laparoscopic and hepatobiliary skills , and some have published their experience with starting an milr program from scratch . the learning - curve effect has not been studied extensively in laparoscopic liver surgery . numerous single - institution series have shown an improvement in outcomes when the latter experience is compared to the early experience [ 124 , 125 ] . the first detailed analysis evaluating the learning curve effect in milr was published by vigano et al . in 2009 , revealing significantly improved operative time , conversion rate , blood loss , morbidity , and hospital stay progressively over time as experience and volume increased , despite an increase in operative complexity over time . the shape of the learning curve was , not surprisingly , similar to those reported regarding laparoscopic colectomy . these results suggest that milr is reproducible in selected high - volume centers , by surgeons with advanced laparoscopic and hepatobiliary training . although the liver is the most common site of colorectal metastasis , another 3.5% of patients with colon and 11.5% of patients with rectal cancer will develop lung metastasis , most of whom have both liver and lung diseases . although early ( prior to 2001 ) studies of curative - intent metastasectomy in patients with combined hepatic and pulmonary colorectal metastases found widely variable 5-year survival rates , ranging from 11% to 44% [ 129132 ] , more recent retrospective studies have produced improved 5-year survival rates as high as 64% [ 133137 ] in patients with colorectal pulmonary metastases . although fewer data are available regarding minimally invasive treatment of pulmonary as compared to hepatic colorectal metastases , large recent series have shown that pulmonary metastasectomies may be performed with minimally invasive video - assisted thoracic surgery . recently studied a series of 78 patients with 198 colorectal lung metastases treated with rfa , reporting a 5-year and median survival of 35% and 38 months , respectively . large series of patients with crc have revealed that 4% to 19% of patients have colorectal carcinomatosis at the time of the crc resection or in follow up , although as many as 80% of patients who die of crc have developed carcinomatosis by the time of their death [ 139 , 140 ] . in a review of nearly 3000 cases of crc in singapore , 349 ( 13% ) had carcinomatosis , 61% ( 214 ) of whom had disease that was synchronous and 39% ( 135 ) metachronous with their initial presentation of crc . the proportions of crlm patients with no carcinomatosis , with metachronous carcinomatosis , and with synchronous carcinomatosis were 10% , 33% , and 42% , respectively , respectively . an increasing body of data , including randomized controlled data , suggests that complete surgical eradication of metastatic peritoneal disease with cytoreduction and hyperthermic intraperitoneal chemotherapy ( hipec ) is beneficial to selected patients with colon cancer with carcinomatosis , with 5-year survival rates > 50% recently reported . although the presence of crlm is often considered a contraindication to cytoreduction and hipec , and similarly the presence of carcinomatosis a contraindication to lr , several recent studies [ 144147 ] have evaluated the combination of cytoreduction , hipec , and liver resection as an aggressive emerging option for highly selected crc patients with both crlm and peritoneal carcinomatosis . elias et al . evaluated a series of 24 such patients with a mean peritoneal cancer index ( pci ) of 8.6 ( range : 225 ) , half of whom underwent a major hepatectomy , and reported one postoperative death , a morbidity of 58% , and an overall 2-year survival of 61% . patients with 3 crlm had significantly worse survival compared with patients who had <3 crlm . more recently chua et al . evaluated a series of 16 patients with both crlm and carcinomatosis treated by combined cytoreduction , hipec , and liver resection and reported a 2-year survival of 65% ; neither survival nor perioperative factors such as morbidity and los were different compared with patients who had isolated carcinomatosis without crlm , although those with both crlm and carcinomatosis had significantly lower pci compared with patients with isolated carcinomatosis . most patients with crlm and peritoneal carcinomatosis are not , however , currently candidates for aggressive surgical resection of their crlm and these emerging data should be interpreted with caution . minimally invasive techniques have been used to perform cytoreduction to palliate metastatic disease to the ovaries , to completely remove primary ovarian carcinomas with limited peritoneal dissemination , and to do staged laparoscopic hipec following open cytoreduction . the results of an ongoing protocol on laparoscopic cytoreduction and hipec in patients with limited peritoneal dissemination appear promising and compare favorably to those patients having an open cytoreductive procedure , suggesting that combining milr and minimally invasive cytoreduction and hipec is on the horizon . the number of cases of milr performed in the world has increased exponentially in recent years , and many centers are now performing major , complex resections . in the absence of randomized trials comparing olr and milr , which is not likely obtainable , nonrandomized data suggest that milr produces similar or improved morbidity , mortality , los , and cost compared with olr , although significant selection bias exists . because randomized data will be difficult or impossible to obtain , patient registries should be used to track safety and efficacy outcomes . training of surgeons similarly should become more formalized , including an independent process for surgeon credentialing .

minimally invasive techniques used in the evaluation and treatment of colorectal liver metastases ( crlms ) include ultrasonography ( us ) , computed tomography , magnetic resonance imaging , percutaneous and operative ablation therapy , standard laparoscopic techniques , robotic techniques , and experimental techniques of natural orifice endoscopic surgery . laparoscopic techniques range from simple staging laparoscopy with or without laparoscopic intraoperative us , through intermediate techniques including simple liver resections ( lrs ) , to advanced techniques such as major hepatectomies . hereins , we review minimally invasive evaluation and treatment of crlm , focusing on a comparison of open lr ( olr ) and minimally invasive lr ( milr ) . although there are no randomized trials comparing olr and milr , nonrandomized data suggest that milr compares favorably with olr regarding morbidity , mortality , los , and cost , although significant selection bias exists . the future of milr will likely include expanding criteria for resectability of crlm and should include both a patient registry and a formalized process for surgeon training and credentialing .

1. Introduction 2. Minimally Invasive Evaluation 3. Minimally Invasive Treatment Techniques 4. Comparison of OLR and MILR for CRLM 5. Future Directions and Controversies 6. Summary

there are estimated to be 334 000 new cases of crc in europe and 142 570 new cases in the united states , and 36% of these patients will succumb to their crc . of all patients with crc , approximately 65% develop distant metastasis , and the commonest location ( 40% ) is the liver . the proportion of patients who presented with synchronous versus metachronous colorectal liver metastases ( crlms ) in france was equal in a recent epidemiologic study : the proportion of patients with synchronous crlm and the 5-year rate of metachronous crlm were both 14% . since the first report of a laparoscopic liver resection ( lr ) in 1992 ( for crlm ) [ 8 , 9 ] , the field of minimally invasive liver resection ( milr ) has seen tremendous advances , paralleling those of open liver resection ( olr ) , with increasing safety and efficacy . transabdominal us is a widely available , inexpensive , and noninvasive technique of evaluating for crlm but , compared with other modalities , has the lowest sensitivity and negative predictive value , excepting contrast - enhanced ultrasound , which by some studies is as sensitive as ct but which is not available in the united states . a recent meta - analysis of diagnostic imaging of crlm , evaluating 39 articles ( 3391 patients ) , found mri to be the optimal first - line modality , with a per - patient sensitivity of 88% . while early ( 1990s ) studies showed staging laparoscopy ( sl ) to be superior to preoperative imaging in detecting unresectable or extrahepatic disease , thereby sparing as many as 34% of patients a laparotomy , when combined with intraoperative ultrasound ( ious ) , even in an era of markedly improved axial imaging ( 2000s ) , sl was able to detect unresectable disease and potentially prevent unnecessary laparotomies in 1025% of patients with primary and secondary hepatic malignancies [ 1416 ] . compared data from preoperative imaging using multidetector ct ( mdct ) and mri to intraoperative findings using ious and bimanual palpation and found that ious provided useful information regarding additional crlm in only 2.6% of patients . other groups , however , have consistently found that 10% of additional small tumors that were missed by axial imaging may be detected [ 18 , 19 ] and the preoperative treatment plan may change in nearly half of cases when a complete evaluation is performed , including exposure of the entire surface of the liver and porta hepatis , an ious scan of all 8 liver segments , porta hepatis and the paraceliac nodal bed , and a thorough evaluation of the entire peritoneal cavity to detect extrahepatic metastases . whether done laparoscopically or open , a complete us evaluation of the liver should include four steps : ( 1 ) an identification of intrahepatic vascular anatomy , ( 2 ) identification and characterization of known lesions , ( 3 ) a search for previously unrecognized lesions , and ( 4 ) the planning of a treatment strategy , which may include resection , ablation , or both . intrahepatic tumors are evaluated for size , number , location , relationship to biliary and vascular structures , and echogenicity ( most crlms are hypoechoic ( 42% ) or isoechoic ( 43% ) , while a minority ( 15% ) are hyperechoic ) . standard ablative techniques include both chemical and thermal ablation and are increasingly used , both in isolation for patients with unresectable crlm and in combination with lr . thermal ablation includes radiofrequency ablation ( rfa ) , microwave , laser , and cryoablation . although operative ablation of crlm is often done in combination with both milr and olr , percutaneous ablation of crlm is appropriate in cases in which no resection is planned and offers an attractive minimally invasive treatment option for such patients . however administered , rfa is relatively safe and less invasive than formal hepatic resection , but several notable complications may occur , including hepatic failure , hydrothorax , intraperitoneal bleeding , hepatic abscess , bile duct leaks , and tumor seeding [ 2428 ] . given the frequent association of hcc with cirrhosis , complications such as bleeding , liver failure , and death may be more common following ablation of hcc than crlm [ 24 , 27 ] . in a recent report of 100 patients undergoing rfa for crlm , there was no procedure - related morality and the major complication rate was 8% . like with rfa , however , no randomized trials support its use over other techniques . , rfa versus mwa ) and route ( e.g. ablation of dome lesions or left lateral segment lesions , for instance , could expose the diaphragm and heart to thermal injury and serious morbidity , and such lesion may be better treated with an open or laparoscopic as opposed to percutaneous technique . it offers many of the same benefits of rfa and mwa , such as preservation of liver parenchyma , but at a potentially increased cost , given that the complication rates may be higher compared with rfa : morbidity 1040% and mortality 05% . complications include hepatic / iceball fracture ( 19% ) , hemorrhage ( 3.7% ) , coagulopathy ( 3.8% ) , biliary fistula ( 2.9% ) , and organ failure . its reversible counterpart , reversible electroporation , is a common laboratory technique that has been employed for decades to transiently render cells widely permeable to allow entry of large molecules such as drugs and genes by applying an electrical field to cells that are otherwise not permeable to the molecules of interest . it does have the theoretical advantages , however , of being very fast ( micro- to milliseconds ) , preserving connective tissue architecture thereby allowing ablation close to vital structures in the liver hilum for example , and unlike thermal techniques it is not likely affected by blood flow . as with many new techniques , all cases of ire performed should be registered to document the role and safety of ire , as well as to identify important questions for study in clinical trials , and indeed such a registry is underway at the university of louisville . surgical resection is the standard of care for crlm , but rfa has produced comparable outcomes for limited disease , with some important caveats . although rfa has demonstrated 5-year survival rates as high as 30% in some studies [ 31 , 32 ] and numerous other studies have attempted to compare resection and ablation [ 4347 ] , recent analyses using propensity score methodology [ 48 , 49 ] have shown that comparison of survival rates following rfa and resection is not reliable , due to major differences in clinicopathologic characteristics , rfa technologies and expertise , and analysis of margin status ( namely , lack of pathologic analysis after rfa since no specimen is available ) . until randomized controlled trials comparing rfa and surgical resection are available , the question of their comparability will likely remain unanswered . in a recent systematic review performed by the american society of clinical oncology in 2009 regarding rfa for crlm , over 400 published articles were reviewed , 46 identified as having over 10 patients with adequate followup and perioperative data to analyze . there was a wide variability in the reported 5-year survival ( 1455% ) and local tumor recurrence ( 3.660% ) . rfa is an operator - dependent procedure , whether open , laparoscopic , or percutaneous , and requires careful technique and patient selection to achieve optimal outcomes . although initial laparoscopic liver surgery was typically limited to wedge resections of benign lesions that were easily accessible [ 8 , 9 , 56 ] , more advanced liver resections are now performed laparoscopically , including totally laparoscopic right , left , and central hepatectomies , extended right and left hepatectomies , and posterosuperior resections [ 5763 ] . many surgeons wishing to perform milr begin a stepwise process of beginning with easy minor resections ( figure 1 ) , later using a hand port [ 6467 ] or hybrid technique for complex resections before finally attempting totally laparoscopic major resections . the main advantages of hals are tactile feedback , including ability to palpate the liver , tumor , and nodes , facile liver mobilization and retraction , quick and easy hemostasis with digital compression in cases of unexpected hemorrhage , and the multiple additional uses of the hand - port incision , including placement of additional instruments and removal of the specimen . the hybrid approach , like the hals approach , is meant to combine the advantages of laparoscopic surgery ( decreased postoperative pain and improved cosmesis , largely due to the avoidance of a subcostal incision ) with the safety , ease , and accessibility provided in open procedures . robotic surgery overcomes certain limitations encountered with the laparoscopic technique , such as 2-dimensional images and linear instruments , and offers clear advantages such as 3-dimensional imaging , tremor filtration , higher magnification , and articulating instruments with seven degrees of freedom . disadvantages inherent in the complex robotic technology include significant expense of the acquisition and maintenance of a robotic system , longer operating times , a significant learning curve to becoming proficient with the setup and utilization of the instruments , a loss of tactile feedback , and the requirement of a skilled assistant , as both the assistant and console surgeon perform integral parts of the hepatic transection [ 69 , 70 ] . reported 70 patients , 66 of whom underwent successful robotic resection and 16 of whom had colorectal metastases ( 4 minor resections and 12 major ) . the average surgical margin for colorectal metastases was 25 mm and the overall morbidity was 21% with a mortality of 0% and a median length of stay ( los ) of 7 days . even very complex cases , such as single - stage , combined liver and colon resection for synchronous crlm , have been performed with robotassisted laparoscopy . given that a number of studies suggest that regional ( as opposed to systemic ) chemotherapy may improve the response crlm and possibly improve survival in patients with resectable and unresectable crlm [ 7680 ] , the principle rational for hai catheter is that crlms derive 80% to 100% of their blood supply from the hepatic artery , as opposed to the portal vein . this allows for a high concentration of chemotherapeutic agents to be delivered directly into the tumors , with maximal effect on the metastatic lesion , minimal parenchymal and systemic toxicity , and minimal loss of activity due to the first - pass hepatic extraction . other transarterial therapies include transarterial embolization ( tae ) , transarterial chemoembolization ( tace ) , and intra - arterial radiotherapy ( iart ) , among others , all of which share with hai the advantages associated with delivery of therapy via the hepatic arterial system . there is not an overabundance of high - quality data supporting either tae or tace over the other , and , when compared , both have produced median survival results of 812 months [ 86 , 87 ] . neither tace nor tae is as well studied as the treatment of crlm compared with hepatocellular carcinoma , and more data are needed to define this role . as minimally invasive techniques , surgeon familiarity with those techniques , and minimally invasive instruments particularly coagulation and stapling devices have improved , the number of patients undergoing milr has increased exponentially in the recent years ( figure 2 ) . indeed , similar concerns were weighed against laparoscopic colon resection until randomized trials supported equivalent safety , negative - margins status , and disease - free survival [ 94 , 95 ] . in the absence of randomized controlled trials comparing milr with olr , several observational studies have compared these modalities , including cohort studies , case - controlled studies , and intention - to - treat studies [ 96103 ] ; although they show similar rates of complications and survival , many of these contain mixed patient populations including benign and various malignant histologies . forty - five percent of patients underwent a major ( 3 segments ) hepatectomy , with no mortalities and a 12% complication rate ; actuarial survival at 1 , 3 , and 5 years was 88% , 69% , and 50% , respectively , although no comparison to an open cohort was made . compared matched cases of laparoscopic and open hepatectomy performed for crlm and reported comparable 5-year survival rates of 64% and 56% for laparoscopic and open lr , respectively , thus supporting comparable oncologic outcomes . while the majority of the laparoscopic resections reported in the literature are minor ( <3 segments ) , dagher and colleagues studied a series of 210 major hepatectomies from 5 institutions , with 114 for malignant disease , reporting a mortality of 1% ( non - liver - related ) and an overall morbidity of 22% ( 8.1% liver - related : 6.2% bile leak , 1.4% ascites , and 0.5% hemorrhage ) . positive margins were identified in only 3 patients ( 2.6% ) , which compares favorably to the rate of margin positivity in olr for crlm which has had a wide variance from 5 to 24% [ 107109 ] . although margin status and oncologic outcomes appear favorable these results should be interpreted with caution , since selection bias is inherent in nonrandomized studies and patients deemed laparoscopic candidates may represent a different and more favorable group . randomized data comparing milr and olr are , unfortunately , unlikely to be obtained because of difficulty in defining end points ( safety versus cost versus efficacy ) , none of which were deemed good end points for such a trial in the recent international consensus louisville statement , because of heterogeneity of the patient population and because of the length of time needed to accumulate enough patients . although initial nonrandomized data suggest that milr is likely a safe , oncologically sound means of managing crlm , ongoing critical review , multidisciplinary teams and participation in prospective data collection or trials will continue to define optimal approach to crlm . in a retrospective comparison of open versus laparoscopic left lateral segmentectomy , vanounou et al . at the university of pittsburgh used deviation - based cost modeling to analyze open and laparoscopic liver resections ( for both benign and malignant diseases ) and found that the overall los was 2 days shorter for the laparoscopic cases , which was associated with a per - patient cost reduction of $ 2939 compared to open cases ; when only malignant cases were considered , however , that cost reduction was attenuated at $ 1527 per patient . , for instance , showed that only in studies published after 2003 and in studies including 20 laparoscopic procedures did the operative blood loss , los , and complications decrease for laparoscopic liver surgery when compared to open lr . as the number of milr cases performed increases worldwide at a tremendous rate ( figure 2 ) and as the milr community increasingly recognizes the infeasibility of performing randomized trials of milr , there have been many calls for an international registry [ 64 , 92 ] of milr to maintain a record for monitoring outcomes of efficacy and patient safety , as has already been achieved for natural orifice transluminal endoscopic surgery , an experimental modality much more widely used for cholecystectomy ( 85% of all notes procedures ) than for milr [ 120 , 121 ] . training and credentialing of milr are currently performed at the local level and left to individual institutions . at a minimum a strong combined expertise in major laparoscopic surgery and advanced hepatobiliary techniques , including knowledge and skill in the use of intraoperative ultrasound , is required of a surgeon who wishes to begin the learning curve for milr [ 64 , 117 ] . currently , however , there are no clear , widely accepted criteria that define this required expertise . a certification process for milr has also yet to be defined and the american hepato - pancreato - biliary association does not currently require a specific number of milr cases for the training of hepatobiliary surgeons . many surgeons , however , recognize the need for a systematic progression from basic to advanced laparoscopic and hepatobiliary skills then to combined advanced laparoscopic and hepatobiliary skills , and some have published their experience with starting an milr program from scratch . in 2009 , revealing significantly improved operative time , conversion rate , blood loss , morbidity , and hospital stay progressively over time as experience and volume increased , despite an increase in operative complexity over time . these results suggest that milr is reproducible in selected high - volume centers , by surgeons with advanced laparoscopic and hepatobiliary training . although fewer data are available regarding minimally invasive treatment of pulmonary as compared to hepatic colorectal metastases , large recent series have shown that pulmonary metastasectomies may be performed with minimally invasive video - assisted thoracic surgery . the proportions of crlm patients with no carcinomatosis , with metachronous carcinomatosis , and with synchronous carcinomatosis were 10% , 33% , and 42% , respectively , respectively . although the presence of crlm is often considered a contraindication to cytoreduction and hipec , and similarly the presence of carcinomatosis a contraindication to lr , several recent studies [ 144147 ] have evaluated the combination of cytoreduction , hipec , and liver resection as an aggressive emerging option for highly selected crc patients with both crlm and peritoneal carcinomatosis . evaluated a series of 24 such patients with a mean peritoneal cancer index ( pci ) of 8.6 ( range : 225 ) , half of whom underwent a major hepatectomy , and reported one postoperative death , a morbidity of 58% , and an overall 2-year survival of 61% . evaluated a series of 16 patients with both crlm and carcinomatosis treated by combined cytoreduction , hipec , and liver resection and reported a 2-year survival of 65% ; neither survival nor perioperative factors such as morbidity and los were different compared with patients who had isolated carcinomatosis without crlm , although those with both crlm and carcinomatosis had significantly lower pci compared with patients with isolated carcinomatosis . minimally invasive techniques have been used to perform cytoreduction to palliate metastatic disease to the ovaries , to completely remove primary ovarian carcinomas with limited peritoneal dissemination , and to do staged laparoscopic hipec following open cytoreduction . the results of an ongoing protocol on laparoscopic cytoreduction and hipec in patients with limited peritoneal dissemination appear promising and compare favorably to those patients having an open cytoreductive procedure , suggesting that combining milr and minimally invasive cytoreduction and hipec is on the horizon . the number of cases of milr performed in the world has increased exponentially in recent years , and many centers are now performing major , complex resections . in the absence of randomized trials comparing olr and milr , which is not likely obtainable , nonrandomized data suggest that milr produces similar or improved morbidity , mortality , los , and cost compared with olr , although significant selection bias exists . training of surgeons similarly should become more formalized , including an independent process for surgeon credentialing .